Anti-B7H3 Antibody and Its Use

Abstract

Provided are an antibody that binds to B7H3 or an antigen-binding portion thereof, a method for producing such an antibody, and a method for treating a disorder associated with B7H3 using such an antibody or an antigen-binding portion thereof.

Description

Technical Field

[0001] Cross-reference This application claims the benefits of International Patent Application PCT / CN2022 / 093524 filed on May 18, 2022, and incorporates the same herein by reference in its entirety.

[0002] Sequence Listing This application is filed in electronic format together with the sequence listing. The entire content of this sequence listing is incorporated herein by reference.

[0003] The present disclosure generally relates to anti-B7H3 antibodies or antigen-binding portions thereof, methods for preparing the same, and uses thereof.

Background Art

[0004] B7H3, also known as CD276 and B7RP-2, is a B7 family member and shares 20% - 27% amino acid identity with other B7 family members. B7H3 transcripts are ubiquitously expressed, but their expression on normal tissues and immune cells is restricted and maintained at low levels, and highly expressed in multiple human malignancies including melanoma, breast cancer, prostate cancer, etc. B7-H3 has been reported to be expressed not only on the membrane, in the cytoplasm or in the nucleus of cancer cells, but also on tumor-associated vascular structures. B7H3 was not constitutively expressed on T cells, NK cells, and APCs including DCs and macrophages, but this expression can be induced by GM-CSF, IFNγ, etc. on APCs.

[0005] B7-H3 is a promising anti-cancer target. Although various immune functions including stimulation of T cell proliferation, inhibition, and inhibition of NK cell function have been demonstrated, the detailed function of B7-H3 remains unclear. It was assumed that various receptors capable of competing with the binding to B7H3 on tumors exist on immune cells. In addition to this immune checkpoint function, high levels of B7H3 expression have been reported to correlate with poor prognosis in cancer and enhance cell proliferation, migration, invasion, angiogenesis, metastatic ability, and anti-cancer drug resistance.

[0006] Similar to other B7 family molecules, B7H3 is a type I transmembrane glycoprotein with an extracellular domain containing an IgV-IgC domain pair. Different from the mouse B7H3 gene that has only a single copy of the IgV-IgC domain pair, human B7H3 has been found to have two isoforms. One isoform contains a single copy (named 2IgB7H3), and the other isoform has two IgV-IgC domain pairs (named 4IgB7H3) resulting from gene duplication and alternative splicing. 4IgB7H3, rather than 2IgB7H3, is the major isoform expressed not only on immune cells but also on malignant cells, indicating that 4IgB7H3 may play a characteristically important role in tumor formation and cancer immunity.

[0007] Because B7H3 is highly expressed on many tumor cells, therapeutic molecules targeting B7H3 have been developed to treat related indications. However, the development of novel anti-B7H3 antibodies still has room for improvement and clinical needs.

Summary of the Invention

Problems to be Solved by the Invention

[0008] These and other objects are provided by the present disclosure that broadly targets compounds, methods, compositions, and articles of manufacture that improve the effectiveness of antibodies. The advantages provided by the present disclosure are widely applicable in the fields of antibody-based therapy and diagnosis and can be used in combination with antibodies that react with diverse targets.

Means for Solving the Problems

[0009] In one aspect, the present disclosure provides an antibody or an antigen-binding portion thereof that specifically binds to a B7H3 antigen. The B7H3 antigen can be human B7H3, mouse B7H3, and / or cynomolgus monkey B7H3 or a fragment thereof (e.g., extracellular domain). Preferably, the B7H3 antigen is a human 4IgB7H3 protein or its extracellular domain.

[0010] In some embodiments, the antibody or antigen-binding portion thereof disclosed herein comprises a heavy chain CDR1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an 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, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0011] In some embodiments, the antibody or antigen-binding portion thereof disclosed herein comprises a heavy chain CDR1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and KSSQSLLX 1 a light chain CDR1 (LCDR1) comprising the amino acid sequence of SSNQKNYLA, wherein X 1 can be any amino acid other than N, and comprises LCDR1, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0012] Preferably, the above X 1 is Q, and LCDR1 comprises the amino acid sequence of SEQ ID NO: 4.

[0013] In some embodiments, the antibody or antigen-binding portion thereof disclosed herein comprises a heavy chain CDR1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and KSSQSLLNX 2 a light chain CDR1 (LCDR1) comprising the amino acid sequence of SNQKNYLA, wherein X can be any amino acid other than S, and comprises LCDR1, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0014] Preferably, the above X 2is P, and LCDR1 comprises the amino acid sequence of SEQ ID NO: 18.

[0015] In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the VH (A) the amino acid sequence set forth 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 region compared to SEQ ID NO: 8 or 10, and / or the VL (A) the amino acid sequence set forth 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.

[0016] In some embodiments, the antibody or antigen-binding portion thereof comprises a VH and / or a VL that comprises 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 derived from the human germline sequence is replaced by a different amino acid at the corresponding position in the parental antibody.

