Anti-B7H3 antibodies and uses thereof

Abstract

The present disclosure provides antibodies, or antigen-binding portions thereof, that bind to B7H3, methods of producing the antibodies, and methods of treating diseases associated with B7H3 using the antibodies, or antigen-binding portions thereof.

Description

[0001] Cross-references

[0002] This application claims priority to international application PCT / CN2022 / 093524 filed on May 18, 2022, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application submits a sequence listing in electronic form. The entire contents of the sequence listing are hereby incorporated by reference. Technical Field

[0005] The present disclosure generally relates to anti-B7H3 antibodies or antigen-binding portions thereof, methods of making the same, and uses thereof. Background Art

[0006] B7H3, also known as CD276 and B7RP-2, is a member of the B7 family and has 20%-27% amino acid identity with other B7 family members. Although B7H3 transcripts are ubiquitously expressed, they are limited and maintained at low levels on normal tissues and immune cells, and are overexpressed in a variety of human malignancies (including melanoma, breast cancer, prostate cancer, etc.). It is reported that B7-H3 is expressed on the membrane, cytoplasm, or nucleus of cancer cells, but is also expressed on tumor-associated vasculature. B7H3 is not constitutively expressed on T cells, NK cells, and APCs (including DCs and macrophages), but its expression on APCs can be induced by GM-CSF, IFNγ, etc.

[0007] B7-H3 is a promising anticancer target. The exact function of B7-H3 is still unclear, but its different immune functions have been confirmed, including stimulation, inhibition of T-cell proliferation, and inhibition of NK cell function. It is speculated that there are different receptors on immune cells that compete for binding to B7H3 on tumors. In addition to its immune checkpoint function, high expression levels of B7H3 have been reported to be associated with poor prognosis in cancer and also enhance cell proliferation, migration, invasion, angiogenesis, metastatic ability, and anticancer drug resistance.

[0008] Similar to other B7 family molecules, B7H3 is a type 1 transmembrane glycoprotein whose extracellular domain contains an IgV-IgC domain pair. Unlike the mouse B7H3 gene, which has only a single copy of the IgV-IgC domain pair, human B7H3 was found to have two isoforms. One isoform contains a single copy of the IgV-IgC domain pair (named 2IgB7H3), and the other isoform has two IgV-IgC domain pairs (named 4IgB7H3), which are generated by gene duplication and differential splicing. 4IgB7H3 is the major isoform expressed on immune cells as well as malignant cells, rather than 2IgB7H3, suggesting that 4IgB7H3 may play a significant and important role in tumor development and cancer immunity.

[0009] Since B7H3 is overexpressed on many tumor cells, therapeutic molecules targeting B7H3 are being developed to treat related indications. However, there is still room for improvement and clinical need for the development of novel anti-B7H3 antibodies. Summary of the invention

[0010] The present disclosure provides these and other objects, and broadly relates to compounds, methods, compositions and articles of manufacture that provide antibodies with improved therapeutic efficacy. The benefits provided by the present disclosure are broadly applicable to the field of antibody therapy and diagnosis, and can be used in combination with antibodies reactive with a variety of targets.

[0011] In one aspect, the present disclosure provides an antibody or antigen binding portion thereof that specifically binds to B7H3. The B7H3 antigen can be human B7H3, mouse B7H3 and / or cynomolgus monkey B7H3 or a fragment thereof (e.g., an extracellular domain). Preferably, the B7H3 antigen is a human 4IgB7H3 protein or an extracellular domain thereof.

[0012] In some embodiments, an antibody, or antigen binding portion thereof, as disclosed herein comprises:

[0013] A heavy chain CDR1 (HCDR1) comprising the amino acid sequence of SEQ ID No: 1; a HCDR2 comprising the amino acid sequence of SEQ ID No: 2; a HCDR3 comprising the amino acid sequence of SEQ ID No: 3; and

[0014] A light chain CDR1 (LCDR1) comprising the amino acid sequence of SEQ ID No: 4 or 7; a LCDR2 comprising the amino acid sequence of SEQ ID No: 5; and a LCDR3 comprising the amino acid sequence of SEQ ID No: 6.

[0015] In some embodiments, an antibody, or antigen binding portion thereof, as disclosed herein comprises:

[0016] A heavy chain CDR1 (HCDR1) comprising the amino acid sequence of SEQ ID No: 1; a HCDR2 comprising the amino acid sequence of SEQ ID No: 2; a HCDR3 comprising the amino acid sequence of SEQ ID No: 3; and

[0017] A light chain CDR1 (LCDR1) comprising the amino acid sequence of KSSQSLLX1SSNQKNYLA, wherein X1 can be any amino acid except N; a LCDR2 comprising the amino acid sequence of SEQ ID No: 5; and a LCDR3 comprising the amino acid sequence of SEQ ID No: 6.

[0018] Preferably, X1 as described above is Q, and LCDR1 comprises the amino acid sequence of SEQ ID No:4.

[0019] In some embodiments, an antibody, or antigen binding portion thereof, as disclosed herein comprises:

[0020] A heavy chain CDR1 (HCDR1) comprising the amino acid sequence of SEQ ID No: 1; a HCDR2 comprising the amino acid sequence of SEQ ID No: 2; a HCDR3 comprising the amino acid sequence of SEQ ID No: 3; and

[0021] A light chain CDR1 (LCDR1) comprising the amino acid sequence of KSSQSLLNX2SNQKNYLA, wherein X can be any amino acid except S; a LCDR2 comprising the amino acid sequence of SEQ ID No: 5; and a LCDR3 comprising the amino acid sequence of SEQ ID No: 6.

[0022] Preferably, X2 as described above is P, and LCDR1 comprises the amino acid sequence of SEQ ID No:18.

[0023] In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises:

[0024] (A) the amino acid sequence shown in SEQ ID No: 8 or 10;

[0025] (B) an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to SEQ ID No: 8 or 10; or

[0026] (C) an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid additions, deletions and / or substitutions in the framework regions compared to SEQ ID No: 8 or 10;

[0027] and / or VL contains:

[0028] (A) the amino acid sequence shown in SEQ ID No: 9 or 11;

[0029] (B) an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to SEQ ID No: 9 or 11; or

[0030] (C) an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid additions, deletions and / or substitutions in the framework regions compared to SEQ ID No: 9 or 11.

[0031] In some embodiments, the antibody or antigen binding portion thereof comprises a VH and / or VL comprising one or more amino acid substitutions in the framework region. In some embodiments, at least one of the amino acid substitutions is a back mutation in which an amino acid from a human germline sequence is substituted with a different amino acid at the corresponding position in the parent antibody.

[0032] In some embodiments, at least one of the amino acid substitutions removes a potential post-translational modification site or a glycosylation site. Such amino acid modifications may be in a CDR region or a framework region.

[0033] In some embodiments, the antibody, or antigen binding portion thereof, is a full length antibody, ScFv, Fab, F(ab')2, or Fv fragment.

[0034] In some embodiments, the antibody further comprises an immunoglobulin constant region, such as a human IgG constant region, optionally a human IgG1 constant region. In some embodiments, the IgG1 constant region comprises one or more modifications, such as a LALA mutation.

[0035] In some embodiments, the antibody, or antigen-binding portion thereof, is a chimeric antibody or a humanized antibody.

[0036] In some embodiments, the antibody, or antigen-binding portion thereof, comprises:

[0037] (a) a heavy chain comprising the amino acid sequence of SEQ ID No: 12 and a light chain comprising the amino acid sequence of SEQ ID No: 13; or

[0038] (b) A heavy chain comprising the amino acid sequence of SEQ ID No: 14 and a light chain comprising the amino acid sequence of SEQ ID No: 15.

[0039] In one aspect, the disclosure provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding an antibody, or antigen-binding portion thereof, as defined above.

[0040] In one aspect, the present disclosure provides a vector comprising a nucleic acid molecule as defined above.

[0041] In one aspect, the present disclosure provides a host cell comprising a nucleic acid molecule or a vector as defined above.

[0042] In one aspect, the present disclosure provides a pharmaceutical composition comprising an antibody, or antigen-binding portion thereof, as defined above and a pharmaceutically acceptable carrier.

[0043] In one aspect, the present disclosure provides a method for producing an antibody or antigen binding portion thereof as defined above, the method comprising the steps of:

[0044] - culturing the host cell under conditions suitable for expression of the antibody or antigen-binding portion thereof; and

[0045] -Isolating the antibody or antigen-binding portion thereof from the host cell.

[0046] In one aspect, the present disclosure provides a method for modulating a B7H3-related immune response in a subject, the method comprising administering to the subject an antibody or antigen-binding portion thereof or a pharmaceutical composition as defined above.

[0047] In one aspect, the present disclosure provides a method for preventing or treating a B7H3-related disease in a subject, the method comprising administering to the subject an effective amount of the antibody or antigen-binding portion thereof or the pharmaceutical composition as defined above.

[0048] In some embodiments, the B7H3-related disease is selected from cancer, autoimmune diseases and infectious diseases. The cancer can be selected from breast cancer, neurological tumors, melanoma, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, prostate cancer, ovarian cancer, cervical cancer, glioblastoma, esophageal cancer, bladder cancer, renal cell carcinoma, endometrial cancer, skin cancer, testicular cancer, thyroid cancer, urothelial carcinoma, lymphoma such as non-Hodgkin's lymphoma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma and multiple myeloma.

[0049] In some embodiments, the antibody, or antigen-binding portion thereof, is administered in combination with cellular immunotherapy, chemotherapeutic agents, radiation therapy, targeted therapy, and / or other agents used in cancer immunotherapy.

[0050] In one aspect, the present disclosure provides an antibody or antigen-binding portion thereof as defined above for use in treating or preventing a B7H3-associated disease in a subject.

[0051] In one aspect, the present disclosure provides use of an antibody or antigen-binding portion thereof as defined above for the preparation of a medicament for treating or preventing a B7H3-related disease in a subject.

[0052] In one aspect, the present disclosure provides a kit comprising an antibody, or antigen-binding portion thereof, as defined above.

[0053] The foregoing is an overview and therefore necessarily includes simplifications, generalizations, and omissions of detail; therefore, those skilled in the art will appreciate that the overview is merely illustrative and is not intended to be limiting in any way. The overview is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The SDS-PAGE results of W301088-1.145.16-z3-p1-uIgG1KV320 are shown. M: PageRuler TM Unstained protein ladder; Lane 1: W301088-1.145.16-z3-p1-uIgG1KV320, non-reducing; Lane 2: W301088-1.145.16-z3-p1-uIgG1KV320, reducing; Gel information: NuPAGE, Novex 4-12% Bis-Tris gel.

[0055] Figure 2 HLPC-SEC results of W301088-1.145.16-z3-p1-uIgG1KV320 are shown.

[0056] Figure 3 The DSF curve of W301088-1.145.16-z3-p1-uIgG1KV320 is shown.

[0057] Figure 4 The HIC-HPLC curve of W301088-1.145.16-z3-p1-uIgG1KV320 is shown.

[0058] Figure 5 DLS-k of W301088-1.145.16-z3-p1-uIgG1KV320 is shown. D picture.

[0059] Figure 6 The FACS binding results of the antibodies to MCF-7 cells expressing human B7H3 are shown.

[0060] Figure 7 Shown are the FACS binding results of antibodies to cynomolgus monkey B7H3-transfected cells.

[0061] Figure 8A-8CBinding of W301088-1.145.16-xIgG1KV320 (A), W301088-1.145.16-z3-pl-uIgG1KV320 (B), and MacroGenics' Enochumab (C) to human 4IgB7H3 is shown.

[0062] Figure 9A-9C Binding of W301088-1.145.16-xIgG1KV320 (A), W301088-1.145.16-z3-pl-uIgG1KV320 (B), and MacroGenics' Enochumab (C) to human 2IgB7H3 is shown.

[0063] Figure 10A-10C Binding of W301088-1.145.16-xIgG1KV320 (A), W301088-1.145.16-z3-p1-uIgG1KV320 (B), and MacroGenics' enoxitumomab (C) to cynomolgus monkey B7H3 is shown.

[0064] Fig.11 Internalization of different antibodies by MCF-7 cells expressing human B7H3 is shown.

[0065] Figure 12A-12B The alignment of the VH and VL regions between W301088-1.145.16-xIgG1KV320 and W301088-1.145.16-z3-p1-uIgG1KV320 is shown. DETAILED DESCRIPTION

[0066] Although the present disclosure can be implemented in many different forms, disclosed herein are specific illustrative embodiments thereof that illustrate the principles of the present disclosure. It should be emphasized that the present disclosure is not limited to the specific embodiments illustrated. In addition, the section headings used herein are only for organizational purposes and should not be interpreted as limiting the subject matter described.

[0067] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. More specifically, as used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "one", "a", "the", "said" include plural references. Thus, for example, reference to "a protein" includes multiple proteins; reference to "a cell" includes a mixture of cells, etc. In this application, unless otherwise indicated, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms (such as "including" and "containing") is not restrictive. In addition, the scope provided in the specification and the appended claims includes both endpoints and all points between the endpoints.

[0068] In the context of the present disclosure, the term "about" represents an accuracy interval, and those of ordinary skill in the art understand that the technical effect of the feature in question is still ensured in this accuracy interval. The term generally represents a deviation from the indicated value of ±5%, such as ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, and such as ±0.01%. Those of ordinary skill in the art will understand that this specific deviation of the numerical value of a given technical effect will depend on the nature of the technical effect. For example, a natural technical effect or a biotechnological effect may generally have a larger deviation than an artificial or engineered technical effect.

[0069] Generally, nomenclature used in connection with and techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, e.g., Abbas et al., Cellular and Molecular Immunology, 6th ed., WB Saunders Company (2010); Sambrook J. and Russell D., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The nomenclature, laboratory procedures, and techniques used in connection with the analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those well known and commonly used in the art. In addition, the contents of all references, patents, and published patent applications cited throughout this application are incorporated herein by reference.

[0070] definition

[0071] In order to better understand the present disclosure, the following definitions and explanations of relevant terms are provided.

[0072] The term "antibody" or "Ab" herein is used in the broadest sense, covering various antibody structures, including polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies). Natural intact antibodies are typically Y-shaped tetrameric proteins that include two heavy chains (H) and two light chains (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. The light chains of antibodies can be classified into kappa light chains and lambda light chains. The heavy chains can be classified into μ, δ, γ, α, and ε, which define the isotypes of antibodies as IgM, IgD, IgG, IgA, and IgE, respectively. In the light and heavy chains, the variable region is connected to the constant region via a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H ) and the heavy chain constant region (C H ). The heavy chain constant region consists of three domains (C H 1. C H 2 and C H 3). Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L ). The VH and VL regions can be further divided into hypervariable regions (called complementarity determining regions (CDRs)) separated by relatively conserved regions (called framework regions (FRs)). H and V L It consists of three CDRs and four FRs from the N-terminus to the C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region (V H and V L) respectively form an antigen binding site. The scope of the framework region and CDR can be accurately identified using methods known in the art, such as by Kabat definition, Dr. Martin website definition, Chothia definition, AbM definition, EU definition and Contact definition, all of which are well known in the art. See, e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Bulletin No. 91-3242; Martin A, "Antibody bioinformatics website of Dr. Andrew Martin's lab at UCL", last updated on July 31, 2018; Chothia et al. (1989), Nature, Vol. 342: 877; Chothia, C. et al. (1987), J. Mol. Biol., Vol. 196: 901-917; Al-lazikani et al. (1997), J. Molec. Biol., Vol. 273: 927-948; Edelman et al., Proc Natl Acad Sci USA, May 1969, Vol. 63, No. 1: pp. 78-85; and Almagro, J. Mol. Recognit., Vol. 17: pp. 132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs.The correspondence or comparison between the numbers according to different definitions can be found, for example, at http: / / www.imgt.org / (see also Giudicelli V et al., "IMGT, the international ImMunoGeneTics database", Nucleic Acids Res., (1997) Vol. 25: pp. 206-211; Lefranc MP et al., "Unique database numbering system for immunogenetic analysis", Immunol Today (1997), Vol. 18: p. 509; and Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains", Dev Comp Immunol. (2003), Vol. 27: pp. 55-77). The antibodies may be of different antibody isotypes, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0073] The term "antigen binding portion" or "antigen binding fragment" of an antibody is used interchangeably in the context of the present application and refers to a polypeptide comprising a full-length antibody fragment that retains the ability to specifically bind to an antigen that specifically binds to a full-length antibody and / or competes with a full-length antibody for binding to the same antigen. Generally, see Fundamental Immunology, Chapter 7, Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference for all purposes. Antigen binding fragments of an antibody can be derived from a full-length antibody molecule, for example, using any suitable standard technique (such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding antibody variable domains and optional constant domains). Such DNA is known and / or easily obtained from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated, for example, chemically or by using molecular biology techniques, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add or delete amino acids, etc.