[0017] In some embodiments, the antibody or antigen-binding portion thereof comprises at least one amino acid modification (e.g., an amino acid substitution) in which a potential post-translational modification site or glycosylation site is removed. Such amino acid modifications can be present in the CDR region or the framework region.

[0018] In some embodiments, the antibody or antigen-binding portion thereof is a full-length antibody, ScFv, Fab, F(ab’)2 or Fv fragment.

[0019] In some embodiments, the antibody further comprises an immunoglobulin constant region, such as a human IgG constant region, and may comprise a human IgG1 constant region. In some embodiments, the IgG1 constant region comprises one or more modifications, such as the LALA mutation.

[0020] In some embodiments, the antibody or antigen-binding portion thereof is a chimeric antibody or a humanized antibody.

[0021] In some embodiments, the antibody or antigen-binding portion thereof (a) the amino acid sequence of the heavy chain of SEQ ID NO: 12 and the amino acid sequence of the light chain of SEQ ID NO: 13, or (b) the amino acid sequence of the heavy chain of SEQ ID NO: 14 and the amino acid sequence of the light chain of SEQ ID NO: 15.

[0022] In one aspect, the present disclosure provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the antibody or antigen-binding portion thereof as defined above.

[0023] In one aspect, the present disclosure provides a vector comprising the nucleic acid molecule as defined above.

[0024] In one aspect, the present disclosure provides a host cell comprising the nucleic acid molecule or vector as defined above.

[0025] In one aspect, the present disclosure provides a pharmaceutical composition comprising the antibody or antigen-binding portion thereof as defined above and a pharmaceutically acceptable carrier.

[0026] A method for producing the antibody or antigen-binding portion thereof as defined above, In one aspect, - culturing a host cell under conditions suitable for the expression of the antibody or antigen-binding portion thereof; and - isolating the antibody or antigen-binding portion thereof from the host cell. The present disclosure provides a method comprising

[0027] In one aspect, the present disclosure provides a method for modulating an immune response related to B7H3 in a subject, the method comprising administering to the subject an antibody or an antigen-binding portion thereof or a pharmaceutical composition as defined above.

[0028] In one aspect, the present disclosure provides a method for preventing or treating a disorder related to B7H3 in a subject, the method comprising administering to the subject an effective amount of an antibody or an antigen-binding portion thereof or a pharmaceutical composition as defined above.

[0029] In some embodiments, the disorder related to B7H3 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 cancer, lymphoma, e.g., non-Hodgkin lymphoma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and multiple myeloma.

[0030] In some embodiments, the antibody or an antigen-binding portion thereof is administered in combination with other substances for use in adoptive cell therapy, chemotherapeutic agents, radiotherapy, targeted therapy, and / or cancer immunotherapy.

[0031] In one aspect, the present disclosure provides an antibody or an antigen-binding portion thereof as defined above for use in the treatment or prevention of a disorder related to B7H3 in a subject.

[0032] In one aspect, the present disclosure provides the use of an antibody or an antigen-binding portion thereof as defined above in the manufacture of a medicament for treating or preventing a disorder related to B7H3 in a subject.

[0033] In one aspect, the present disclosure provides a kit comprising an antibody or an antigen-binding portion thereof as defined above.

[0034] The foregoing is a summary and, as such, includes simplifications, generalizations, and omissions as necessary. Consequently, those skilled in the art will understand that the summary is merely illustrative and is not intended to be limiting in any way. This summary 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**

[0035]

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Mode for Carrying Out the Invention

[0036] The present disclosure can be implemented in many different forms, and specific exemplary embodiments thereof that illustrate the principles of the present disclosure are disclosed herein. It should be emphasized that the present disclosure is not limited to the specific exemplary embodiments illustrated. Moreover, any section headings used herein are for mere organizational purposes and should not be construed as limiting the subject matter described.

[0037] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include singulars. More specifically, as used in this specification and the appended claims, the singular forms of "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a plurality of proteins, and reference to "a cell" includes a mixture of cells. In this application, when the term "or" is used, unless stated otherwise, it means "and / or". Moreover, the term "comprising" and other forms, such as "comprises" and "comprised", are used without limitation. Additionally, ranges presented in this specification and the appended claims include both endpoints and all points between the endpoints.

[0038] In the context of the present disclosure, the term "about" represents an interval of precision understood by those of ordinary skill in the art within which the technical effect of the characteristic in question is still guaranteed. This term typically indicates a deviation from the indicated numerical value by ±5%, for example, ±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 for example, ±0.01%. As understood by those of ordinary skill in the art, such specific deviations in the numerical values for a given technical effect depend on the nature of the technical effect. For example, natural or biological technical effects may generally have such larger deviations than those for artificial or engineering technical effects.