[0074] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., isolated complementary determining regions (CDRs), such as CDR3 peptides) or restricted FR3-CDR3-FR4 peptides. As used herein, other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, double antibodies, three antibodies, four antibodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also encompassed within the expression "antigen-binding fragment". In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. The variable domains and constant domains may be directly linked to each other, or may be linked by all or part of a hinge or connecting region. The hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which results in a flexible or semi-flexible connection between adjacent variable domains and / or constant domains in a single polypeptide molecule.

[0075] The term "variable region" or "variable domain" with respect to an antibody as used herein refers to an antibody variable region or fragment thereof comprising one or more CDRs. Although a variable domain may comprise a complete variable region (such as a HCVR or LCVR), it is also possible to comprise less than a complete variable region but still retain the ability to bind to an antigen or form an antigen binding site.

[0076] As used herein, the term "constant region" refers to an immunoglobulin constant region, which comprises CH1, CH2 and CH3 domains (and optional hinge regions) in the heavy chain, and constant domains in the light chain. Antibody heavy and light chain constant regions are well known in the art, such as those provided in the IMGT database (www.imgt.org) or at www.vbase2.org / vbstat.php., and these two sources are incorporated herein by reference. "Fc" about an antibody refers to the part of the antibody that comprises the second (CH2) and third (CH3) constant regions of the first heavy chain that are bound to the second and third constant regions of the second heavy chain via disulfide bonds. The Fc region may also comprise part or all of the hinge region. The Fc region of an antibody is responsible for various effector functions, such as ADCC and CDC, but does not play a role in antigen binding. The ability of an antibody to start and regulate effector functions through its Fc domain is a key component of its in vivo protective activity. While the neutralizing activity of antibodies was previously thought to be solely a result of Fab-antigen interactions, it has become apparent that their in vivo activity is highly dependent on the interaction of the IgG Fc domain with its cognate receptor, the Fcγ receptor (FcγR), expressed on the surface of effector leukocytes.

[0077] The term "B7H3" (also referred to as CD276 antigen) refers to a type 1 transmembrane protein, which is a member of the B7 family with an extracellular domain consisting of a single IgV-IgC domain pair. The B7 family protein contains extracellular IgV-like and IgC-like domains with short cytoplasmic tails. B7H3 is an immune checkpoint molecule and is abnormally overexpressed in many types of cancer. When referring to the amino acid sequence of a B7H3 protein, it includes a full-length B7H3 protein (such as human 4IgB7H3 protein or human 2IgB7H3 protein) or an extracellular domain of B7H3 (B7H3 ECD) or a fragment containing B7H3 ECD; a fusion protein of B7H3 ECD. Exemplary sequences of B7H3 proteins can be found in Uniprot ID: Q5ZPR3 (human 4IgB7H3), Genebank accession numbers NP_001019907 (human), NP_001316557 (human), NP_001316558 (human), NP_079516 (human), and NP_598744 (mouse). Cynomolgus monkey B7H3 has approximately 97% and 88% amino acid sequence identity with human and mouse B7H3, respectively.

[0078] As used herein, the term "antibody that binds to B7H3" or "anti-B7H3 antibody" includes antibodies that specifically recognize B7H3 protein and antigen-binding fragments thereof, as well as antibodies that specifically bind to B7H3 protein and antigen-binding fragments thereof. As used herein, the term "anti-B7H3 antibody" includes both monovalent antibodies with monospecificity and bispecific antibodies that include a first antigen-binding site that binds to B7H3 and a second antigen-binding site that binds to a second (target) antigen.

[0079] As used herein, the term "monoclonal antibody" or "mAb" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope.

[0080] As used herein, the term "chimeric antibody" refers to an antibody consisting of the original antigen-binding variable domains of an antibody (e.g., mouse) and constant domains from a different species (e.g., human). Preferably, the term "chimeric antibody" as used herein refers to an antibody consisting of the original antigen-binding variable domains of an antibody (e.g., mouse) and constant domains from a different species (e.g., human).

[0081] The term "humanized antibody" is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species (such as rat / mouse) have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences. For example, certain residues in the framework regions may be backmutated to the original sequence to maintain affinity.

[0082] The term "operably linked" refers to the juxtaposition of two or more biological sequences of interest, with or without spacers or linkers, in a manner that allows them to function in a desired manner. When applied to polypeptides, it is intended to mean that the polypeptide sequences are linked in a manner that allows the linked product to have the desired biological function. For example, an antibody variable region can be operably linked to a constant region in order to provide a stable product with antigen binding activity. The term can also be applied to polynucleotides. For example, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.), it is intended to mean that the polynucleotide sequences are linked in a manner that allows the polypeptide to be regulatedly expressed from the polynucleotide.

[0083] As used herein, the term "K D ” is intended to refer to the dissociation constant for a specific antibody-antigen interaction, which is given by k d With k a The ratio (i.e. k d / k a ) and expressed as a molar concentration (M). As used herein, the term "k a" is intended to refer to the association rate of a particular antibody-antigen interaction, and as used herein, the term "k d ” is intended to refer to the dissociation rate of a specific antibody-antigen interaction. The K of an antibody D The value can be determined using methods well known in the art. D A preferred method is to use surface plasmon resonance, preferably using a biosensor system such as system.

[0084] As used herein, the term "high affinity" for IgG antibodies refers to the strength of the binding interaction between the antigen and the antibody. Various methods for measuring affinity have been established in the art, such as surface plasmon resonance (SPR), FACS affinity testing, FACS binding testing, and ELISA binding. In some embodiments, the K of an antibody as disclosed herein for a target antigen as determined by SPR is D 1×10 -9 M or less, more preferably 5×10 -10 M or less, even more preferably 1×10 -10 M or less, even more preferably 9×10 -11 M or less, and even more preferably 8×10 -11 M or lower.

[0085] As used herein, the term “EC 50 ” (also called “half-maximal effective concentration”) refers to the concentration of a drug, antibody, or toxic agent that induces a response that is median between baseline and maximum after a specified exposure time. In the context of this application, EC 50 The unit is "nM".

[0086] As used herein, the term "isolated" refers to a state obtained from a natural state by artificial means. If a certain "isolated" substance or component exists in nature, it may be because its natural environment has changed, or the substance has been separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity separated from this natural state is called an isolated polynucleotide or polypeptide. The term "isolated" does not exclude mixed artificial or synthetic substances, nor does it exclude other impure substances that do not affect the activity of the isolated substance.

[0087] As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to a B7H3 protein is substantially free of antibodies that specifically bind to antigens other than the B7H3 protein). However, an isolated antibody that specifically binds to a human B7H3 protein may have cross-reactivity with other antigens (such as B7H3 proteins from other species). In addition, an isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0088] As used herein, the term "vector" refers to a nucleic acid delivery vehicle that may have a polynucleotide inserted therein. When a vector allows the expression of a protein encoded by a polynucleotide inserted therein, the vector is referred to as an expression vector. A vector may have genetic material elements carried, which are expressed by transformation, transduction or transfection into a host cell. Vectors are well known to those skilled in the art, and include, but are not limited to, plasmids; bacteriophages; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector may contain a variety of elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, a vector may contain an origin of replication.

[0089] As used herein, the term "host cell" refers to a cell system that can be engineered to produce a target protein, protein fragment or peptide. Host cells include, but are not limited to, cultured cells, such as cultured mammalian cells derived from rodents (rat, mouse, guinea pig or hamster), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; or human tissue or hybridoma cells, yeast cells and insect cells, as well as cells contained in transgenic animals or cultured tissues. The term covers not only specific test cells, but also the offspring of such cells. Because certain modifications may appear in offspring due to mutations or environmental influences, such offspring may be different from parental cells, but are still included in the scope of the term "host cell".

[0090] As used herein, the term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing and comparing the sequences. "Identity percentage" means the percentage of identical residues between the amino acids or nucleotides in the molecules being compared, and is calculated based on the size of the smallest molecule in the molecules being compared. For these calculations, preferably the gaps (if any) in the comparison are solved by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I (Griffin, AM and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math., Vol. 48: p. 1073.

[0091] As used herein, the term "immunogenicity" refers to the ability to stimulate the formation of specific antibodies or sensitized lymphocytes in the body. It not only refers to the characteristics of antigens stimulating specific immune cells to activate, proliferate, and differentiate to ultimately produce immune effector substances such as antibodies and sensitized lymphocytes, but also refers to the specific immune response of antibodies or sensitized T lymphocytes that can be formed in the body's immune system after the antigen stimulates the body. Immunogenicity is the most important characteristic of an antigen. Whether an antigen can successfully induce the generation of an immune response in a host depends on three factors: the characteristics of the antigen, the reactivity of the host, and the immune means.

[0092] As used herein, the term "transfection" refers to the process of introducing a nucleic acid into a eukaryotic cell, particularly a mammalian cell. Protocols and techniques for transfection include, but are not limited to, lipid transfection and chemical and physical methods (such as electroporation). Many transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology, Vol. 52: p. 456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene, Vol. 13: p. 197. In a specific embodiment of the present disclosure, the human B7H3 gene can be transfected into 293F or CHO cells.

[0093] As used herein, the term "SPR" or "surface plasmon resonance" refers to and includes an optical phenomenon that allows analysis of real-time biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further description, see U. et al. (1993) Ann. Biol. Clin. Vol. 51: pp. 19-26; U. et al. (1991), Biotechniques, Vol. 11: pp. 620-627; Johnsson, B. et al. (1995), J. Mol. Recognit. Vol. 8: pp. 125-131; and Johnnson, B. et al. (1991), Anal. Biochem., Vol. 198: pp. 268-277.

[0094] As used herein, the term "fluorescence activated cell sorting" or "FACS" refers to a special type of flow cytometry. A method is provided for sorting a heterogeneous mixture of biological cells into two or more containers one cell at a time, based on the specific light scattering and fluorescence characteristics of each cell (FlowMetric, "Sorting Out FluorescenceActivated Cell Sorting", retrieved on 2017-11-09). Instruments for performing FACS are known to those skilled in the art and are commercially available to the public. Examples of such instruments include FACS Star Plus, FACScan, and FACSort instruments from Becton Dickinson (FosterCity, Calif.); Epics C from Coulter Epics Division (Hialeah, Fla.); and MoFlo from Cytomation (Colorado Springs, Colo.).

[0095] The term "subject" includes any human or non-human animal, and in some embodiments, the subject is preferably a human.

[0096] As used herein, the term "B7H3-associated disease" refers to any condition or disease caused by, exacerbated by, or otherwise associated with increased or decreased (usually increased) expression or activity of B7H3 (eg, human B7H3).

[0097] As used herein, the term "cancer" refers to any tumor or malignant cell growth, proliferation or metastasis mediated by solid tumors and non-solid tumors such as leukemia and resulting in a medical condition.

[0098] As used herein in the context of treating a disease, the term "treatment," "treating," or "treated" generally relates to treatment and therapy (whether human or animal) that achieves some desired therapeutic effect, such as inhibition of disease progression, and includes a reduction in the rate of progression, cessation of the rate of progression, regression of the disease, improvement of the disease, and cure of the disease. Treatment as a preventive measure (i.e., prevention, prevention) is also included. For cancer, "treatment" may refer to inhibiting or slowing tumor or malignant cell growth, proliferation, or metastasis, or some combination thereof. For tumors, "treatment" includes removing all or part of a tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof.

[0099] As used herein, the term "effective amount" relates to an amount of an active compound or a material, composition or dosage form comprising an active compound that is effective to produce some desired therapeutic effect commensurate with a reasonable benefit / risk ratio when administered according to a desired treatment regimen. For example, when used in conjunction with the treatment of a B7H3-associated disease or condition, an "effective amount" refers to an amount or concentration of an antibody or antigen-binding portion thereof that is effective to treat the disease or condition.

[0100] As used herein, the terms "prevent," "prevention," or "preventing," when referring to certain disease conditions in mammals, means preventing or delaying the onset of the disease, or preventing the manifestation of clinical or subclinical symptoms thereof.

[0101] As used herein, the term "pharmaceutically acceptable" means that the vehicle, diluent, excipient and / or salt thereof is chemically and / or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient.

[0102] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an active agent, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences, edited by Gennaro AR, 19th edition, Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; ionic strength enhancers may include, but are not limited to, sodium chloride.

[0103] As used herein, the term "adjuvant" refers to a non-specific immunopotentiator that, when delivered to the body together with an antigen or delivered to the body in advance, can enhance the body's immune response to the antigen or change the type of immune response. There are a variety of adjuvants, including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., Freund's complete adjuvant and Freund's incomplete adjuvant), Corynebacterium brevis, lipopolysaccharide, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvants are more commonly used in clinical trials.

[0104] Antibodies that specifically bind to B7H3

[0105] The present disclosure provides antibodies that specifically bind to B7H3, such as human B7H3, mouse B7H3, cynomolgus monkey B7H3, and their ECD domains. As used herein, the term "antibody" includes full-length immunoglobulins and antibody portions thereof that bind to the same antigen. The antibody can be, for example, a monoclonal, polyclonal, chimeric, humanized, or single-chain antibody. In some embodiments, the antibody portion is a Fab fragment or a F(ab')2 fragment. In some embodiments, the antibody portion retains the ability to specifically bind to B7H3.

[0106] Recombinant anti-B7H3 antibodies (such as chimeric and humanized monoclonal antibodies comprising both human and non-human portions) that can be prepared using standard recombinant DNA techniques are within the scope of the present disclosure. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques, such as the methods described in the following documents: U.S. Pat. No. 7,112,421; Better et al. (1988), Science, Vol. 240: pp. 1041-1043; or Liu et al. (1987), Proc. Natl. Acad. Sci. USA, Vol. 84: pp. 3439-3443. Also included in the present disclosure are humanized monoclonal antibodies that are further modified / optimized, for example, via affinity maturation, back mutations, and removal of post-translational modification sites.

[0107] Preferably, the antibodies or antigen binding portions thereof as disclosed herein may bind to human 4IgB7H3 with high affinity. The antibodies or antigen binding portions thereof as disclosed herein may bind to human 2IgB7H3 with much lower affinity. The antibodies or antigen binding portions thereof as disclosed herein may bind to human 4IgB7H3 with high affinity, but bind to human 2IgB7H3 with much lower affinity.

[0108] The binding of the disclosed antibodies to B7H3 can be assessed using one or more techniques well known in the art, such as ELISA or flow cytometry. In some embodiments, the antibodies can be tested by flow cytometry assays in which the antibodies react with a cell line expressing human B7H3 (such as MCF-7 cancer cells or CHOK1 cells that have been transfected to express B7H3 on their cell surfaces). Additionally or alternatively, the binding of the antibodies can be tested in a BIAcore binding assay, including binding kinetics (e.g., K D In some other embodiments, the antibody is tested by ELISA in which the antibody reacts with soluble B7H3 protein.

[0109] The present disclosure provides a 1×10 -8 M or lower K D , 1×10 -9 M or lower K D , 5×10-10 M or lower K D , 1×10 -10 M or lower K D ,9×10 -11 M or lower K D , 8×10 -11 M or lower K D , or 7×10 -11 M or lower K D Antibodies that bind to human 4IgB7H3 protein, as measured by surface plasmon resonance (SPR). Antibodies as disclosed herein can also bind to human 4IgB7H3 protein at 5×10 -9 M or higher K D , 1×10 -8 M or higher K D , 2×10 -8 M or higher K D , 3×10 -8 M or higher K D , or 4×10 -8 M or higher K D In some embodiments, the antibodies herein bind to human 2IgB7H3 at a K of 1:1. D K values ​​that are 500 times smaller, 100 times smaller, or 50 times smaller D These values ​​were used to compare the K D The value can be measured by surface plasmon resonance (SPR).