[0039] Generally, the cell culture and tissue culture, molecular biology, immunology, microbiology, genetics, and protein chemistry and nucleic acid chemistry, nomenclature used in connection with hybridization, and these techniques described in this specification are well-known and commonly used nomenclature and techniques in the art. The methods and techniques of the present disclosure are generally carried out according to conventional methods well-known in the art, as described in various general and more specific references cited and discussed throughout this specification, unless otherwise indicated. For example, see Abbas et al., Cellular and Molecular Immunology, 6th ed., W.B. Saunders Company (2010); Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (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, N.Y. (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The nomenclature used in connection with analytical chemistry, organic synthetic chemistry, and pharmaceutical chemistry and pharmaceutical manufacturing chemistry described in this specification, as well as these test methods and test techniques, are well-known and commonly used nomenclature and techniques in the art. Further, the contents of all references, patents, and patent application publications cited throughout this application are incorporated herein by reference in their entirety.

[0040] Definition For a better understanding of the present disclosure, the definitions and explanations of related terms are presented as follows.

[0041] As used herein, the terms "antibody" or "Ab" are used in a broad sense and encompass various antibody structures, including polyclonal antibodies, monospecific antibodies, and multispecific antibodies (e.g., bispecific antibodies). A naturally intact antibody is generally a Y-shaped tetrameric protein in which two heavy polypeptide chains (H) and two light polypeptide chains (L) are linked by disulfide bonds and non-covalent interactions. The light chains of an antibody can be classified into κ light chains and λ light chains. The heavy chains can be classified into μ, δ, γ, α, and ε, whereby the isotypes of the antibody are defined as IgM, IgD, IgG, IgA, and IgE, respectively. In light and heavy chains, the variable region is linked to the constant region via a "J" region of about 12 or more amino acids, and the heavy chain further includes a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H ) and a 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 a light chain constant region (C L ). The V H region and the V L region can be further separated into highly variable regions (called complementarity-determining regions (CDRs)), between which relatively conserved regions (called framework regions (FRs)) are arranged. V H and V L each consist of three CDRs and four FRs in the following order from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (V H and V LEach forms an antigen-binding site. The ranges of the framework regions and CDRs can be identified in detail using methods known in the art, for example, by the Kabat definition, the definition of the website by Dr. Martin, the Chothia definition, the AbM definition, the EU definition, and the contact definition, all of which are well-known in the art. See, for example, Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Martin A. "Antibody bioinformatics website of Dr. Andrew Martin's lab at UCL," last updated on 31 July 2018; Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; Edelman et al., Proc Natl Acad Sci U S A. 1969 May;63(1):78-85; and Almagro, J. Mol. Recognit. 17:132-143 (2004). Also see hgmp.mrc.ac.uk and bioinf.org.uk / abs.The correspondence or alignment between numberings according to various 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) 25:206-11; Lefranc MP et al., Unique database numbering system for immunogenetic analysis. Immunol Today (1997) 18:509; 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) 27:55-77). The antibody can be an antibody of various antibody isotypes, for example, an IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody.

[0042] The terms "antigen-binding portion" or "antigen-binding fragment" of an antibody are used interchangeably in the context of this application and refer to a polypeptide that includes a fragment of a full-length antibody, retains the ability to specifically bind to an antigen to which the full-length antibody specifically binds, and / or competes with the full-length antibody for binding to the same antigen. Generally, reference is made to Fundamental Immunology, Ch. 7 (Paul, W., ed., the second edition, Raven Press, N.Y. (1989)), which is hereby incorporated by reference in its entirety for all purposes. An antigen-binding fragment of an antibody can be obtained from a full antibody molecule using, for example, any suitable standard technique, such as proteolytic techniques, or recombinant genetic modification techniques that include manipulation and expression of DNA encoding antibody variable domains and any constant domains. Such DNA is known and / or can be readily obtained, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated by chemical or molecular biological techniques to, for example, arrange one or more variable domains and / or constant domains in a suitable conformation, or to introduce codons, generate cysteine residues, modify, add or delete amino acids, etc.

[0043] 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 regions of an antibody (e.g., isolated complementarity-determining regions (CDRs), e.g., CDR3 peptides) or FR3-CDR3-FR4 restricted peptides. Also included within the expression “antigen-binding fragment” as used herein are other modified molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetra-bodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark IgNAR variable domains. In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently attached to at least one constant domain. The variable and constant domains may be directly linked to each other or may be linked either by a full or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, thereby creating a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule.

[0044] As used herein, the term “variable region” or “variable domain” with respect to an antibody refers to an antibody variable region or fragment thereof that includes one or more CDRs. A variable domain may include an intact variable region (e.g., HCVR or LCVR), but may also include less than an intact variable region, yet still retain the ability to bind to an antigen or form an antigen-binding site.

[0045] The term "constant region", as used herein, refers to the immunoglobulin constant region, which includes the CH1 domain, CH2 domain, CH3 domain (and any hinge region) in the heavy chain and the constant domain in the light chain. The heavy chain constant region and light chain constant region of an antibody are well known in the art and are provided, for example, in the IMGT database (www.imgt.org) or www.vbase2.org / vbstat.php, both of which are incorporated herein by reference. "Fc" with respect to an antibody refers to the portion of the antibody that includes the second constant region (CH2) and the third constant region (CH3) of the first heavy chain and is linked via disulfide bonds to the second constant region and the third constant region of the second heavy chain. Also, the Fc region may include 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 function in antigen binding. The ability of an antibody to initiate and regulate effector functions via the Fc domain is a key component of its in vivo defensive activity. Antibody neutralizing activity has previously been thought to be simply a result of Fab-antigen interactions, but it has now been revealed that these in vivo activities are highly dependent on the interaction between the IgG Fc domain and its cognate receptor, the Fcγ receptor (FcγR), which is expressed on the surface of effector leukocytes.