[0110] In some embodiments, the antibodies of the present disclosure are capable of activating antibodies with an EC less than 5 nM, less than 4 nM, less than 3 nM, or less than 2 nM as determined by FACS. 50 Binding to cell lines expressing human or cynomolgus monkey B7H3.

[0111] Antibody variable domains and CDRs

[0112] In some embodiments, the antibody or its antigen binding portion is a chimeric antibody or a mouse antibody that specifically binds to B7H3, preferably human 4IgB7H3. In some other embodiments, the antibody or its antigen binding portion is a humanized antibody that specifically binds to B7H3, preferably human 4IgB7H3. In some other embodiments, the humanized antibody or its antigen binding portion comprises one or more back mutations in the framework region. In some other embodiments, the humanized antibody or its antigen binding portion comprises one or more modifications at potential post-translational modification (PTM) sites, for example, to remove NG, NS and DG in CDR, to remove NXS and NXT (X can be any amino acid other than P) over the entire length.

[0113] In some embodiments, an antibody or antigen binding portion thereof as disclosed herein comprises: one or more heavy chain CDRs (HCDRs) selected from at least one of the following groups:

[0114] (i) comprising the HCDR1 of SEQ ID No: 1 or a HCDR1 having an amino acid addition, deletion or substitution that differs from the amino acid sequence of SEQ ID No: 1 by no more than 2 amino acids (e.g. 2 amino acids or 1 amino acid);

[0115] (ii) comprising the HCDR2 of SEQ ID No: 2 or a HCDR2 having an amino acid addition, deletion or substitution that differs from the amino acid sequence of SEQ ID No: 2 by no more than 2 amino acids (e.g. 2 amino acids or 1 amino acid); and

[0116] (iii) comprising the HCDR3 of SEQ ID No: 3 or a HCDR3 having an amino acid addition, deletion or substitution that differs from the amino acid sequence of SEQ ID No: 3 by no more than 2 amino acids (e.g. 2 amino acids or 1 amino acid); and / or

[0117] One or more light chain CDRs (LCDRs) selected from at least one of the following groups:

[0118] (i) LCDR1 comprising SEQ ID No: 4, 7 or 18, or a LCDR1 having an amino acid addition, deletion or substitution that differs from the amino acid sequence of SEQ ID No: 4, 7 or 18 by no more than 2 amino acids (e.g. 2 amino acids or 1 amino acid);

[0119] (ii) LCDR2 comprising SEQ ID No: 5 or a LCDR2 having an amino acid addition, deletion or substitution that differs from the amino acid sequence of SEQ ID No: 5 by no more than 2 amino acids (e.g. 2 amino acids or 1 amino acid); and

[0120] (iii) LCDR3 comprising SEQ ID No: 6 or an LCDR3 having an amino acid addition, deletion or substitution that differs from the amino acid sequence of SEQ ID No: 6 by no more than 2 amino acids (eg, 2 amino acids or 1 amino acid).

[0121] In some embodiments, the amino acid substitutions in the CDRs are conservative substitutions. In some embodiments, the antibodies or antigen-binding portions thereof as described above may contain: HCDR1 comprising or consisting of SEQ ID NO: 1; HCDR2 comprising or consisting of SEQ ID NO: 2; HCDR3 comprising or consisting of SEQ ID NO: 3; LCDR1 comprising or consisting of SEQ ID NO: 7; LCDR2 comprising or consisting of SEQ ID NO: 5; and LCDR3 comprising or consisting of SEQ ID NO: 6. Specifically, the antibody may also contain one or more modifications in the CDRs to remove potential PTM sites. In some embodiments, the antibody may contain one or more modifications in the CDRs to remove potential PTM sites. NS Compared to the LCDR1 shown in SEQ ID NO: 7 ("KSSQSLL"), the antibody comprises one or more modifications in LCDR1 to remove the potential PTM site "NS" in the sequence. In some preferred embodiments, compared to the LCDR1 shown in SEQ ID NO: 7 ("KSSQSLL") NS In some more preferred embodiments, the antibody comprises a substitution at position 8 or 9 of the amino acid sequence of LCDR1 compared to LCDR1 (SEQ ID NO: 4, "KSSQSLL"). QS SNQKNYLA") comprises an N to Q substitution at position 8 of the amino acid sequence of. In some other embodiments, the antibody comprises an N to Q substitution at position 8 of LCDR1 (i.e., SEQ ID NO: 18, "KSSQSLL NP SNQKNYLA") comprises an S to P substitution at position 9 of the amino acid sequence. Those skilled in the art will appreciate that other types of substitutions may be selected as long as the binding affinity to B7H3 is substantially retained.

[0122] In some embodiments, an antibody or antigen-binding portion thereof as disclosed herein may contain: a HCDR1 comprising or consisting of SEQ ID NO: 1; a HCDR2 comprising or consisting of SEQ ID NO: 2; a HCDR3 comprising or consisting of SEQ ID NO: 3; a LCDR1 comprising or consisting of SEQ ID NO: 4; a LCDR2 comprising or consisting of SEQ ID NO: 5; and a LCDR3 comprising or consisting of SEQ ID NO: 6.

[0123] In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region and the light chain variable region comprise HCDR1-3 and LCDR1-3, respectively, as described above.

[0124] In some embodiments, the heavy chain variable region of the antibody, or antigen binding portion thereof, comprises:

[0125] (i) an amino acid sequence of SEQ ID No: 8 or 10; (ii) an amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID No: 8 or 10; or (iii) an amino acid sequence that has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) additions, deletions and / or substitutions of amino acids compared to SEQ ID No: 8 or 10.

[0126] In some embodiments, amino acid substitutions may be conservative substitutions.

[0127] In some embodiments, the light chain variable region of the antibody, or antigen binding portion thereof, comprises:

[0128] (i) an amino acid sequence of SEQ ID No: 9 or 11; (ii) an amino acid sequence that is at least 85%, at least 90% or at least 95% identical to SEQ ID No: 9 or 11; or (iii) an amino acid sequence that has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid additions, deletions and / or substitutions compared to SEQ ID No: 9 or 11.

[0129] In some embodiments, amino acid substitutions may be conservative substitutions.

[0130] In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises:

[0131] (i) an amino acid sequence of SEQ ID No: 8 or 10; or (ii) an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid substitutions in the framework regions compared to SEQ ID No: 8 or 10;

[0132] and / or VL contains:

[0133] (i) an amino acid sequence of SEQ ID No: 9 or 11; or (ii) an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5 or more) amino acid substitutions in the framework regions compared to SEQ ID No: 9 or 11;

[0134] In some embodiments, amino acid substitutions may be conservative substitutions.

[0135] In some embodiments, the antibody or antigen binding portion thereof comprises HCDR1, HCDR2 and HCDR3 of the VH region shown in SEQ ID No: 8 or 10, and LCDR1, LCDR2 and LCDR3 of the VL region shown in SEQ ID No: 9 or 11.

[0136] Those skilled in the art will appreciate that the precise numbering and position of CDRs may be different in different numbering systems. However, it should be understood that the disclosure of variable heavy chain and / or variable light chain sequences includes the disclosure of related (intrinsic) CDRs. Therefore, the disclosure of each variable heavy chain region is the disclosure of heavy chain CDRs (e.g., HCDR1, HCDR2, and HCDR3), and the disclosure of each variable light chain region is the disclosure of light chain CDRs (e.g., LCDR1, LCDR2, and LCDR3). The available comparison of CDR numbering is as follows, see Lafranc et al., Dev. Comp. Immunol. Vol. 27 (Issue 1): pp. 55-77 (2003):

[0137]

[0138]

[0139] Amino acids may be assigned to each CDR according to one or a combination of the numbering schemes provided above, all of which are well known in the art. See Kabat et al. (1991), Sequences of Proteins of Immunological Interest (5th edition), US Dept. of Health and Human Services, PHS, NIH, NIH Bulletin No. 91-3242; Chothia et al., 1987, PMID: 3681981; Martin A, "Amibodybioinformatics website of Dr. Andrew Martin's lab at UCL", last updated on July 31, 2018; Chothia et al., 1989, PMID: 2687698; MacCallum et al., 1996, PMID: 8876650; Dubel, ed. (2007), Handbook of Therapeutic Antibodies, 3rd edition, Wily-VCH Verlag GmbH and Co; and http: / / www.imgt.org / .

[0140] The variable region and CDR in the antibody sequence can be identified according to the general rules developed in the art (as described above, such as Kabat, Martin and IMGT numbering systems) or by comparing the sequence with a database of known variable regions. The method for identifying these regions is described in Kontermann and Dubel, ed., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Exemplary databases of antibody sequences are described in "Abysis" website: www.bioinf, org.uk / abs (maintained by ACMartin, Department of Biochemistry & Molecular Biology University College London, London, England) and VBASE2 website: www.vbase2.org, as described in Retter et al., Nucl. Acids Res., No. 33 (database issue): D671-D674 (2005), and can be accessed through these websites. For example, the sequence can be analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT and the Protein Data Bank (PDB) with structural data from the PDB. See the chapter "Protein Sequence and Structure Analysis of Antibody Variable Domains" in the book "Antibody Engineering Lab Manual" by Dr. Andrew C. R. Martin (Duebel, S. and Kontermann, R. eds., Springer-Verlag, Heidelberg, ISBN-13:978-3540413547, also available on the website bioinforg.uk / abs). The Abysis database website also includes general rules that have been developed for identifying CDRs, which can be used according to the teachings of this article. Two antibodies with the same VH and / or VL CDRs mean that their CDRs are the same when determined by the same method (e.g., Kabat method, Dr Martin method, Chothia method or IMGT numbering as known in the art).

[0141] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., Vol. 4: pp. 11-17 (1988)) incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol., Vol. 48: pp. 444-453 (1970)) incorporated into the GAP program in the GCG software package (see http: / / www.gcg.com), using a Blossum62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6.

[0142] Additionally or alternatively, the protein sequences of the present disclosure may also be used as a "query sequence" to perform searches against public databases, e.g., to identify related sequences. Such searches may be performed using the XBLAST program (version 2.0) of Altschul et al. (1990), J. Mol. Biol., Vol. 215: pp. 403-410. BLAST protein searches may be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the antibody molecules of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST may be utilized as described in Altschul et al. (1997), Nucleic Acids Res., Vol. 25 (Issue 17): pp. 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) may be used. See www.ncbi.nlm.nih.gov.

[0143] In other embodiments, the CDR amino acid sequence may be at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the corresponding sequences described above. In other embodiments, the amino acid sequence of the variable region may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the corresponding sequences set forth above.

[0144] As described above, the addition, deletion and / or substitution of at least one of the amino acids in the VH or VL region may not be in any sequence in the CDR sequence, but in the framework (FRW) sequence. For example, the isolated antibody or antigen-binding portion thereof may comprise one or more amino acid substitutions in a framework sequence, such as FRW1, FRW2, FRW3 and / or FRW4 in the VH or VL region.

[0145] In some embodiments, the 6 CDR groups may have a total of 0, 1, 2, 3, 4 or 5 amino acid modifications (preferably amino acid substitutions), as well as changes in the framework regions of the variable heavy and light chain regions, as long as the framework (excluding the CDRs) remains at least about 80%, 85% or 90% identical to the parent antibody (e.g., W301088-1.145.16). Thus, for example, the same CDRs as described herein may be combined with different framework sequences from human germline sequences, as long as the framework regions remain at least 80%, 85% or 90% identical to the human germline sequences.

[0146] In certain embodiments, an isolated antibody or antigen-binding portion thereof as provided herein comprises any suitable framework region (FR) sequence, so long as the antigen-binding domain can specifically bind to B7H3, preferably human Ig4B7H3.

[0147] The framework region of the separated antibody or its antigen-binding portion thereof may contain one or more back mutations, wherein the framework amino acids from the human germline are replaced by the original mouse amino acids at the corresponding positions. The framework region of the separated antibody or its antigen-binding portion thereof may also include one or more conservative substitutions. As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the basic properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which the amino acid residue is replaced by another amino acid residue with a similar side chain, for example, physically or functionally similar to the corresponding amino acid residue (such as having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (such as threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, the corresponding amino acid residue is preferably substituted by another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem., Vol. 32: pp. 1180-1187; Kobayashi et al., Protein Eng., Vol. 12 No. 10: pp. 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA, Vol. 94: pp. 412-417 (1997), which are incorporated herein by reference).

[0148] IgG constant region

[0149] In some embodiments, the antibody further comprises an immunoglobulin constant region, such as a human IgG, IgA, IgM, IgE or IgD constant region. Antibodies as disclosed herein can be various IgG isotypes, such as IgG1, IgG2, IgG3 and IgG4. Preferably, the antibody is an IgG1 isotype and comprises a human IgG1 constant region. An IgG constant region as described herein comprises an Fc region and optionally part or all of the hinge region of an IgG molecule.

[0150] Compared to the wild-type Fc region, the Fc region may include one or more amino acid changes (e.g., insertions, deletions, or substitutions) without changing the desired function. For example, the present disclosure includes antibodies comprising one or more modifications in the Fc region, which may have a modified (e.g., enhanced or weakened) binding interaction between Fc and FcRn. Non-limiting examples of Fc modifications also include, for example: amino acid modifications of Leu234Ala / Leu235Ala (or LALA) in the IgG1 Fc region, which alter antibody-dependent cellular cytotoxicity (ADCC) or other effector functions; mutations of serine ("S") to proline ("P") at position 228 of the amino acid sequence in the human IgG4 Fc region. The S228P mutation reduces Fab arm exchange by stabilizing the disulfide bonds in the core hinge of the IgG4 molecule, and therefore belongs to an IgG4 stabilizing mutation, which helps prevent half-antibody formation. It is known that an increase in binding to FcγRIIIa generally leads to an increase in ADCC. Similarly, reduced binding to FcγRIIb (inhibitory receptor) can be beneficial in some cases. Amino acid replacements for antibodies of the present invention include those listed in U.S. Patent Nos. 8,188,321 (particularly Figure 41) and 8,084,582 and U.S. published application Nos. 20060235208 and 20070148170, all of which are expressly incorporated herein by reference in their entirety. Available specific variants include, but are not limited to, 236A, 239D, 239E, 332E, 332D, 239D / 332E, 267D, 267E, 328F, 267E / 328F, 236A / 332E, 239D / 332E / 330Y, 239D, 332E / 330L, 243A, 243L, 264A, 264V, and 299T. Such modifications may be included in one or two Fc domains of an antibody as disclosed herein.

[0151] The variable region is operably connected to the IgG constant region in the heavy chain and light chain, respectively. In some embodiments, the variable region is directly connected to the IgG constant region. In some other embodiments, the variable region is connected to the IgG constant region via a joint that can include as little as one amino acid. Peptide linkers for constructing polypeptide sequences are well known in the art, such as (GS) n, (GSGGS) n, (GGGGS) n and (GGGS) n joints, wherein n is at least an integer of one (usually 3 to 4 to 5); or a joint derived from an immunoglobulin heavy chain or light chain (such as a sequence derived from a hinge region), and any peptide sequence with sufficient length and flexibility to allow each region to perform its biological function is allowed to be reorganized and attached to the constant region.

[0152] Unless otherwise indicated, the numbering of antibody constant regions herein is based on the EU numbering of Kabat. Thus, the "CH" domains in the context of IgG are as follows: "CH1" refers to positions 118-215 according to the EU index as of Kabat, "hinge" refers to positions 216-230 according to the EU index as of Kabat, "CH2" refers to positions 231-340 according to the EU index as of Kabat, and "CH3" refers to positions 341-447 according to the EU index as of Kabat.

[0153] Functionality of anti-B7H3 antibodies

[0154] Antibodies or antigen-binding portions thereof as disclosed herein are characterized by specific functional characteristics or properties. In some embodiments, antibodies (including both chimeric antibodies and humanized antibodies) have one or more of the following properties:

[0155] (a) specifically binds to cells expressing human 4IgB7H3 with high affinity (e.g., less than 2 nM, as measured by FACS);

[0156] (b) binds to human 2IgB7H3 with a significantly lower affinity than that of human 4IgB7H3, and the difference between the antibody binding to 2IgB7H3 and binding to 4IgB7H3 is significant;

[0157] (c) specifically binds cynomolgus monkey B7H3 with a high affinity (e.g., less than 5 nM, as measured by FACS) that is superior to a benchmark antibody; and

[0158] (d) It showed good internalization ability into cancer cells expressing B7H3.