[0046] The term "B7H3", also known as CD276 antigen, refers to a type I transmembrane protein that is a member of the B7 family and has an extracellular domain composed of a single IgV-IgC domain pair. B7 family proteins contain an extracellular IgV-like domain and an IgC-like domain along with a short cytoplasmic tail. B7H3 is an immune checkpoint molecule and is abnormally highly expressed in many types of cancer. When referring to the amino acid sequence of the B7H3 protein, it includes the full-length B7H3 protein (e.g., human 4IgB7H3 protein or human 2IgB7H3 protein) or the extracellular domain of B7H3 (B7H3 ECD) or a fragment containing B7H3 ECD; a fusion protein of B7H3 ECD. Example sequences of the B7H3 protein can be found in UniprotID: Q5ZPR3 (human 4IgB7H3), Genebank accession numbers NP_001019907 (human), NP_001316557 (human), NP_001316558 (human), NP_079516 (human), and NP_598744 (mouse). Cynomolgus B7H3 shares approximately 97% and 88% amino acid sequence identity with human and mouse B7H3, respectively.

[0047] The term "antibody that binds to B7H3" or "anti-B7H3 antibody", as used herein, includes an antibody and antigen-binding fragment thereof that specifically recognizes the B7H3 protein, and an antibody and antigen-binding fragment thereof that specifically binds to the B7H3 protein. As used herein, the expression "anti-B7H3 antibody" includes both a monospecific monovalent antibody and a bispecific antibody that includes a first antigen-binding site that binds to B7H3 and a second antigen-binding site that binds to a second (target) antigen.

[0048] The term "monoclonal antibody" or "mAb", as used herein, refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibodies exhibit a single binding specificity and binding affinity for a particular epitope.

[0049] As used herein, the term "chimeric antibody" refers to an antibody that contains or consists of the original antigen-binding variable domain of an antibody (e.g., murine) together with the constant domain from a different species (e.g., human). Preferably, as used herein, the term "chimeric antibody" refers to an antibody consisting of the original antigen-binding variable domain of an antibody (e.g., murine) and the constant domain from a different species (e.g., human).

[0050] As used herein, the term "humanized antibody" is intended to refer to an antibody in which the CDR sequences derived from the germline of another mammalian species, e.g., rat / mouse, are grafted onto a human framework sequence. Further modifications within the framework region can be made within the human framework sequence. For example, certain residues of the framework region can be reverted to the original sequence to maintain affinity.

[0051] As used herein, the term "operably linked" refers to two or more biological sequences of interest being located in proximity to each other, with or without a spacer or linker, in a relationship such that they can function as intended. When used with respect to a polypeptide, it is intended to mean that the polypeptide sequences are linked such that the resulting product can have the intended biological function. For example, an antibody variable region can be operably linked to a constant region to yield a stable product having antigen-binding activity. This term can also be used with respect to polynucleotides. In some cases, 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 such that regulation of polypeptide expression from the polynucleotide is possible.

[0052] "K D ", as used herein, is intended to refer to the dissociation constant of a particular antibody-antigen interaction, which is the ratio of k d to k a (i.e., k d / k a) obtained and expressed as molar concentration (M). "k a " When used in this specification, the term is intended to refer to the association rate of a particular antibody-antigen interaction, while the term "k d " When used in this specification, the term is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The K D value of the antibody can be determined using methods well established in the art. A preferred method for determining the K D of an antibody is by using surface plasmon resonance, preferably by using a biosensor system, such as a Biacore® system.

[0053] The term "high affinity" with respect to an IgG antibody, when used in this specification, refers to the strength of the binding interaction between an antigen and an antibody. In measuring affinity, various methods have been established in the art, such as surface plasmon resonance (SPR), FACS affinity assay, FACS binding assay, and ELISA binding. In some embodiments, the antibodies disclosed herein have a K -9 of 1×10 -10 M or less, more preferably 5×10 -10 M or less, even more preferably 1×10 -11 M or less, even more preferably 9×10 -11 M or less, and even more preferably 8×10 D when determined by SPR.

[0054] The term "EC 50 " When used in this specification, also referred to as the "half maximal effective concentration", refers to the concentration of a drug, antibody or poison that induces a response halfway between the baseline value and the maximum value after a particular exposure time. In the context of this application, EC 50 is expressed in units of "nM".

[0055] As used herein, the term "isolated" refers to a state obtained by artificial means from a natural state. When a particular "isolated" substance or component occurs in nature, this isolation can be accomplished by changing the natural environment, or by separating the substance from the natural environment, or both. For example, a particular polynucleotide or polypeptide that occurs naturally within a particular living organism, and the same polynucleotide or polypeptide of high purity isolated from such a natural state, is referred to as an isolated polynucleotide or polypeptide. The term "isolated" does not exclude mixtures of artificial or synthetic substances, nor other impurities that do not affect the activity of the isolated substance.

[0056] As used herein, the term "isolated antibody" is intended to refer to an antibody that substantially excludes other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to the B7H3 protein substantially excludes antibodies that specifically bind to antigens other than the B7H3 protein). However, an isolated antibody that specifically binds to the human B7H3 protein may have cross-reactivity to other antigens, e.g., B7H3 proteins from other species. Moreover, an isolated antibody may substantially exclude other cellular materials and / or chemical substances.

[0057] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector enables the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can carry genetic material elements that are expressed in a host cell by transformation, transduction, or transfection of the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages such as λ 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, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40). A vector may contain multiple 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.

[0058] As used herein, the term "host cell" refers to a cell line that can be modified to produce a protein, protein fragment, or peptide of interest. Host cells include, but are not limited to, cultured cells such as mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; or human tissues or hybridoma cells, yeast cells, and insect cells, as well as cells contained in transgenic animals or cultured tissues. The term includes not only the particular subject cells but also the progeny of such cells. Because such progeny may have certain modifications in subsequent generations due to either mutation or environmental influence, they may not be identical to the parent cells, but are still within the scope of the term "host cell".

[0059] 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 alignment and comparison of the sequences. "Percent identity" means the percentage of identical residues between amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest molecule to be compared. In such calculations, gaps in the alignment (if any) are preferably designated by a particular mathematical model or computer program (i.e., an "algorithm"). Methods available for calculating the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A. M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., 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. 48:1073.

[0060] As used herein, the term "immunogenicity" refers to the ability to stimulate the formation of specific antibodies or sensitized lymphocytes in a living body. This not only refers to the property of an antigen that ultimately generates immune effector substances, such as antibodies and sensitized lymphocytes, by stimulating and activating, proliferating, and differentiating specific immune cells, but also refers to a specific immune response in which antibodies or sensitized T lymphocytes can be formed in the immune system of a living body after stimulation by the antigen. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce the generation of an immune response in a host depends on three factors: the properties of the antigen, the reactivity of the host, and the means of immunity.

[0061] As used herein, the term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Transfection protocols and techniques include, but are not limited to, lipid transfection and chemical and physical methods, such as electroporation. A number of transfection techniques are well known in the art and are also disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; 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 13:197. In certain embodiments of the present disclosure, the human B7H3 gene can be transfected into 293F or CHO cells.

[0062] The term "SPR" or "surface plasmon resonance", as used herein, refers to and includes, for example, an optical phenomenon that enables real-time analysis of biomolecular specific interactions by detecting changes in protein concentration in a biosensor matrix using, for example, a BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden, and Piscataway, N.J.). For further explanation, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0063] The term "fluorescence-activated cell sorting" or "FACS", as used herein, refers to a specialized type of flow cytometry that provides a method 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 Fluorescence Activated Cell Sorting". Retrieved 2017-11-09). Devices for performing FACS are known to those of skill in the art and are generally commercially available. Examples of such devices include the FACS Star Plus, FACScan, and FACSort devices from Becton Dickinson (Foster City, Calif.), the Epics C from Coulter Epics Division (Hialeah, Fla.), and the MoFlo from Cytomation (Colorado Springs, Colo.).

[0064] The term "subject" includes any human or non-human animal. In some embodiments, the subject is preferably human.

[0065] As used herein, the term "disorder associated with B7H3" refers to any symptom or disease caused by, exacerbated by, or otherwise associated with an increase or decrease (generally an increase) in the expression or activity of B7H3 (e.g., human B7H3).

[0066] As used herein, the term "cancer" refers to solid tumors and non-solid tumors, such as leukemia, mediated by the growth, proliferation, or metastasis of any tumor or malignant cell.

[0067] As used herein, the terms "treatment", "treating", or "treated", when used in the context of treating a symptom, generally relate to treatments and therapies, whether for a human or an animal, in which some desired therapeutic effect, such as inhibition of the progression of the symptom, is achieved, and include reduction of the rate of progression, arrest of the rate of progression, regression of the symptom, recovery of the symptom, and cure of the symptom. Treatment as a prophylactic measure (i.e., prophylaxis, prevention) is also included. In cancer, "treating" can refer to attenuation or slowing of the growth, proliferation, or metastasis of a tumor or malignant cells, or some combination thereof. In a tumor, "treatment" includes removal of all or part of the tumor, inhibition or slowing of tumor growth and metastasis, prevention or delay of tumorigenesis, or some combination thereof.

[0068] As used herein, the term "effective amount" relates to the amount of an active compound, or a material, composition, or dosage form containing an active compound, which, when administered according to a desired treatment regimen, is effective in producing some desired therapeutic effect and is commensurate with a reasonable benefit-risk ratio. By way of example, an "effective amount", when used in connection with the treatment of a disease or symptom associated with B7H3, refers to an amount or concentration of an antibody or antigen-binding portion thereof that is effective to treat or prevent this disease or symptom.