[0159] Nucleic acid molecules encoding the antibodies of the present disclosure

[0160] In some aspects, the present disclosure relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a heavy chain variable region and / or a light chain variable region of an isolated antibody as disclosed herein. In some embodiments, the isolated nucleic acid molecule herein comprises a nucleic acid sequence as shown in SEQ ID No: 16. In some embodiments, the isolated nucleic acid molecule herein comprises a nucleic acid sequence as shown in SEQ ID No: 17.

[0161] Nucleic acids of the present disclosure can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as further described below), cDNAs encoding the light and heavy chains of antibodies prepared by hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding such antibodies can be recovered from gene libraries.

[0162] The isolated nucleic acid encoding the VH region can be converted into a full-length heavy chain gene by operably linking the nucleic acid encoding the VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2 and CH3). The sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat et al. (1991), supra), and DNA fragments covering these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but more preferably an IgG1 or IgG4 constant region.

[0163] The isolated nucleic acid encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene) by operably linking the DNA encoding the VL to another DNA molecule encoding the light chain constant region CL. The sequence of the human light chain constant region gene is known in the art (see, e.g., Kabat et al., supra), and the DNA fragments covering these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region can be a kappa or lambda constant region.

[0164] Once the DNA fragments encoding the VH region and VL segment are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes or scFv genes. In these operations, the DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein (such as, an antibody constant region or a flexible linker). As used herein, the term "operably linked" is intended to refer to the connection of two DNA fragments so that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0165] In some aspects, the disclosure relates to vectors comprising nucleic acid sequences as disclosed herein. The vector in the context of the disclosure may be any suitable vector, including chromosomal, non-chromosomal and synthetic nucleic acid vectors (containing a nucleic acid sequence of a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In some embodiments, the nucleic acid encoding the B7H3 antibody is contained in a naked DNA or RNA vector that includes, for example, a linear expression element (as described in, for example, Sykes and Johnston, Nat Biotech, Vol. 17: pp. 355-359 (1997)); a compacted nucleic acid vector (as described in, for example, US 6,077,835 and / or WO 00 / 70087); a plasmid vector such as pBR322, pUC 19 / 18 or pUC 118 / 119; a "Midge" minimal size nucleic acid vector (as described in, for example, Schakowski et al., Mol Ther, Vol. 3: pp. 793-800 (2001)), or as a precipitation nucleic acid vector construct, such as a CaP04 precipitation construct (as described in, for example, WO200046147; Benvenisty and Reshef, PNAS USA, Vol. 83: pp. 9551-9555 (1986); Wigler et al., Cell, Vol. 14: p. 725 (1978); and Coraro and Pearson, Somatic Cell Genetics, Vol. 7: p. 603 (1981). Such nucleic acid vectors and their uses are well known in the art (see, for example, US 5,589,466 and US 5,973,972).

[0166] In one embodiment, the vector is suitable for expressing antibodies in bacterial cells. Examples of such vectors include expression vectors, such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem, Vol. 264: pp. 5503-5509 (1989)), pET vectors (Novagen, Madison WI), etc. The vector may also be or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be used. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters such as alpha factors, alcohol oxidases, and PGHs (reviewed in: F. Ausubel et al., eds., Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987) and Grant et al., Methods in Enzymol, Vol. 153: pp. 516-544 (1987)).

[0167] The vector may also or alternatively be a vector suitable for expression in mammalian cells, for example a vector comprising glutamine synthetase as a selectable marker such as that described in Bebbington (1992), Biotechnology (NY), Vol. 10: pp. 169-175.

[0168] Nucleic acid and / or vector can also comprise the nucleic acid sequence of coding secretion / localization sequence, and this secretion / localization sequence can target polypeptide such as nascent polypeptide chain to periplasmic space or target cell culture medium.Such sequence is well known in the art, and comprises secretion leader peptide or signal peptide.

[0169] The vector may include any suitable promoter, enhancer and other expression promoting elements, or may be associated with them. Examples of these elements include strong expression promoters (e.g., human CMV IE promoter / enhancer and RSV, SV40, SL3-3, MMTV and HIV LTR promoters), effective polyadenylic acid termination sequences, plasmid product replication origins in Escherichia coli, antibiotic resistance genes as selectable markers and / or convenient cloning sites (e.g., polylinkers). Nucleic acid may also include inducible promoters, rather than constitutive promoters such as CMV IE.

[0170] In some aspects, the disclosure relates to a host cell comprising the vector described above.

[0171] Thus, the present disclosure also relates to recombinant eukaryotic or prokaryotic host cells, such as transfectomas, that produce the antibodies of the present disclosure.Antibodies can be expressed in recombinant eukaryotic or prokaryotic host cells to produce the antibodies of the present disclosure.

[0172] Examples of host cells include yeast, bacteria, plants, and mammalian cells. Mammalian host cells for expressing the antibodies of the present disclosure include Chinese hamster ovary (CHO cells) (including dhfr CHO cells, described in Urlaub and Chasin (1980), Proc. Natl. Acad. Sci USA, Vol. 77: pp. 4216-4220, used with a DHFR selectable marker, e.g., as described in RJ Kaufman and PA Sharp (1982), J. Mol. Biol. Vol. 159: pp. 601-621), COS cells, and SP2 cells. Specifically, another expression system used with NSO myeloma cells is the GS gene expression system disclosed in WO87 / 04462, WO 89 / 01036, and EP 338,841.Also included are SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 cells or subcloned 293 cells for growth in suspension culture, Graham et al., J. Gen Virol., Vol. 36: p. 59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA, Vol. 77: p. 4216); mouse testicular Sertoli cells (TM4, Mather, 1980, Biol. Reprod., Vol. 23: pp. 243-251); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., 1982, Annals NY Acad. Sci., Vol. 383: pp. 44-68); MRC 5 cells; FS4 cells; mouse myeloma cells, such as NSO (e.g., RCB0213, 1992, Bio / Technology, Vol. 10: p. 169) and SP2 / 0 cells (e.g., SP2 / 0-Ag14 cells, ATCC CRL 1581); rat myeloma cells, such as YB2 / 0 cells (e.g., YB2 / 3HL.P2.G11.16Ag.20 cells, ATCC CRL 1662); PER.C6 cells; and human hepatoma cell line (Hep G2). CHO cells are one type of cell line that can be used herein, with CHO-K1, DUK-B11, CHO-DP12, CHO-DG44 (Somatic Cell and Molecular Genetics, Vol. 12: p. 555 (1986)) and Lec13 being exemplary host cell lines. In the case of CHO-K1, DUK-B11, DG44 or CHO-DP12 host cells, these host cells can be altered so that they lack the ability to express fucosylated proteins in the cells. In some embodiments, the host cell herein is selected from CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6 or NSO cells or lymphocytes.

[0173] Prokaryotes suitable for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia, e.g., Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces.

[0174] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for bispecific antibody encoding vectors. Saccharomyces cerevisiae or common baker's yeast is the most commonly used among the lower eukaryotic host microorganisms. However, many other genera, species and strains are generally available and can be used herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as, for example, K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesia (EP 244,234); Neurosporacrassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as, for example, Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger.

[0175] In some embodiments, the host cell may comprise the first and second nucleic acid constructs stably integrated into the cell genome. In another embodiment, the disclosure provides a cell comprising a non-integrated nucleic acid such as a plasmid, cosmid, phagemid or linear expression element comprising the first and second nucleic acid constructs as described above.

[0176] In even another aspect, the disclosure relates to a transgenic non-human animal or plant comprising nucleic acids encoding one or two sets of human heavy chains and human light chains, wherein the animal or plant produces an antibody of the disclosure.

[0177] In another aspect, the present disclosure relates to a hybridoma producing an antibody of the present disclosure as defined herein. In even another aspect, the present disclosure relates to a transgenic non-human animal or plant comprising a nucleic acid encoding one or two sets of human heavy chains and human light chains, wherein the animal or plant produces an antibody of the present disclosure.

[0178] In one aspect, the present disclosure relates to an expression vector comprising:

[0179] (i) a nucleic acid sequence encoding an antibody or antigen binding portion thereof according to any one of the embodiments disclosed herein, or a VH or VL domain of an antibody;

[0180] (ii) a nucleic acid sequence encoding the amino acid sequence of a heavy chain or a light chain;

[0181] (iii) a nucleic acid sequence encoding the amino acid sequence of a constant region; or

[0182] (iv) a combination of two or more of the above nucleic acid sequences.

[0183] In one aspect, the disclosure relates to nucleic acid constructs encoding one or more of the amino acid sequences shown in the Sequence Listing.

[0184] In one aspect, the disclosure relates to a method for producing an antibody according to any one of the embodiments disclosed herein, the method comprising the steps of: culturing a host cell comprising an expression vector or more than one expression vector as disclosed herein, expressing an antibody, and purifying the antibody from a culture medium. In one aspect, the disclosure relates to a host cell comprising an expression vector as defined above. In one embodiment, the host cell is a recombinant eukaryotic, recombinant prokaryotic, or recombinant microbial host cell.

[0185] Pharmaceutical composition

[0186] In some aspects, the present disclosure relates to a pharmaceutical composition comprising at least one antibody, or antigen-binding portion thereof, as disclosed herein and a pharmaceutically acceptable carrier.

[0187] Components of the composition

[0188] The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical composition of the present disclosure may also be administered with, for example, another immunostimulant, anticancer agent, antiviral agent or vaccine in a combined therapy so that additional or synergistic effects can be achieved. Pharmaceutically acceptable carriers may include, for example, pharmaceutically acceptable liquids, gels or solid carriers, aqueous media, non-aqueous media, antimicrobials, isotonic agents, buffers, antioxidants, anesthetics, suspensions / dispersants, chelating agents, diluents, adjuvants, excipients or nontoxic adjuvants, other carriers known in the art, components or various combinations of more carriers.

[0189] Suitable components can include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers or stabilizers such as sugar and cyclodextrin. Suitable antioxidants can include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butyl methyl anisole, butylated hydroxytoluene and / or propyl gallate. According to the present disclosure, the solvent containing the antibody or Fab of the present disclosure can include one or more antioxidants such as methionine, and the reducing antibody or its Fab can be oxidized. Redox can prevent or reduce the reduction of binding affinity, thereby enhancing antibody stability and the shelf life of extension. Therefore, in some embodiments, the present disclosure provides a composition comprising one or more antibodies or their Fabs and one or more antioxidants (such as methionine). The present disclosure also provides methods in which antibodies or antigen-binding fragments thereof are mixed with one or more antioxidants (such as methionine) so that the antibodies or antigen-binding fragments thereof can be prevented from oxidation to extend their shelf life and / or increase activity.

[0190] To further illustrate, pharmaceutically acceptable carriers can include, for example: aqueous carriers such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection; non-aqueous carriers such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents at bacteriostatic or fungistatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifiers such as polysorbate 80 (TWEEN-80); chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethanol; polyethylene glycol; propylene glycol; sodium hydroxide; hydrochloric acid; citric acid or lactic acid. Antimicrobial agents used as carriers can be added to pharmaceutical compositions in multiple dose containers, including phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients can include, for example, water, saline, glucose, glycerol or ethanol. Suitable nontoxic auxiliary substances can include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubilizers or reagents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrins.

[0191] Administration, formulation and dosage

[0192] Pharmaceutical compositions of the present disclosure can be administered to a subject in need in vivo by various approaches, including but not limited to oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, local, transdermal and intrathecal, or otherwise by implantation or suction. This theme composition can be formulated into preparations in solid, semisolid, liquid or gaseous form, including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants and aerosols. Suitable preparations and routes of administration can be selected according to expected application and treatment regimen.

[0193] Suitable preparations for enteral administration include hard or soft gelatin capsules, pills, tablets (including coated tablets), elixirs, suspensions, syrups or inhalants and controlled release forms thereof.

[0194] Preparations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the active ingredient is dissolved, suspended or otherwise provided (e.g., in liposomes or other microparticles). Such liquids may also contain other pharmaceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, antibacterial agents, suspending agents, thickeners, and solutes that make the preparation isotonic with the blood (or other relevant body fluids) of the intended recipient. Examples of excipients include, for example, water, alcohol, polyols, glycerol, vegetable oils, etc. Examples of isotonic carriers suitable for such preparations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, specific dosing regimens (including dosage, time, and repetition) will depend on specific individuals and the individual's medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance, etc.).

[0195] The frequency of administration can be determined and adjusted during the course of treatment and is based on reducing the number of proliferative or tumorigenic cells, maintaining a reduction in such tumor cells, reducing the proliferation of tumor cells, or delaying the development of metastases. In some embodiments, the administered dose can be adjusted or reduced to control potential side effects and / or toxicity. Alternatively, a continuous sustained release formulation of the therapeutic composition of the present invention may be suitable.

[0196] It will be appreciated by those skilled in the art that the appropriate dosage may vary from patient to patient. Determining the optimal dosage will generally involve a balance between the level of therapeutic beneficial effects and any risks or harmful side effects. The selected dosage level will depend on a variety of factors, including but not limited to the activity of the specific compound; route of administration; time of administration; excretion rate of the compound; duration of treatment; other drugs, compounds and / or materials used in combination; severity of the condition; and the species, sex, age, weight, condition, general health and previous medical history of the patient. The amount and route of administration of the compound will ultimately be determined by the physician, veterinarian or clinician, but will generally be selected to achieve a local concentration at the site of action, thereby achieving the desired effect without causing substantial adverse or harmful side effects.

[0197] Generally, the antibodies or antigen-binding portions thereof disclosed herein can be administered in a variety of ranges. These ranges include about 5 μg / kg body weight to about 100 mg / kg body weight per dose; about 50 μg / kg body weight to about 5 mg / kg body weight per dose; about 100 μg / kg body weight to about 10 mg / kg body weight per dose. Other ranges include about 100 μg / kg body weight to about 20 mg / kg body weight and about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose. In certain embodiments, the dosage is at least about 100 μg / kg body weight, at least about 250 μg / kg body weight, at least about 750 μg / kg body weight, at least about 3 mg / kg body weight, at least about 5 mg / kg body weight, at least about 10 mg / kg body weight per dose.

[0198] In any case, the antibodies or antigen-binding portions thereof of the present disclosure are preferably administered to subjects in need as needed. The determination of the frequency of administration can be made by a person skilled in the art, such as an attending physician, based on considerations of the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health status of the subject being treated, etc.

[0199] In certain preferred embodiments, the treatment process involving antibodies or antigen-binding portions thereof of the present disclosure will include multiple doses of selected drug products over a period of weeks or months. More specifically, antibodies or antigen-binding portions thereof of the present disclosure can be administered once a day, once every two days, once every four days, once a week, once every ten days, once every two weeks, once every three weeks, once a month, once every six weeks, once every two months, once every ten weeks, once every three months, or with less frequent administration. In this regard, it should be understood that dosages can be changed or intervals adjusted based on patient responses and clinical practice.

[0200] For individuals who have been given one or more administrations, the dosage and regimen of the disclosed therapeutic composition can also be determined empirically. For example, an individual can be given an increasing dose of a therapeutic composition produced as described herein. In a selected embodiment, the dosage can be gradually increased or decreased or reduced based on empirically determined or observed side effects or toxicity, respectively. In order to evaluate the efficacy of the selected composition, markers of specific diseases, disorders or conditions can be tracked as described above. For cancer, these markers include direct measurement of tumor size via palpation or visual observation, indirect measurement of tumor size by X-ray or other imaging techniques; such as improvement assessed by direct tumor biopsy and microscopy of tumor samples; indirect tumor markers (such as PSA for prostate cancer) or measurement of tumorigenic antigens identified according to the methods described herein, relief of pain or paralysis; improvement of speech, vision, breathing or other disabilities associated with tumors; increased appetite; or improvement of quality of life or extension of survival measured by recognized tests. It is obvious to those skilled in the art that the dosage will vary according to the individual, the type of tumor condition, the stage of the tumor condition, whether the tumor condition has begun to metastasize to other locations of the individual, and the treatment used in the past and at present.