[0069] As used herein, the terms "prevent", "prevention" or "preventing", when used with respect to a particular medical condition in a mammal, refer to preventing or delaying the onset of a disease or preventing the manifestation of its clinical or subclinical symptoms.

[0070] As used herein, the term "pharmaceutically acceptable" means that a medium, diluent, excipient, and / or salts thereof are chemically and / or physically compatible with the other ingredients in the formulation and physiologically compatible with the recipient.

[0071] 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 the subject and the active substance. This is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, a phosphate buffer may be mentioned as a pH adjuster, but is not limited thereto; a cationic, anionic or nonionic surfactant, such as Tween-80, may be mentioned as a surfactant, but is not limited thereto; and sodium chloride may be mentioned as an ionic strength enhancer, but is not limited thereto.

[0072] As used herein, the term "adjuvant" refers to a non-specific immunostimulant that, when delivered to a living body together with or prior to an antigen, can enhance the immune response to the antigen or change the type of immune response in the living body. There are various 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 parvum, lipopolysaccharide, cytokines, etc. Freund's adjuvant is currently the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

[0073] Antibodies that specifically bind to B7H3 In the present disclosure, antibodies that specifically bind to B7H3, such as human B7H3, mouse B7H3, cynomolgus monkey B7H3, and their ECD domains, are provided. The term "antibody" as used herein includes both full-length immunoglobulins and antibody portions that bind to the same antigen. Antibodies can be, for example, monoclonal, polyclonal, chimeric, humanized, or single-chain antibodies. In some embodiments, the antibody portion is a Fab fragment or an F(ab’)2 fragment. In some embodiments, the antibody portion retains the ability to specifically bind to B7H3.

[0074] Recombinant anti-B7H3 antibodies, such as chimeric antibodies and humanized monoclonal antibodies that include both human and non-human portions, can be made using standard recombinant DNA techniques and are within the scope of the present disclosure. Such chimeric antibodies and humanized monoclonal antibodies can be generated by recombinant DNA techniques, such as those described in U.S. Patent No. 7,112,421; Better et al. (1988) Science 240:1041-1043; or Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443. For example, further modified / optimized humanized monoclonal antibodies are also included in the present disclosure by affinity maturation, back mutations, and removal of post-translational modification sites.

[0075] Preferably, the antibodies or antigen-binding portions thereof disclosed herein can bind to human 4IgB7H3 with high affinity. The antibodies or antigen-binding portions thereof disclosed herein can bind to human 2IgB7H3 with a much lower affinity. The antibodies or antigen-binding portions thereof disclosed herein bind to human 4IgB7H3 with high affinity but can bind to human 2IgB7H3 with a much lower affinity.

[0076] The binding of the antibodies of the present disclosure to B7H3 can be evaluated using one or more techniques well established in the art, such as ELISA or flow cytometry as examples. In some embodiments, the antibodies can be assayed by a flow cytometry assay that reacts the antibody with a cell line expressing human B7H3, such as MCF-7 cancer cells or CHOK1 cells transfected to express B7H3 on the cell surface. Additionally or alternatively, the binding of the antibodies, including binding kinetics (e.g., K D values), can be assayed by a BIAcore binding assay. In some other embodiments, the antibodies are assayed by ELISA that reacts the antibody with soluble B7H3 protein.

[0077] In the present disclosure, when measured by surface plasmon resonance (SPR), 1×10 -8K of less than M D , 1×10 -9 K of less than M D , 5×10 -10 K of less than M D , 1×10 -10 K of less than M D , 9×10 -11 K of less than M D , 8×10 -11 K of less than M D or 7×10 -11 K of less than M D to provide an antibody that binds. Also, the antibodies disclosed herein, when measured by surface plasmon resonance (SPR), bind to human 2IgB7H3 protein with a K -9 of 5×10 D M or more -8 K of 1×10 D M or more -8 K of 2×10 D M or more -8 K of 3×10 D M or more -8 or 4×10 D K of M or more. In some embodiments, the antibodies herein bind to human 4IgB7H3 with a K D value that is less than 1 / 500, less than 1 / 100, or less than 1 / 50 of the binding value to human 2IgB7H3. Such K D values for comparison can be measured by surface plasmon resonance (SPR).

[0078] In some embodiments, the antibodies of the present disclosure can bind to human or cynomolgus monkey B7H3-expressing cell lines with an EC 50 of less than 5 nM, less than 4 nM, less than 3 nM, or less than 2 nM as determined by FACS.

[0079] Antibody variable domains and CDRs In some embodiments, the antibody or antigen-binding portion thereof is a chimeric antibody or a murine antibody that specifically binds to B7H3, preferably human 4IgB7H3. In some further embodiments, the antibody or antigen-binding portion thereof is a humanized antibody that specifically binds to B7H3, preferably human 4IgB7H3. In some still further embodiments, the humanized antibody or antigen-binding portion thereof comprises one or more revertant mutations in the framework region. In some still further embodiments, the humanized antibody or antigen-binding portion thereof comprises one or more modifications at potential post-translational modification (PTM) sites, such as removing NG, NS, and DG in the CDR and NXS and NXT (where X can be any amino acid other than P) in the full length.