[0201] Compatible formulations for parenteral administration (e.g., intravenous injection) will include an antibody or antigen-binding portion thereof as disclosed herein at a concentration of about 10 μg / mL to about 100 mg / mL. In certain selected embodiments, the concentration of the antibody or antigen-binding portion thereof will include about 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 700 μg / mL, 800 μg / mL, 900 μg / mL, or 1 mg / mL. In other preferred embodiments, the concentration of the antibody, or antigen-binding portion thereof, will comprise about 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 14 mg / mL, 16 mg / mL, 18 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, or 100 mg / mL.

[0202] Applications of the present disclosure

[0203] In some aspects, the disclosure provides a method for preventing or treating a B7H3-related disease, disorder or condition in a subject, the method comprising administering a therapeutically effective amount of an antibody as disclosed herein to a subject (e.g., a human) in need of treatment. The disease or disorder may be cancer, an autoimmune disease, or an infectious disease.

[0204] A variety of cancers involving B7H3 (whether malignant or benign and whether primary or secondary) can be treated or prevented using the methods provided by the present disclosure. The cancer can be a solid cancer or a hematological malignancy. Examples of such cancers include lung cancers such as bronchial cancers (e.g., squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondromatous hamartoma (non-cancerous) and sarcoma (cancerous); heart cancers such as myxoma, fibroma and rhabdomyoma; bone cancers such as osteochondroma, enchondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma, giant cell tumor of bone, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, Ewing's tumor (Ewing's sarcoma) and reticulum cell sarcoma; brain cancers such as gliomas (e.g., glioblastoma multiforme), anaplastic astrocytoma, astrocytoma, oligomyeloma; Synaptoglioma, medulloblastoma, chordoma, neurilemoma, ependymoma, meningioma, pituitary adenoma, pinealoma, osteoma, hemangioblastoma, craniopharyngioma, germ cell tumor, teratoma, dermoid cyst and hemangioma; cancers of the digestive system such as colon cancer, leiomyoma, squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, gastric adenocarcinoma, intestinal lipoma, intestinal fibroma, intestinal fibroma, giant polyp of the large intestine and colorectal cancer; liver cancer such as hepatocellular adenoma, hemangioma, hepatocellular carcinoma, fibrolamellar hepatocellular carcinoma, bile duct carcinoma, hepatoblastoma and angiosarcoma; kidney cancer such as renal adenocarcinoma, renal cell carcinoma, adrenal tumor and transitional cell carcinoma of the renal pelvis; bladder cancer; hematological cancers such as acute lymphocytic (lymphoblastic) leukemia, acute myeloid (myeloid, myeloid, myeloblastic, myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., Sezary syndrome and hairy cell leukemia), chronic myeloid (myeloid, myeloid, granulocytic) leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, B-cell lymphoma, mycosis fungoides, and myeloproliferative disorders (including myeloproliferative disorders such as polycythemia vera, myelofibrosis, thrombocythemia, and chronic myeloid leukemia); skin cancers such as basal cell carcinoma, squamous cell carcinoma, melanoma, Kaposi's sarcoma, and Paget's disease; head and neck cancer; eye cancers; Cancers of the male reproductive system such as benign prostatic hyperplasia, prostate cancer, and testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, and choriocarcinoma); breast cancer; cancers of the female reproductive system such as cancer of the uterus (endometrial cancer), cancer of the cervix (cervical cancer), cancer of the ovaries (ovarian cancer), cancer of the vulva, cancer of the vagina, cancer of the fallopian tubes, and hydatidiform moles; thyroid cancer (including papillary, follicular, undifferentiated, or medullary carcinoma); pheochromocytoma (adrenal gland); noncancerous growths of the parathyroid glands; pancreatic cancer; and hematological cancers such as leukemia, myeloma, non-Hodgkin's lymphoma, and Hodgkin's lymphoma.In some specific embodiments, the cancer is selected from neurological tumors, melanoma, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer and ovarian cancer.

[0205] In some embodiments, examples of cancer include, but are not limited to, B cell cancers, including B cell lymphomas (including low grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); L); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as associated with brain tumors), B-cell proliferative disorders, and Meigs syndrome. More specific examples include, but are not limited to, relapsed or refractory NHL, frontline low-grade NHL, stage III / IV NHL, chemotherapy-resistant NHL, precursor B lymphoblastic leukemia and / or lymphoma, small lymphocytic lymphoma, B-cell chronic lymphocytic leukemia and / or prolymphocytic leukemia and / or small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, immunocytoma and / or lymphoplasmacytic lymphoma, lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone MALT lymphoma, nodal marginal zone lymphoma, hairy cell leukemia, plasmacytoma and / or plasma cell myeloma, low-grade / follicular lymphoma, intermediate-grade / follicular NHL, mantle cell lymphoma, follicle center lymphoma (follicular), intermediate-grade diffuse NHL, diffuse Large B-cell lymphoma, aggressive NHL (including aggressive frontline NHL and aggressive relapsed NHL), relapsed or refractory NHL after autologous stem cell transplantation, primary mediastinal large B-cell lymphoma, primary effusion lymphoma, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, bulky NHL, Burkitt's lymphoma, precursor (peripheral) large granular lymphocytic leukemia, mycosis fungoides and / or Sezary syndrome, cutaneous lymphoma, anaplastic large cell lymphoma, angiocentric lymphoma.

[0206] In some embodiments, examples of cancer include, but are not limited to, B cell proliferative disorders, which further include, but are not limited to, lymphomas (e.g., B cell non-Hodgkin lymphoma (NHL)) and lymphocytic leukemias. Such lymphomas and lymphocytic leukemias include, for example, a) follicular lymphoma, b) small, non-cleaving cell lymphoma / Burkitt lymphoma (including endemic Burkitt lymphoma, sporadic Burkitt lymphoma, and non-Burkitt lymphoma), c) marginal zone lymphoma (including extranodal marginal zone B cell lymphoma (mucosa-associated lymphoid tissue lymphoma, MALT), nodal marginal zone B cell lymphoma, and splenic marginal zone lymphoma), d) mantle cell lymphoma (MCL), e) large cell lymphoma (including B cell diffuse large cell lymphoma ( DLCL), diffuse mixed cell lymphoma, immunoblastic lymphoma, primary mediastinal B-cell lymphoma, angiocentric lymphoma-pulmonary B-cell lymphoma), f) hairy cell leukemia, g) lymphocytic lymphoma, Waldenstrom's macroglobulinemia, h) acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, i) plasma cell neoplasms, plasma cell myeloma, multiple myeloma, plasmacytoma, and / or j) Hodgkin's disease.

[0207] In some other embodiments, the disease or condition is an autoimmune disease. Examples of autoimmune diseases that can be treated with antibodies or antigen-binding portions thereof as disclosed herein include autoimmune encephalomyelitis, lupus erythematosus, and rheumatoid arthritis, etc. The antibodies or antigen-binding portions thereof can also be used to treat or prevent infectious diseases, inflammatory diseases (such as allergic asthma), and chronic graft-versus-host disease.

[0208] Combined use

[0209] The antibody can be used in combination with different anticancer agents, cytotoxic agents, chemotherapeutic agents or cellular immunotherapy. For example, the antibody of the present disclosure can be administered in combination with anti-PD-1 / PD-L1 antibodies. The antibody of the present disclosure can also be administered in combination with CART therapy.

[0210] Cellular Immunotherapy

[0211] In some embodiments, antibodies as disclosed herein are used in combination with cell immunotherapy, which is also referred to as adoptive cell therapy. As is generally known, cell immunotherapy is a form of treatment that utilizes cells of the human immune system to eliminate cancer. Some of these methods involve directly separating our own immune cells and simply expanding their number (e.g., by activating and expanding the patient's immune cells in vitro and infusing them into the patient), while other methods involve (via gene therapy) genetically engineering immune cells to enhance their anti-cancer capabilities. Cell immunotherapy can be deployed in different ways, including but not limited to tumor-infiltrating lymphocytes (TIL) therapy, engineered T cell receptor (TCR-T) therapy, chimeric antigen receptor (CAR) T cell therapy, CARNK cell therapy, natural killer (NK) cell therapy.

[0212] In some embodiments, antibodies can be used in combination with additional anti-tumor therapies such as tumor-infiltrating lymphocytes (TIL) therapy, T cell receptor T cell (TCR-T) therapy, chimeric antigen receptor (CAR) T cell therapy and NK cell therapy, as well as targeted therapy and chemotherapy. The administration of additional anti-tumor therapies can be performed before, after, or simultaneously with the administration of an antibody or pharmaceutical composition as disclosed herein.

[0213] Chemotherapy

[0214] The term "anticancer agent" or "antiproliferative agent" means any agent that can be used to treat a cell proliferative disorder such as cancer, and includes, but is not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, debulking agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy and anti-metastatic agents and immunotherapeutic agents. It should be understood that in selected embodiments as described above, such anticancer agents may comprise a conjugate and may be associated with the disclosed site-specific antibodies prior to administration. More specifically, in certain embodiments, the selected anticancer agent is linked to an unpaired cysteine ​​of an engineered antibody to provide an engineered conjugate as described herein. Therefore, it is explicitly contemplated that such engineered conjugates are within the scope of the present disclosure. In other embodiments, the disclosed anticancer agent will be administered in combination with a site-specific conjugate comprising a different therapeutic agent as shown above.

[0215] As used herein, the term "cytotoxic agent" means a substance that is toxic to cells and reduces or inhibits the function of cells and / or causes cell destruction. In certain embodiments, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, small molecule toxins or enzymatically active toxins of bacteria (e.g., diphtheria toxin, Pseudomonas endotoxins and exotoxins, Staphylococcus enterotoxin A), enzymatically active toxins of fungi (e.g., alpha-sarcin, restrictocin), enzymatically active toxins of plants (e.g., abrin, ricin, modicin, mistletoe lectin, pokeweed antiviral protein, saporin, gelonin, momordica charantia toxin, trichosanthin, hordeoside toxin, Aleurites fordii proteins, dianthin proteins, pokeweed proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curculin, crotonin, chlorambu officinalis inhibitor, gelonin, mitegellin, restrictocin, phenomycin, neomycin, and trichothecenes), or enzymatically active toxins of animals (e.g., cytotoxic RNases, such as extracellular pancreatic RNase; DNase I, including fragments and / or variants thereof).

[0216] For the purposes of this disclosure, "chemotherapeutic agents" include compounds (e.g., cytotoxic agents or cytostatic agents) that non-specifically reduce or inhibit the growth, proliferation and / or survival of cancer cells. Such chemical agents are generally directed to the intracellular processes necessary for cell growth or division, and are therefore particularly effective for cancer cells that generally grow and divide rapidly. For example, vincristine depolymerizes microtubules, thereby inhibiting cells from entering mitosis. Generally speaking, chemotherapeutic agents may include any chemical agent that inhibits or is designed to inhibit cancer cells or cells that may become cancer cells or produce tumorigenic offspring (e.g., TICs). Such chemotherapeutic agents are generally administered in combination, such as in the regimen of CHOP or FOLFIRI, and are generally the most effective.

[0217] Examples of anticancer agents that can be used in combination with the site-specific constructs of the present disclosure (either as a component of a site-specific conjugate or in an unconjugated state) include, but are not limited to, alkylating agents, alkyl sulfonates, aziridines, ethyleneimine and methylmelamine, polyacetyl, camptothecin, bryostatin, carlatin, CC-1065, nostoc, dolestacin, duocarmycin, acanthosiphon, scutellarin, scutellarin, spongestatin, nitrogen mustards, antibiotics, enediyne antibiotics, danemycin, bisphosphonates, esperamicins, chromophores of enediyne antibiotics, aclarubicin, actinomycin, anthramycin, azaserine, bleomycin, actinomycin C, caraminomycin, carmomycin, chromomycin, actinomycin D, daunorubicin, detoxibacin, 6-diazo-5-oxo-L-norleucine, Doxorubicin, epirubicin, esorubicin, idarubicin, mexilomycin, mitomycin, mycophenolic acid, nogamycin, olivetomycin, peplomycin, porfibrinocin, puromycin, triferric doxorubicin, rhodorubicin, streptozotocin, streptozotocin, tuberculocidin, ubenimex, zoloft, daunorubicin; antimetabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogs, purine analogs, androgens, antiadrenal agents, folic acid supplements such as folate Acid, aceglucuronide, aldehyde phosphoramide glycoside, aminolevulinic acid, eniluracil, amsacrine, amustine, bisantrene, edatrexa, defofamine, colcemid, ferrugin, eflavourine, elliptinium acetate, epothilone, ethoxyquin, gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansinoid alkaloids, mitoxantrone, mitoxantrone, mopidarol, diamine nitracrine, pentostatin, mustard, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazide, methylbenzylhydrazine, polysaccharide complex (JHS Natural Products, Eugene, OR), razoxane; rhizoxin; sizoran; spirogermanamine; tricholomanic acid; triazoline quinone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucosin A, baculosporin A, and serpentin); ethyl carbamates; vindesine; dacarbazine; mannitol mustard; dibromomannitol; dibromodulanol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Vinorelbine; mitoxantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine; ibandronate; irinotecan (Camptosar, CPT-11), topoisomerase inhibitor RFS2000; difluoromethylornithine; retinoids; capecitabine; combretin; folinic acid; oxaliplatin; inhibitors of PKC-α, Raf, H-Ras, EGFR and VEGF-A that reduce cell proliferation; pharmaceutically acceptable salts, acids or derivatives of any of the above substances. Also included in this definition are anti-hormonal agents that act to regulate or inhibit the effects of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit the enzyme aromatase (which regulates estrogen production in the adrenal glands), and antiandrogens; as well as troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, ribozymes such as VEGF expression inhibitors and HER2 expression inhibitors; vaccines, rIL-2; Topoisomerase 1 inhibitors; rmRH; vinorelbine and esperamycin and pharmaceutically acceptable salts, acids or derivatives of any of the above substances.

[0218] Used in combination with radiation therapy

[0219] The present disclosure also provides a combination of antibodies and radiotherapy (i.e., any mechanism for inducing DNA damage locally in tumor cells, such as gamma radiation, X-rays, UV radiation, microwaves, electron emission, etc.). It is also contemplated that a combination therapy using directed delivery of radioisotopes to tumor cells is used, and the disclosed antibodies can be used in combination with targeted anticancer agents or other targeted means. Typically, radiotherapy is administered in a pulsed manner over a period of about 1 week to about 2 weeks. Radiotherapy can be administered to a subject with head and neck cancer for about 6 to 7 weeks. Optionally, radiotherapy can be administered as a single dose or as multiple doses in sequence.

[0220] Drug Packs and Test Kits

[0221] Also provided are pharmaceutical packages and kits comprising one or more containers, the one or more containers comprising one or more doses of antibodies or antigen-binding portions thereof. In certain embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising, for example, an antibody or an antigen-binding portion thereof, with or without one or more additional agents. For other embodiments, such a unit dose is provided in a single-use prefilled syringe for injection. In other embodiments, the composition contained in the unit dose may include saline, sucrose, etc.; a buffer such as phosphate, etc.; and / or formulated within a stable and effective pH range. Alternatively, in certain embodiments, the composition may be provided as a lyophilized powder, which may be reconstituted after adding a suitable liquid such as sterile water or a saline solution. In certain preferred embodiments, the composition comprises one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. Any label on or associated with the container indicates that the encapsulated composition is used to treat a selected tumor disease condition.

[0222] The present disclosure also provides a kit for producing a single-dose or multi-dose administration unit of an antibody and optionally one or more anticancer agents. The kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed of a variety of materials such as glass or plastic, and contains a pharmaceutically effective amount of the disclosed antibody or its antigen-binding portion. In other preferred embodiments, the container comprises a sterile inlet (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). Such kits typically contain a pharmaceutically acceptable formulation of an antibody in a suitable container and, optionally, one or more anticancer agents in the same or different containers. The kit may also contain other pharmaceutically acceptable formulations for diagnosis or combined therapy. For example, in addition to the antibody or its antigen-binding portion of the present disclosure, such kits may contain any one or more anticancer agents in a series of anticancer agents, such as chemotherapeutic or radiotherapeutic drugs; anti-angiogenic agents; anti-metastatic agents; targeted anticancer agents; cytotoxic agents; and / or other anticancer agents.