[0080] In some embodiments, the antibody or antigen-binding portion thereof disclosed herein (i) HCDR1 comprising SEQ ID NO: 1, or HCDR1 having an amino acid sequence derived from SEQ ID NO: 1 and differing by no more than 2 amino acids (e.g., 2 or 1 amino acid) in amino acid addition, deletion, or substitution, (ii) HCDR2 comprising SEQ ID NO: 2, or HCDR2 having an amino acid sequence derived from SEQ ID NO: 2 and differing by no more than 2 amino acids (e.g., 2 or 1 amino acid) in amino acid addition, deletion, or substitution, and (iii) HCDR3 comprising SEQ ID NO: 3, or HCDR3 having an amino acid sequence derived from SEQ ID NO: 3 and differing by no more than 2 amino acids (e.g., 2 or 1 amino acid) in amino acid addition, deletion, or substitution is selected from at least one of the group consisting of one or more heavy chain CDRs (HCDRs), and / or (i) LCDR1 comprising SEQ ID NO: 4, 7, or 18, or LCDR1 having an amino acid sequence derived from SEQ ID NO: 4, 7, or 18 and differing by no more than 2 amino acids (e.g., 2 or 1 amino acid) in amino acid addition, deletion, or substitution, (ii) LCDR2 containing SEQ ID NO: 5, or an LCDR2 that differs by 2 or fewer amino acids (e.g., 2 amino acids or 1 amino acid) in terms of amino acid addition, deletion, or substitution in the amino acid sequence derived from SEQ ID NO: 5, and (iii) LCDR3 containing SEQ ID NO: 6, or an LCDR3 that differs by 2 or fewer amino acids (e.g., 2 amino acids or 1 amino acid) in terms of amino acid addition, deletion, or substitution in the amino acid sequence derived from SEQ ID NO: 6 comprises one or more light chain CDRs (LCDRs) selected from at least one of the group consisting of

[0081] In some embodiments, the amino acid substitutions in the CDRs are conservative substitutions. In some embodiments, the above antibody or its antigen-binding portion may comprise or consist of 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 further comprise one or more modifications in the CDRs so as to remove potential PTM sites. In some embodiments, the antibody comprises one or more modifications in LCDR1 as compared to LCDR1 shown in SEQ ID NO: 7 ("KSSQSLLNSSNQKNYLA") so as to remove the "NS" which is a potential PTM site within the sequence. In some preferred embodiments, the antibody comprises one substitution at the 8th or 9th position of the amino acid sequence of LCDR1 as compared to LCDR1 shown in SEQ ID NO: 7 ("KSSQSLLNSSNQKNYLA"). In some more preferred embodiments, the antibody comprises a substitution from N to Q at the 8th position of the amino acid sequence of LCDR1 (i.e., SEQ ID NO: 4, "KSSQSLLQSSNQKNYLA"). In some other embodiments, the antibody comprises a substitution from S to P at the 9th position of the amino acid sequence of LCDR1 (i.e., SEQ ID NO: 18, "KSSQSLLNPSNQKNYLA"). Those skilled in the art will understand that other types of substitutions can be selected as long as the binding affinity for B7H3 is substantially maintained.

[0082] In some embodiments, the antibodies or antigen-binding portions thereof disclosed herein comprise, consist of, or consist essentially of an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, an HCDR3 comprising SEQ ID NO: 3, an LCDR1 comprising SEQ ID NO: 4, an LCDR2 comprising SEQ ID NO: 5, and an LCDR3 comprising SEQ ID NO: 6.

[0083] In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), and these heavy chain variable region and light chain variable region each comprise the above HCDR1-3 and LCDR1-3.

[0084] In some embodiments, the heavy chain variable region of the antibody or antigen-binding portion thereof is (i) the 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 having an addition, deletion, and / or substitution of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acids compared to SEQ ID NO: 8 or 10.

[0085] In some embodiments, the amino acid substitution may be a conservative substitution.

[0086] In some embodiments, the light chain variable region of the antibody or antigen-binding portion thereof is (i) the 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 having an addition, deletion, and / or substitution of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acids compared to SEQ ID NO: 9 or 11.

[0087] In some embodiments, the amino acid substitution may be a conservative substitution.

[0088] In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), and this VH (i) the 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 region as compared to SEQ ID NO: 8 or 10, and / or this VL (i) the 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 region as compared to SEQ ID NO: 9 or 11.

[0089] In some embodiments, the amino acid substitution can be a conservative substitution.

[0090] 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.

[0091] As will be understood by those skilled in the art, the exact numbering and placement of the CDRs can vary between different numbering schemes. However, it should be understood that the disclosure of a heavy or light variable sequence includes the disclosure of the relevant (native) CDRs. Thus, the disclosure of each heavy variable region is the disclosure of the heavy chain CDRs (e.g., HCDRl, HCDR2, and HCDR3), and the disclosure of each light variable region is the disclosure of the light chain CDRs (e.g., LCDRl, LCDR2, and LCDR3). A useful comparison of CDR numbering is as follows; see Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003).