[0223] More specifically, the kit may have a single container containing the disclosed antibody or antigen-binding portion thereof with or without additional components, or the kit may have different containers for each desired agent. Alternatively, the antibody and any optional anticancer agent of the kit may be stored separately in different containers before administration to the patient. The kit may also include a second / third container device for containing sterile, pharmaceutically acceptable buffers or other diluents such as bacteriostatic water for injection (BWFI), phosphate buffered saline (PBS), Ringer's solution, and glucose solution.

[0224] When the components of the kit are provided in one or more liquid solutions, the liquid solution is preferably an aqueous solution, and particularly preferably a sterile aqueous solution or a saline solution. However, the components of the kit can be provided as dry powders. When the reagents or components are provided as dry powders, the powder can be reconstituted by adding a suitable solvent. It is contemplated that the solvent can also be provided in another container.

[0225] As briefly noted above, the kit may also contain a device for administering the antibody or antigen-binding portion thereof and any optional components to a patient, e.g., one or more needles, intravenous bags or syringes, or even eye drops, pipettes or other such similar devices, from which the formulation can be injected or introduced into an animal or administered to a diseased area of ​​the body. The kits of the present disclosure also typically include a device for containing vials and the like, as well as other components for commercial sale in a tightly sealed manner, such as, for example, an injection or blow molded plastic container in which the desired vials and other equipment are placed and retained.

[0226] SEQUENCE LISTING SUMMARY

[0227] The attached sequence table of the application includes many amino acid sequences and nucleotide sequences. Tables A, B and C below provide a sequence overview of the antibodies exemplified herein, including W301088-1.145.16, its chimeric form W301088-1.145.16-xlgG1KV320 and humanized form W301088-1.145.16-z3-p1-ulgG1KV320. W301088-1.145.16-xlgG1KV320 has the same variable region as the W301088-1.145.16 parent mouse clone.

[0228] Table A. Amino acid sequence of W301088-1.145.16-xIgG1KV320 (i.e., chimeric antibody), with CDR portions underlined

[0229]

[0230] Table B. Amino acid sequence of W301088-1.145.16-z3-pl-uIgG1KV320 (i.e., the final lead antibody), in which the CDR part is underlined

[0231]

[0232]

[0233] Table C. Nucleotide sequences encoding the VH and VL regions of W301088-1.145.16-z3-p1-uIgG1KV320

[0234]

[0235] Example

[0236] The present disclosure, thus generally described, will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present disclosure. The examples are not intended to represent that the experiments below are all or the only experiments performed.

[0237] Example 1

[0238] Preparation of antigens, reference antibodies and cell lines

[0239] 1.1 Preparation of commercial materials

[0240] Table 1. Information of commercially available materials

[0241]

[0242]

[0243] 1.2 Antigen production

[0244] The amino acid sequence encoding the extracellular domain of human 4IgB7H3 (Uniprot ID: Q5ZPR3, amino acids 29-461) was first codon optimized for mammalian expression and then synthesized by Sangon Biotech (Shanghai, China). The DNA fragment was subcloned into a pcDNA3.3 expression vector with 6xHis at the C-terminus and then transfected into Expi293F cells (Invitrogen, A14635). After five days of incubation, the supernatant was purified using a Ni column (Cytiva, 173712). The eluted protein was dialyzed into PBS via a dialysis bag (Spectrum-888-10987, MWCO 3.5kDa). The protein concentration was determined by the absorbance at 280nm on a NanoDrop device. 2 μg of purified protein was electrophoresed on an SDS-PAGE gel (Invitrogen) with and without a reducing agent. The purity of the purified protein was quantified by HPLC-SEC using a TSKgel G3000SWXL size exclusion column (Tosoh, 008541). The purified antibodies were stored at -80°C. Human 2IgB7H3 and cynomolgus monkey B7H3 were purchased from ACRO (Catalog No. B73-H52E2) and ACRO (Catalog No. B73-C52Ha).

[0245] 1.3 Generation of benchmark antibody (BMK)

[0246] The anti-B7H3 antibody enoxicam (MacroGenics, US20120294796A1: Seq 117, 119) was used as a benchmark antibody. The nucleic acid sequence encoding the variable domain of the antibody was first codon-optimized for mammalian expression and then synthesized by Sangon Biotech (Shanghai, China). The DNA fragment was then subcloned into a modified pcDNA3.4 expression vector with the constant region of human IgG1. Plasmids containing VH and VL genes were co-transfected into Expi293F cells (Invitrogen, A14635). After five days of incubation, the supernatant was purified using a protein A column (Cytiva, 175438). The eluted protein was dialyzed into PBS via a dialysis bag (Spectrum, 888-10987). The protein concentration was determined by absorbance at 280 nm on a NanoDrop device. 2 μg of purified protein was electrophoresed on SDS-PAGE gel (Invitrogen) with and without reducing agent. The purity of the purified antibody was quantified by HPLC-SEC using a TSKgel G3000SWXL size exclusion column (Tosoh, 008541). The purified antibody was stored at -80°C.

[0247] 1.4 Establishment of stable cell lines / cell pools

[0248] Generate a cell line expressing human 4IgB7H3. In brief, according to the manufacturer's protocol, CHOK1 cells were transfected with a pcDNA3.3 expression vector containing full-length 4IgB7H3 using Lipofectamine2000 transfection kit (Invitrogen, 11668027). 48 hours after transfection, cells were subcultured to a selective medium (F12-K containing 10% FBS and 15 μg / mL blasticidin) in a T75 flask. After two or three subcultures, a cell line (W3XX088-CHOK1.hPro 1.2A5) expressing human 4IgB7H3 was obtained by blasticidin selection and limiting dilution, and expression levels were determined by FACS using anti-B7H3 antibodies.

[0249] Generate a cell pool expressing cynomolgus monkey B7H3. Briefly, FlipinCHO cells were transfected with pcDNA5 / pOG44 expression vector containing full-length cynomolgus monkey B7H3 using Lipofectamine2000 transfection kit (Invitrogen-11668027) according to the manufacturer's protocol. 48 hours after transfection, cells were subcultured to selective medium (F12 containing 10% FBS and 600 μg / mL hygromycin B) in T75 flasks. After two or three passages of selection, the cells were selected by hygromycin B and BD FACS MelodyT M Cell sorting was used to obtain a high-expressing and stable cell pool (WBP3XX088-FlpinCHO.cProl.pool).

[0250] Example 2

[0251] Generation of WBP301088 antibody

[0252] 2.1 Animal immunization

[0253] Four Balb / c mice aged 6-8 weeks were immunized with DNA plasmids encoding human 4IgB7H3, and each mouse was immunized with 200 μg-400 μg of DNA plasmids encoding human 4IgB7H3. The adjuvant mixture includes Adju-Phos, CpG-ODN and GM-CSF. Animals were injected subcutaneously, intramuscularly and hydrodynamically in the tail vein once every other week. Blood was collected from mice after 3 injections (first blood draw) and 5 injections (second blood draw). Serum titers were measured by ELISA and FACS.

[0254] ELISA assay is used to measure serum antibody titers against 4IgB7H3 antigen. Plates were coated with 100 μL His tag at 0.5 μg / mL (Nunc), overnight at 4°C, and then blocked with blocking buffer (2% BSA in PBS) at ambient temperature for 1 hour. The plates were then washed and incubated with 1 μg / mL antigen at ambient temperature for 1 hour. After washing, mouse serum was serially diluted 1:3 in blocking buffer starting from a 1:100 dilution and incubated at ambient temperature for 2 hours. The plates were then washed and subsequently incubated with secondary goat anti-mouse IgG-Fc-HRP (Bethyl, A90-231P) for 1 hour. After washing, TMB substrate was added and the interaction was terminated with 2M HCl. The absorbance at 450 nm was read using a microplate reader (Molecular Device). Serum titers were measured under 2 times background.

[0255] Table 2. Summary of serum titers tested for human 4IgB7H3 binding by ELISA

[0256]

[0257] FACS assay was used to measure serum antibody titers against 4IgB7H3 antigen. Briefly, cells engineered to express B7H3 were cultured at 1×10 5 The cells were seeded into a 96-well U-bottom plate at a density of 10 cells / well, centrifuged at 1500 rpm for 4 minutes at 4°C, and the supernatant was removed. Mouse serum was diluted in 1×PBS / 1% BSA starting with a 1:100 dilution and serially diluted 1:3 to resuspend the cells and incubated at 4°C for 1 hour. The cells were washed twice with 180 μL 1×PBS / 1% BSA. The secondary antibody goat anti-mouse IgG-Fc Alexa 647 (Jackson, 109-605-098) was added to the resuspended cells and incubated at 4°C in the dark for 30 minutes, then washed with 180 μL 1xPBS / 1% BSA. Finally, the cells were resuspended in 100 μL 1xPBS / 1% BSA, and the fluorescence intensity was measured by FACS (BD Canto II) and analyzed by FlowJo version software. Serum titers were determined at 2 times the background.

[0258] Table 3. Summary of serum titer testing on human 4IgB7H3 cell line by FACS

[0259] Titer Mouse #1 Mouse #2 Mouse #3 Mouse #4 Second blood draw 218700 1968300 1968300 Close to 1968300

[0260] When the serum titer was high enough, 400 μg DNA was used before fusion via tail vein hydrodynamics and 4 × 10 6 MCF-7 cells were administered via footpad and subcutaneous administration for the final boost. Lymph nodes and spleen were used for cell fusion.

[0261] 2.2 Hybridoma production

[0262] Lymph nodes and spleens from immunized mice were homogenized and filtered to remove blood clots and cell debris. Sp2 / 0 myeloma cells growing in logarithmic growth were collected and centrifuged. B cells were fused with Sp2 / 0 myeloma cells in a 1:1 ratio in an electrofusion solution according to a general electrofusion procedure. The fused cells were suspended in DMEM medium supplemented with 20% FBS and 1X HAT and then transferred to a 96-well plate (CORNING). The fused cells were cultured in an incubator set at 37°C and 5% CO2 for 10 to 14 days.

[0263] 2.3 Antibody Screening

[0264] First Screen: A cell-based ELISA assay was used as the first screen to test the binding of hybridoma supernatants to human 4IgB7H3. Briefly, CHOK1 cells transfected with human 4IgB7H3 were cultured at 5 × 103 The density of cells / well was inoculated in a 96-well plate (CORNING) and then kept in an incubator set to 37°C, 5% CO2 for 2 days. The plate was then washed and incubated with hybridoma supernatant for 1 hour at ambient temperature. The plate was then washed and subsequently incubated with secondary goat anti-mouse IgG-Fc-HRP (Bethyl, A90-231P) for 1 hour. After washing, TMB substrate was added and the interaction was terminated with 2M HCl. The absorbance at 450nm was read using a microplate reader (Molecular Device).

[0265] Secondary screening: To confirm the binding of hybridoma supernatants to human and cynomolgus monkey antigens, FACS was performed. The parental CHOK1 cell line and the 4IgB7H3-transfected CHOK1 cell line were stained with CellTrace dyes (Violet and FarRed, respectively), while the B7H3-transfected FlipinCHO cell pool was prepared without dye. After incubation at ambient temperature for 15 minutes and washing, the three types of cells were mixed in equal amounts to 1×10 6 The three cell mixtures were diluted to 1×10 5 The cells were transferred to a 96-well U-bottom plate (BD) at a density of 10 cells / well. The hybridoma supernatant was then transferred to the plate and incubated at 4°C for 1 hour. After washing, the secondary antibody goat anti-mouse IgG Fc PE (Jackson, 115-115-164) was added and incubated with the cells at 4°C in the dark for 0.5 hours. The cells were then washed, resuspended in 1×PBS / 1% BSA, and then analyzed by flow cytometry.

[0266] To test the binding of hybridoma supernatants to human 2IgB7H3, conventional ELISA assays were used. At ambient temperature, the plate was pre-coated with 100 μL / well of human 2IgB7H3 (0.2 μL g / mL) for 1 hour. After blocking with 200 μL / well of 1×PBS / 2%BSA for 1 hour, the plate was washed 3 times with 1×PBST. The hybridoma supernatant was added to the plate at a volume of 100 μL / well and incubated at ambient temperature for 2 hours. After the plate was washed 3 times with 1×PBST, 100 μL / well of goat anti-mouse IgG-Fc HRP (Bethyl, A90-231P) was added to the plate and incubated at ambient temperature for 1 hour. After washing 6 times, the color was developed by distributing TMB substrate at 100 μL / well for 5 minutes to 10 minutes, and then the reaction was stopped by adding 100 μL / well of 2M HCl. Read the absorbance at 450 nm using a microplate reader.

[0267] The selected positive cell lines screened by FACS were further confirmed by SPR (surface plasmon resonance). SPR allows real-time, label-free detection of biomolecular interactions. SPR occurs when polarized light strikes a conductive surface at the interface between two media. This generates an electronic charge density wave called a plasma that reduces the intensity of the reflected light at a specific angle called the resonance angle in proportion to the mass on the sensor surface.

[0268] Biacore 8K was used to grade the binding of supernatants to human 4IgB7H3. Activators were prepared by mixing 400mM EDC and 100mM NHS (GE) immediately before injection. The CM5 sensor chip was activated with an activator and kept for 420 seconds. Goat anti-mouse Fc IgG (30μg / mL, in 10mM NaAc, pH 4.5) was then injected into the channel at a flow rate of 10μL / min for 420 seconds. The chip was deactivated with 1M ethanolamine-HCl. The supernatant was injected into the channel at a flow rate of 10μL / min for 30 seconds. 200nM human 4IgB7H3 analyte was injected into the channel at a flow rate of 30μL / min, maintaining a binding period of 120 seconds, followed by a dissociation of 300 seconds. Glycine (10mM, pH 1.5) was injected after the dissociation period as a regeneration buffer. The sensorgram was subtracted from the reference channel and the buffer channel. The data were then fit with a 1:1 Langmuir binding model.

[0269] pHrodo iFL dyes can be conjugated to biomolecules such as antibodies and become highly fluorescent only when present in acidic environments such as cellular lysosomes and endosomes. This enables detection of conjugated biomolecules in live cell assays. AffiniPure F(ab') labeled with pHrodo iFL Red STP ester amine reactive dye 2 Fragments of goat anti-human IgG Fcγ and anti-mouse IgG Fcγ were purified by Zeba Spin desalting columns according to the manufacturer's instructions. MCF-7 cells (2×10 4 Cells were seeded into 96-well clear bottom black plates pre-coated with 8 μg / mL poly-D-lysine (cells / well). The cells were grown overnight in an incubator set at 37°C, 5% CO2. The next day, WBP301088-1 positive cell line supernatant was added to the corresponding wells at a volume of 100 μL. The cells were incubated in an incubator set at 37°C, 5% CO2 for 0.5 hours. Human and mouse IgG1 isotype antibodies were used as negative controls. 50 nM pHrodo iFL Red / F(ab′) was added at a volume of 100 μL / well. 2Goat anti-mouse or anti-human IgG Fcγ conjugate (diluted with complete medium). The plate was then incubated in a dark incubator set at 37°C, 5% CO2 for 5 hours. After incubation, the supernatant was discarded. The plate was washed with complete medium. A mixed dye solution (1 μg / mL Hoechst 33342 and 0.5 μg / mL Calcein AM diluted in DPBS) was added to the plate at a volume of 100 μL / well and kept at ambient temperature for 15 minutes. After incubation, the dye solution was discarded and the plate was washed once with 1 × PBS / 1% BSA. The fluorescence intensity of MCF-7 cells was read and analyzed by the Perkin Elmer Operetta CLS high content analysis system.

[0270] A total of 8460 wells were tested in the primary screening, and 279 hybridomas were selected and amplified in 24-well plates. In the secondary screening, the supernatant from the 24-well plates was used to confirm strong binding to human 4IgB7H3 and cynomolgus monkey B7H3, weak binding to human 2IgB7H3, and strong internalization activity by MCF-7. Based on the secondary screening results, the WBP301088-1.145 hybridoma was selected for subcloning.

[0271] 2.4 Antibody subcloning

[0272] Use selected positive hybridoma cells for subcloning. Count cells growing in logarithmic numbers and add 1.5mL semisolid HAT culture medium. Mix cells gently in a vortex shaker for 5 seconds-10 seconds and then inoculate in a 6-well plate (CORNING). The plate is kept in an incubator set to 37°C, 5% CO2 for 7 to 8 days. Pick each visible single colony into a 96-well plate (CORNING), which has a DMEM culture medium supplemented with 10% FBS. After 2 to 3 days, collect cell supernatants and use the antibody screening scheme as described above, by cell-based ELISA screening (human 4IgB7H3), based on conventional ELISA screening (human 2IgB7H3). After confirmatory screening of human 4IgB7H3 and cynomolgus monkey B7H3 subclones by FACS, collect the culture supernatants of selected single positive clones, and purify antibodies for further characterization. WBP301088-1.145.16 was obtained after subclone screening and confirmatory screening.

[0273] 2.5 Antibody isotyping

[0274] ELISA isotyping was performed in a 96-well high binding plate (Nunc). Each well was coated with 2 μg / mL capture antibodies goat anti-mouse IgG1 (Bethvl, A90-205A), goat anti-mouse IgG2a (Bethyl, A90-207A), goat anti-mouse IgG2b (Bethvl, A90-209A), goat anti-mouse IgG3 (Bethyl, A90-211A), overnight at 4°C, and blocked with 1×PBS / 2% BSA. The hybridoma supernatant was incubated in the coated wells, and the specific binding of the antibody to the capture antibody fixed on the plate was measured using a peroxidase-conjugated goat anti-mouse r / k light chain antibody. The HRP signal was detected by adding TMB substrate, and the reaction was terminated with 2M HCl after 12 minutes. All incubation steps were performed at ambient temperature, and the plates were washed with PBS between each step. The absorbance at 450nm was read using a microplate reader (Molecular Device).

[0275] The isotyping results of WBP301088-1.145.16 are shown in the table below (Table 4).

[0276] Table 4. Isotyping results of WBP301088-1.145.16

[0277] Clone number Isotype WBP301088-1.145.16 IgG2b, kappa

[0278] 2.6 Antibody Production

[0279] When a small amount of purified antibody is needed, T75 flasks (CORNING) are often used to scale up hybridoma cell culture. Monoclonal hybridoma cells are cultured at 5×10 6 Up to 1×10 7 The cells were inoculated into the flasks at 100 cells / T75 flask. The cells were grown continuously for about 7-10 days until the cell viability reached about 30%-40%. The culture supernatant was harvested and cell debris was removed by centrifugation. The supernatant was sterile filtered and stored for antibody purification.

[0280] 2.7 Hybridoma Sequencing

[0281] According to the instructions of TaKaRa MiniBEST Universal RNA Extraction Kit, total RNA of hybridoma cell samples was first extracted. RNA was then converted to cDNA using the SMART RACE cDNA Amplification Kit from Clonetech. VH and VL domain DNA sequences were then amplified from cDNA using 30 PCR cycles, with each cycle denaturing at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and then extending at 72°C for 30 seconds. The PCR products were then subcloned into a TA cloning vector and sent to BiosuneBiotech (Shanghai, China) for sequencing.

[0282] Once the sequencing data confirmed the monoclonality of the hybridoma sample, the DNA sequences of the VH and VL domains were amplified by PCR. Primers were synthesized by Sangon Biotech (Shanghai, China). The DNA fragments were then subcloned into the pcDNA3.4 expression vector of the human IgG1 constant region with the LALA mutation and then sequenced by Tsingke Biotechnology (Beijing, China).

[0283] 2.8 IgG conversion

[0284] Once sequencing data confirmed the monoclonality of the hybridoma cell sample, the amino acid sequences of the VH and VL domains were codon-optimized for mammalian expression and then synthesized by Sangon Biotech (Shanghai, China). The DNA fragments were then subcloned into the pcDNA3.4 expression vector for human IgG1 constant region with LALA mutation.

[0285] In this way, W301088-1.145.16 is converted into human IgG1 with LALA mutation form to obtain chimeric antibody W301088-1.145.16-xIgG1KV320. After protein A purification and buffer exchange, it is characterized by SDS-PAGE and SEC-HPLC. Under reducing conditions, the protein migrates with an apparent molecular weight of 50kDa and 25kDa on SDS-PAGE, corresponding to IgG heavy chain and light chain (data not shown). Purity is higher than 99% (SEC-HPLC).

[0286] Table 5. Summary of chimeric antibody production

[0287]

[0288] 2.9 Humanization

[0289] A local copy of the open source software ANARCI (Dunbar J, Deane CM, "ANARCI: antigen receptor numbering and receptor classification", Bioinformatics, 2015; Vol. 32, No. 5: pp. 298-300) was run to assign Kabat numbers on W301088-1.145.16. CDRs were identified according to the definition of Dr. Martin's website (Martin, 2018, supra) and are shown in Table 6.

[0290] Table 6. CDR ranges defined according to Dr. Martin Antibody Informatics website

[0291]

[0292] Table 7. VH and VL amino acid sequences of W301088-1.145.16, with CDR sequences underlined

[0293]

[0294] Table 8. Identified heavy and light chain CDR sequences of W301088-1.145.16

[0295]

[0296] The VH and VL domain sequences of the mouse antibody W301088-1.145.16 were aligned with the human germline sequence library of the VH and VL domains of IMGT, respectively. The human germline sequence of the VH / VL domain with the minimum number of amino acid differences in the framework relative to the VH / VL domain sequence of W301088-1.145.16 was selected as the humanization template of the VH / VL domain. The combination of IGHV4-31*02 and IGHJ6*01 and the combination of IGKV4-1*01 and IGKJ4*01 are the most homologous human germline sequences to the VH and VL domain sequences of W301088-1.145.16, respectively. The CDRs of W301088-1.145.16 were transplanted into the framework of these two human germline templates to form the germline sequence.

[0297] Several "back mutation" positions in the framework are selected empirically to convert the amino acid in the germline sequence into their corresponding amino acid in the original mouse sequence. A group of humanized variants are designed empirically to study the different combinations of these selected back mutation sites. At 16 positions of the VL domain and 20 positions of the VH domain, the germline sequence is different from the original mouse W301088-1.145.16 sequence. For back mutation, 14 sites in a total of 36 different sites are considered: VH: Q001D, S025T, H040F, K043N, G044K, I048M, V067I, V071R, A085E, V089T, Y091F, VL: I021M, P043S, Y087F. A group of humanized variants are designed empirically to test the different combinations of these back mutations.

[0298] 2.11 PTM Removal

[0299] The VH and VL domain sequences of the humanized variants were scanned for several types of key post-translational modification (PTM) sites: asparagine deamination (NG and NS) in CDRs, aspartate isomerization (DG) in CDRs, unpaired Cs over the entire length, and N-linked glycosylation sites (NXS / T, where X can be any amino acid except P) over the entire length. Point mutations were empirically designed to remove these key PTM sites in the humanized variants to avoid the potential risk of PTM modifications. Finally, we selected PTM removal antibodies that did not affect expression, binding, and thermal stability compared to the parental antibody.

[0300] Since the VL domain sequence of the murine antibody W301088-1.145.16 contains a NSS N-linked glycosylation site, three point mutations were designed, namely VL: N027dQ; VL: S027eP and VL: S027fA (according to Kabat numbering), to remove the critical PTM sites. Together with the humanized variants, they were further modified by k off Hierarchical characterization.

[0301] 2.12 koff classification by SPR

[0302] K analysis of the filtered supernatant was performed on a Biacore 8K instrument. offGrading. First, the CM5 sensor chip was activated with 400mM EDC and 100mM NHS (GE) at a flow rate of 10μL / min for 420 seconds. Then 30μg / mL anti-human Fc IgG (Jackson) in 10mM NaAc (pH 4.5) was injected into channels 1 to 8 at a flow rate of 10μL / min for 420 seconds. Then the chip was deactivated with 1M ethanolamine-HCl (GE) at a flow rate of 10μL / min for 420 seconds. The diluted antibody supernatant was injected into the Fc2 of the channel at a flow rate of 10μL / min. The supernatant of the cell culture without plasmid transfection was used as a negative control and injected into the Fc1 of the channel at a flow rate of 10μL / min. 100nM human 4IgB7H3 and running buffer were injected into the Fc1-Fc2 of the channel at a flow rate of 3% L / min, and the association period was maintained for 180 seconds, followed by a dissociation period of 3600 seconds. 10mM glycine (pH 1.5) was injected after each run to regenerate the chip. The sensorgram was analyzed by the software included with the Biacore 8K device. The sensorgram of the sample was subtracted from the reference channel and the buffer channel. The data was then fitted with a 1:1 Langmuir binding model.

[0303] 2.13 Combination of PTM removal mutations and humanized variants

[0304] will show the optimal k off The rate of PTM removal point mutations is combined to have the optimal k off The humanized variants were subjected to rate point-wise mutagenesis to make the final construct for affinity validation.

[0305] According to k off The final lead was composed of the PTM removal mutation of VL: N027dQ and four selected humanized mutant amino acids S025T, V071R, Y091F, and V089T. The final lead was named W301088-1.145.16-z3-p1-uIgG1KV320.

[0306] Table 9. Heavy and light chain CDR sequences of W301088-1.145.16-z3-p1-uIgG1KV320

[0307]

[0308] 2.14 Production-scale transient transfection and purification

[0309] Using ExpiFectamine TM Transient transfection kit was used to transiently transfect each plasmid at a final concentration of 1 μg / mL into 20 mL of Expi293F cells at a cell density of 3.0 × 10 6Cells / mL were obtained and cell viability was over 95%. Cell cultures were grown in a humidified horizontal shaker with a rotation speed of 150 rpm. The temperature was maintained at 37°C and the CO2 level was maintained at 8%.

[0310] After 5 days of cell culture incubation, the supernatant expressing the target antibody was collected and filtered, and purified using a GE MabSelectSuRE protein A column (Cytiva, 175438). The eluted antibody was dialyzed into PBS via a D-Tube dialyzer Maxi (EMDMillipore71 508, MWCO 3.5 kDa). The antibody concentration was determined by the absorbance at 280 nm on the NanoDrop device. 2 μg of the purified antibody sample was added to a SDS-PAGE gel (Invitrogen NuPAGE) containing and not containing a reducing agent. TM The purity of the purified antibody samples was quantified by HPLC-SEC using a TSKgel G3000SWXL size exclusion column (Tosoh, 008541). The purified antibodies were stored at -80°C.

[0311] Example 3

[0312] In vitro characterization of WBP301088 antibody

[0313] 3.1 Purity testing by SDS-PAGE and SEC-HPLC

[0314] The samples were mixed with loading buffer and heated at 75°C for 10 minutes using a drying bath. After sample loading, SDS-PAGE was run at a constant voltage (200 V) for 35 minutes. The gel was stained and stained with eStain TM L1 was destained. SDS-PAGE results were obtained using the BIO-RAD imaging system.

[0315] The results are summarized in Table 10 below. Under reducing conditions, the protein migrated on SDS-PAGE with apparent molecular weights of 50 kDa and 25 kDa, corresponding to the IgG heavy and light chains, whereas under non-reducing conditions, it migrated with the expected band of approximately 150 kDa ( Figure 1 ).

[0316] 3.2 Purity testing by SEC-HPLC

[0317] The chromatograms were analyzed by Agilent Technologies 1260 Infinity II system (Agilent Technologies, Inc.) equipped with a TSKgel G3000SWXL column (Tosoh Bioscience, 0008541). TM) to detect the purity of each antibody. An appropriate volume of sample (5 μL-100 μL) was injected into the column and separated at a flow rate of 1 mL / min for 20 minutes. The running buffer was 50 mM sodium phosphate, 150 mM NaCl, pH 7.0. Peak retention was detected with UV light at a wavelength of 280 nm. The purity of each antibody was analyzed by integrating all peak areas from 4.5 minutes to 10.5 minutes using SEC-HPLC analysis. The operation and analysis software was OpenLab CDS workstation (v2.3.0.443 or v2.2.0.484).

[0318] The results are summarized in Table 10 below. The purity of the final lead was higher than 99% (SEC-HPLC, Figure 2 ).

[0319] 3.3 Thermal stability of DSF (T m )

[0320] use 7Flex Real-Time PCR System (Applied Biosystems) was used to study the T m (Melting temperature). 19 μL of antibody solution was mixed with 1 μL of SYPRO Orange solution (Invitrogen) and transferred to a 96-well plate (Applied Biosystems). The plate was sealed with an optical adhesive film (Applied Biosystems) and centrifuged at 3,000 rpm for 5 minutes to remove any bubbles. The plate was heated from 26°C to 95°C at a rate of 0.9°C / min, and the resulting fluorescence data was collected. The negative derivative of fluorescence with different temperatures was calculated, and the maximum value was defined as the melting temperature T m If a protein has multiple unfolding transitions, the first T is reported. m , name it T m 1. Data Collection and T m Calculated by Real-time PCR software (v1.3) was used automatically.

[0321] Protein W301088-1.145.16-z3-p1-uIgG1KV320 m The value is defined as the maximum value of the negative derivative of fluorescence with different temperatures, as listed in Table 10 below. The data distribution is shown in Figure 3 , where the dotted line represents T m The position of the 1 value in the fluorescence curve. T of W301088-1.145.16-z3-p1-uIgG1KV320 m 1 is 70.1℃, which shows that it has good thermal stability.

[0322] 3.4 Hydrophobicity detection by HIC-HPLC

[0323] The results were analyzed by HPLC 1260 Infinity II system (Agilent Technologies TM ) The hydrophobic properties of the antibodies were detected using a TSKgel butyl-NPR column (Tosoh-0042168). Each sample was diluted to 0.5 mg / mL with PBS buffer, 20 μL of the diluted sample was injected into the column, and separated for 61 minutes at a flow rate of 0.5 mL / min. The running buffer was 25 mM sodium phosphate, pH 7.0 (buffer A) and 25 mM sodium phosphate, 1.5 M (NH4)2SO4, pH 7.0 (buffer D). The running gradient from 3 minutes to 53 minutes was 0% to 100% buffer D. Peak retention was detected with UV light at 280 nm and 230 nm wavelengths. The retention time was analyzed by integrating all peak areas from 20 minutes to 40 minutes using the HIC-HPLC analysis method. The operation and analysis software was OpenLab CDS workstation (v2.6.0.691).

[0324] The HIC retention time of protein W301088-1.145.16-z3-p1-uIgG1KV320 was 23.52 minutes (Table 10 and Figure 4 ), which indicates that it is an antibody with good hydrophilicity.

[0325] 3.5 Diffusion interaction parameter (k) by DLS D )

[0326] DLS-k was studied using the DynaPro plate reader III (Wyatt Technology). D Measurement results. During sample preparation, the appearance of the samples was recorded when thawing, filtering and concentrating. The samples were concentrated to more than 20 mg / mL and then diluted with PBS buffer to a final concentration of 2.5 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL and 20 mg / mL. 7.5 μL of sample solution was then added to a 1536-well microplate (Aurora, ABI1-00110A). The plate was sealed with a ClearSeal membrane (Hampton Research, HR4-521) and centrifuged at 3,000 rpm for 5 minutes to allow the sample to fall to the bottom of the well. Each sample was tested in duplicate in the well. The plate was placed in the corresponding position and data collection was performed by DYNAMICS operating software (v7.8.1.3). Five acquisitions of each protein sample were collected, with each acquisition time of 5 seconds. For each measurement, the diffusion coefficient was determined and plotted against protein concentration. k DThe values ​​were automatically calculated by the software (v7.8.1.3) and are listed in Table 10. The data distribution is shown in Figure 5 Protein W301088-1.145.16-z3-p1-uIgG1KV320 in PBS D The solubility of the protein was -5.17 mL / g, indicating that it is an antibody with high solubility. During the assay, the appearance of the protein was recorded, and some particles were observed after thawing and gentle shaking. If the buffer was changed to 20 mM His, 8% sucrose, 0.02% PS80, pH 6.5, no particles were observed.

[0327] The above SDS-PAGE, SEC-HPLC, HIC-HPLC, DSF and DLS results are summarized in Table 10.

[0328] Table 10. Summary of analytical data of W301088-1.145.16-z3-p1-uIgG1KV320

[0329]

[0330] 3.6 Human B7H3 binding (FACS)

[0331] FACS was used to detect the binding of antibodies to human B7H3. This method can quantitatively analyze and identify specific molecules expressed on the surface of living cells. Unlabeled cells were used as controls to set thresholds before detection, and the percentage change of each group exceeding the fluorescence intensity threshold was analyzed. MCF-7 cells (1×10 5 cells / well) were incubated with various concentrations of antibodies (serial dilutions 3.16-fold from 50 nM to 0.0005 nM with 1 × DPBS / 1% BSA) at a volume of 100 μL / well in a refrigerator set at 4°C for 1 hour. The anti-human B7H3 reference antibody Enochomab (MacroGeneics) was used as a positive control. Human IgG1 isotype antibody was used as a negative control. After washing the cells with 1×DPBS / 1% BSA, Alexa 647 goat anti-human antibody (diluted 1:500 in 1×DPBS / 1% BSA) was added. The cells were incubated in a refrigerator set at 4°C for 0.5 hour in the dark. The mean fluorescence intensity (MFI) of the cells was measured using a flow cytometer and analyzed using FlowJo. EC was calculated by four-parameter nonlinear regression analysis using GraphPadPrism 7 software. 50 value.

[0332] W301088-1.145.16-z3-p1-ulgG1KV320 showed an EC of 1.08 nM 50The chimeric antibody bound well to MCF-7 cells expressing B7H3, and the maximum MFI was 19900, which was comparable to that of the chimeric antibody and superior to that of the reference antibody enoxitumomab (MacroGenics) (Table 11, Figure 6 ).

[0333] Table 11. FACS binding data for MCF-7 cells expressing human B7H3

[0334]

[0335] Note: NA means not available.

[0336] 3.7 Cynomolgus monkey B7H3 binding (FACS)

[0337] FACS was used to detect the binding of antibodies to cynomolgus monkey B7H3. FlpinCHO cells (W3XX088-FlpinCHO.cProl.pool) were transfected with cynomolgus monkey B7H3 (1×10 5 cells / well) and incubated them with various concentrations of antibodies (serial dilutions 3.16-fold from 50 nM to 0.0005 nM with 1×DPBS / 1% BSA) at a volume of 100 μL / well in a refrigerator set at 4°C for 1 hour. The anti-human B7H3 reference antibody enoxicam (MacroGeneics) was used as a positive control. Human IgG1 isotype antibody was used as a negative control. After washing the cells with 1×DPBS / 1% BSA, Alexa 647 goat anti-human antibody (diluted 1:500 in 1×DPBS / 1% BSA) was added. The cells were incubated in the dark in a refrigerator set at 4°C for 0.5 hour. The mean fluorescence intensity (MFI) of the cells was measured by flow cytometry and analyzed with FlowJo. EC was calculated by four-parameter nonlinear regression analysis using GraphPad Prism 7 software. 50 value.

[0338] W301088-1.145.16-z3-p1-uIgG1KV320 showed an EC of 4.68 nM 50 The binding to cynomolgus monkey transfected cells was good, which was comparable to the chimeric antibody and better than enoximab (MacroGenics) (Table 12, Figure 7 ).

[0339] Table 12. FACS binding data for stable cell pools transfected with cynomolgus monkey B7H3

[0340]

[0341] Note: NA means not available.

[0342] 3.8 Human 4IgB7H3 binding (SPR)

[0343] Use SPR to determine the affinity of the antibody for human 4IgB7H3. The affinity of the antibody for human 4IgB7H3 was determined by Biacore 8K. The activator was prepared by mixing 400mM EDC and 100mM NHS (GE) immediately before injection. The CM5 sensor chip was activated with the activator and kept for 420 seconds. Then goat anti-human Fc IgG (30μg / mL, in 10mM NaAc, pH 4.5) was injected into the channel at a flow rate of 10μL / min for 420 seconds. The chip was deactivated with 1M ethanolamine hydrochloric acid. The antibody diluted in running buffer (1×HBS-EP+) was injected into the channel at a flow rate of 10μL / min for 30 seconds. Seven concentrations (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 2.125 nM and 1.563 nM) of the analyte human 4IgB7H3 were sequentially injected into the channel at a flow rate of 30 μL / min, maintained for 180 seconds of association period, followed by 3600 seconds of dissociation. Glycine (10 mM, pH 1.5) was injected after the dissociation period as a regeneration buffer.

[0344] The sensorgrams of the reference channel and buffer channel were subtracted from the test sensorgram. The experimental data were fitted with a 1:1 binding model. The molecular weight of human 4IgB7H3 used for calculations was 47.8 kDa.

[0345] The experimental data were fitted using the 1:1 binding model included with the Biacore 8K evaluation software. Figure 8A-8C As shown, W301088-1.145.16-z3-p1-uIgG1KV320 showed high binding affinity to human 4IgB7H3. D The value was 7.08E-11M, which was superior to enoxaparin (MacroGenics).

[0346] Table 13. Antibody affinity results for binding to human 4IgB7H3

[0347]

[0348] 3.9 Human 2IgB7H3 Binding (SPR)

[0349] Use SPR to determine the affinity of the antibody for human 2IgB7H3. The affinity of the antibody for human 2IgB7H3 was determined by Biacore 8K. The activator was prepared by mixing 400mM EDC and 100mM NHS (GE) immediately before injection. The CM5 sensor chip was activated with the activator and kept for 420 seconds. Then goat anti-human Fc IgG (30μg / mL, in 10mM NaAc, pH 4.5) was injected into the channel at a flow rate of 10μL / min for 420 seconds. The chip was deactivated with 1M ethanolamine hydrochloric acid. The antibody diluted in running buffer (1×HBS-EP+) was injected into the channel at a flow rate of 10μL / min for 30 seconds. Seven concentrations (500nM, 250nM, 125nM, 62.5nM, 31.25nM, 15.625nM and 7.813nM) of the analyte human 2IgB7H3 were sequentially injected into the channel at a flow rate of 30μL / min, maintained for a 120-second association period, followed by a 300-second dissociation period. Glycine (10mM, pH 1.5) was injected after the dissociation period as a regeneration buffer.

[0350] The sensorgrams of the reference channel and buffer channel were subtracted from the test sensorgram. The experimental data were fitted with a 1:1 binding model. The molecular weight of human 2IgB7H3 used for calculations was 25.2 kDa.

[0351] The experimental data were fitted using the 1:1 binding model included with the Biacore 8K evaluation software. Figure 9A-9C As shown, W301088-1.145.16-z3-p1-uIgG1KV320 showed weaker binding affinity to human 2IgB7H3 compared to the binding to human 4IgB7H3. D The value is 4.64E-08M.

[0352] Table 14. Antibody affinity results for binding to human 2IgB7H3

[0353]

[0354] 3.10 Cynomolgus monkey B7H3 binding (SPR)

[0355] Use SPR to determine the affinity of the antibody for cynomolgus monkey B7H3. The affinity of the antibody for cynomolgus monkey B7H3 was determined by Biacore 8K. The activator was prepared by mixing 400mM EDC and 100mM NHS (GE) immediately before injection. The CM5 sensor chip was activated with the activator for 420 seconds. Then goat anti-human Fc IgG (30μg / mL, in 10mM NaAc, pH 4.5) was injected into the channel at a flow rate of 10μL / min for 420 seconds. The chip was deactivated with 1M ethanolamine hydrochloric acid. The antibody diluted in running buffer (1×HBS-EP+) was injected into the channel at a flow rate of 10μL / min for 30 seconds. The analyte cynomolgus monkey B7H3 at nine concentrations (50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.563 nM, 0.781 nM, 0.391 nM and 0.196 nM) was sequentially injected into the channel at a flow rate of 30 μL / min, maintained for 180 seconds of association period, followed by 3600 seconds of dissociation. Glycine (10 mM, pH 1.5) was injected after the dissociation period as a regeneration buffer.

[0356] The sensorgrams of the reference channel and buffer channel were subtracted from the test sensorgram. The experimental data of 6 analyte concentrations (50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM and 1.563 nM) were fitted with a 1:1 binding model. The molecular weight of cynomolgus monkey B7H3 used for calculations was 49 kDa.

[0357] The experimental data were fitted using the 1:1 binding model included with the Biacore 8K evaluation software. Figure 10A-10C As shown, W301088-1.145.16-z3-p1-uIgG1KV320 showed high binding affinity to cynomolgus monkey B7H3, which was comparable to its binding to human 4IgB7H3. D The value was 5.74E-1 1M, which was superior to enoxaparin (MacroGenics).

[0358] Table 15. Antibody affinity results for binding to cynomolgus monkey B7H3

[0359]

[0360] 3.11 Internalization test (HCS)

[0361] Antibody internalization was measured by operetta CLS (PerkinElmer), a high-content imaging and analysis system that can collect and analyze sample images at high speed and high sensitivity. MCF-7 cells (1.5 × 10 4Cells / well) were seeded into 96-well clear-bottom black plates pre-coated with 8 μg / mL poly-D-lysine. After incubation overnight in an incubator set at 37°C, 5% CO2, various concentrations of antibodies (serial dilutions of 3.16 times from 50nM to 0.0005nM with 1×DPBS / 1%BSA) were incubated in a refrigerator set at 4°C for 2 hours at a volume of 100 μL / well. The anti-human B7H3 reference antibody Enochomab (MacroGeneics) was used as a positive control. Human IgG1 isotype antibody was used as a negative control. After washing the cells with 1×DPBS / 1%BSA, Alexa 647 goat anti-human antibody (diluted 1:500 in 1×DPBS / 1%BSA) was added. The cells were incubated in a refrigerator set at 4°C in the dark for 1 hour. The cells were then washed once and resuspended in 1×DPBS / 1%BSA at 37°C for 2 hours. After incubation, the supernatant was discarded and 100 μL / well Hoechst 33342 (diluted 1:5000 in 1×DPBS) was added and incubated at ambient temperature for 15 minutes. After washing the cells with 1×DPBS, 100 μL / well acid wash buffer (100 mM glycine, 150 mM NaCl, pH 2.5) was added and incubated in a refrigerator set to 4°C for 5 minutes. The cells were washed once with 1×DPBS and fixed with 60 μL 4% PFA. The mean fluorescence intensity (MFI) of the cells was measured and analyzed by operetta CLS. EC was calculated by four-parameter nonlinear regression analysis using GraphPad Prism 7 software. 50 value.

[0362] W301088-1.145.16-z3-p1-uIgG1KV320 showed an EC of 0.51 nM 50 The good internalization ability of the antibodies into MCF-7 cells expressing B7H3 was comparable to that of the chimeric antibodies and superior to that of the reference antibody enoxitumomab (MacroGenics) (Table 16, Fig.11 ).

[0363] Table 16. Internalization of human B7H3 expressed in MCF-7 cells

[0364]

[0365] Note: NA means not available.

[0366] It will also be appreciated by those skilled in the art that the present disclosure may be implemented in other specific forms without departing from its spirit or central attributes. Since the foregoing description of the present disclosure discloses only exemplary embodiments thereof, it will be appreciated that it is within the scope of the present disclosure to envision other variations. Therefore, the present disclosure is not limited to the specific embodiments described in detail herein. Instead, reference should be made to the appended claims as an indication of the scope and content of the present disclosure.

Claims

1. An antibody or antigen-binding portion thereof that binds to B7H3, comprising the following CDRs: A heavy chain CDR1 (HCDR1) comprising the amino acid sequence of SEQ ID No: 1; a HCDR2 comprising the amino acid sequence of SEQ ID No: 2; a HCDR3 comprising the amino acid sequence of SEQ ID No: 3; and A light chain CDR1 (LCDR1) comprising the amino acid sequence of SEQ ID No: 4 or 7; a LCDR2 comprising the amino acid sequence of SEQ ID No: 5; and a LCDR3 comprising the amino acid sequence of SEQ ID No:

6.

2. The antibody or antigen-binding portion thereof of claim 1, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises: (A) the amino acid sequence shown in SEQ ID No: 8 or 10; (B) an amino acid sequence that is at least 85%, at least 90% or at least 95% identical to SEQ ID No: 8 or 10; or (C) an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid additions, deletions and / or substitutions in the framework regions compared to SEQ ID No: 8 or 10; and / or the VL comprises: (A) the amino acid sequence shown in SEQ ID No: 9 or 11; (B) an amino acid sequence that is at least 85%, at least 90% or at least 95% identical to SEQ ID No: 9 or 11; or (C) an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid additions, deletions and / or substitutions in the framework region compared to SEQ ID No: 9 or 11.

3. The antibody or antigen binding portion thereof of claim 2, wherein the VH and / or VL comprises one or more amino acid substitutions in the framework region.

4. The antibody, or antigen binding portion thereof, of claim 3, wherein at least one of the amino acid substitutions is a back mutation in which an amino acid from the human germline sequence is substituted with a different amino acid at the corresponding position in the parent antibody.

5. The antibody or antigen binding portion thereof of claim 3, wherein at least one of the amino acid substitutions removes a potential post-translational modification site.

6. The antibody or antigen binding portion thereof according to any one of the preceding claims, wherein the B7H3 is a human B7H3 protein, a mouse B7H3 protein, a cynomolgus monkey B7H3 protein or an extracellular domain thereof.

7. The antibody or antigen binding portion thereof according to claim 6, wherein the B7H3 is human 4IgB7H3 protein or an extracellular domain thereof.

8. The antibody or antigen binding portion thereof according to any one of the preceding claims, wherein the antibody or antigen binding portion thereof is a full length antibody, scFv, Fab, F(ab')2 or Fv fragment.

9. The antibody, or antigen binding portion thereof, of any preceding claim, wherein the antibody comprises an immunoglobulin constant region, such as a human IgG constant region.

10. The antibody or antigen binding portion thereof according to claim 9, wherein the immunoglobulin constant region is a human IgG1 constant region, optionally comprising one or more modifications such as L234A / L235A (LALA) mutations.

11. The antibody, or antigen binding portion thereof, according to any one of the preceding claims, wherein the antibody, or antigen binding portion thereof, is a chimeric antibody or a humanized antibody.

12. The antibody or antigen binding portion thereof according to any one of the preceding claims, comprising: (a) a heavy chain of the amino acid sequence of SEQ ID No: 12 and a light chain of the amino acid sequence of SEQ ID No: 13; or (b) The heavy chain of the amino acid sequence of SEQ ID No: 14 and the light chain of the amino acid sequence of SEQ ID No:

15.

13. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the VH and / or VL region of the antibody or antigen binding portion thereof according to any one of claims 1-12.

14. The isolated nucleic acid molecule according to claim 13, comprising the nucleic acid sequence shown in SEQ ID No: 16 and / or 17.

15. A vector comprising the nucleic acid molecule according to claim 13 or 14.

16. A host cell comprising the nucleic acid molecule according to claim 13 or claim 14 or the vector according to claim 15.

17. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

18. A method for producing an antibody or an antigen-binding portion thereof, the method comprising the steps of: - culturing the host cell according to claim 16 under conditions suitable for expression of the antibody or antigen-binding portion thereof; as well as - isolating the antibody or antigen-binding portion thereof from the host cell.

19. A method for modulating a B7H3-related immune response in a subject, the method comprising administering to the subject the antibody or antigen-binding portion thereof according to any one of claims 1 to 12 or the pharmaceutical composition according to claim 17.

20. A method for preventing or treating a B7H3-related disease in a subject, the method comprising administering to the subject an effective amount of the antibody or antigen-binding portion thereof according to any one of claims 1 to 12 or the pharmaceutical composition according to claim 15.

21. The method of claim 20, wherein the B7H3-related disease is selected from cancer, autoimmune disease and infectious disease.

22. The method of claim 21, wherein the cancer is selected from breast cancer, neurological tumors, melanoma, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, prostate cancer, ovarian cancer, cervical cancer, glioblastoma, esophageal cancer, bladder cancer, renal cell carcinoma, endometrial cancer, skin cancer, testicular cancer, thyroid cancer, urothelial carcinoma, lymphomas such as non-Hodgkin lymphoma and diffuse large B-cell lymphoma, leukemias such as chronic lymphocytic leukemia, and multiple myeloma.

23. The method of claim 21 or 22, wherein the antibody or antigen binding portion thereof is administered in combination with cellular immunotherapy, chemotherapy, radiation therapy, targeted therapy, and / or other agents used for cancer immunotherapy.

24. The antibody or antigen binding portion thereof according to any one of claims 1 to 12 for use in treating or preventing a B7H3-related disease in a subject.

25. Use of the antibody or antigen binding portion thereof according to any one of claims 1 to 12 for the preparation of a medicament for diagnosing, treating or preventing a B7H3-related disease in a subject.

26. A kit comprising the antibody, or antigen-binding portion thereof, of any one of claims 1 to 12.

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