Table 1

[0092] The assignment of amino acids to each CDR may follow one or a combination of the numbering rules presented above, all of which are well-known in the art. See Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5th Ed.), US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242; Chothia et al., 1987, PMID: 3681981; Martin A. "Antibody bioinformatics website of Dr. Andrew Martin's lab at UCL," last updated on 31 July 2018; Chothia et al., 1989, PMID: 2687698; MacCallum et al., 1996, PMID: 8876650; Dubel, Ed. (2007) Handbook of Therapeutic Antibodies, 3rd Ed., Wily-VCH Verlag GmbH and Co; and http: / / www.imgt.org / .

[0093] The variable regions and CDRs of antibody sequences can be identified according to general rules developed in the art (such as the Kabat numbering system, the Martin numbering system, and the IMGT numbering system presented above), or by aligning the sequences against a database of known variable regions. Methods for identifying such regions are described in Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Examples of databases of antibody sequences are described in Retter et al., Nucl. Acids Res., 33 (Database issue): D671 - D674 (2005), the "Abysis" website at www.bioinf.org.uk / abs (maintained by A.C. Martin of the Department of Biochemistry & Molecular Biology, University College London, London, UK) and the VBASE2 website at www.vbase2.org, and can be accessed through these. For example, sequences may be analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT, and the Protein Data Bank (PDB) with the structural data of the PDB.See Dr. Andrew C. R. Martin's book chapter Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13: 978-3540413547, also available on the website bioinforg.uk / abs). The website of the Abysis database further includes rules developed to identify CDRs, which can be used in accordance with the teachings of this specification. Two antibodies having the same VH CDR and / or VL CDR mean that these CDRs are identical when determined by the same method (e.g., the Kabat method, Dr Martin's method, the Chothia method or the IMGT numbering known in the art).

[0094] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) incorporated in the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percentage of identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) incorporated in the GAP program of the GCG software package (available at http: / / www.gcg.com), using either a Blossum62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0095] In addition or alternatively, the protein sequences of the present disclosure can be further used as "query sequences" for performing searches against public databases, for example, to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to the antibody molecules of the present disclosure. To obtain a gapped alignment for comparison purposes, Gapped BLAST as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402 can be utilized. When using the BLAST program and the Gapped BLAST program, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0096] In other embodiments, the CDR amino acid sequences can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to each of the above sequences. In other embodiments, the amino acid sequences of the variable regions can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to each of the above sequences.

[0097] As described above, at least one addition, deletion, and / or substitution of an amino acid in the VH or VL region can be within the framework (FRW) sequence rather than within any of the CDR sequences. For example, an isolated antibody or an antigen-binding portion thereof can include one or more substitutions of amino acids within the framework sequence, e.g., FRW1, FRW2, FRW3, and / or FRW4 of the VH or VL region. In some embodiments, the framework region of the VH region includes one or more of the following substitutions: S025T, V071R, Y091F, V089T.

[0098] In some embodiments, the set of six CDRs can 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 heavy and light variable regions, as long as the framework (excluding the CDRs) retains at least about 80%, 85%, or 90% identity to the parental antibody (e.g., W301088-1.145.16). Thus, for example, the same CDRs described herein can be combined with various framework sequences derived from the human germline sequence as long as the framework region retains at least 80%, 85%, or 90% identity to the human germline sequence.

[0099] In certain embodiments, the isolated antibody or an antigen-binding portion thereof provided herein includes any suitable framework region (FR) sequence as long as the antigen-binding domain can specifically bind to B7H3, preferably human Ig4B7H3.

[0100] The framework region of an isolated antibody or an antigen-binding portion thereof may contain one or more back mutations, and the framework amino acids derived from the human germline are replaced by the original mouse amino acids at the corresponding positions. Also, the framework region of an isolated antibody or an antigen-binding portion thereof may contain one or more conservative substitutions. The term "conservative substitution" as used herein refers to an amino acid substitution that does not adversely affect and does not cause a change in the essential properties of a protein / polypeptide containing 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 substitutions of amino acids involve substituting an amino acid residue with another amino acid residue having a similar side chain, e.g., a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having chemical properties such as similar size, shape, charge, ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains are defined in the art. Such families include amino acids having basic side chains (e.g., lysine, arginine, and histidine), amino acids having acidic side chains (e.g., aspartic acid and glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids having β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, the corresponding amino acid residue is preferably substituted with another amino acid residue of the same side chain family.Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997), which are incorporated herein by reference).

[0101] IgG constant region In some embodiments, the antibody further comprises an immunoglobulin constant region, e.g., a human IgG, IgA, IgM, IgE or IgD constant region. The antibodies disclosed herein can be of various IgG isotypes, e.g., IgG1, IgG2, IgG3, and IgG4 antibodies. Preferably, the antibody is of the IgG1 isotype and comprises the human IgG1 constant region. The IgG constant regions described herein include the Fc region and may include some or all of the hinge region of the IgG molecule.

[0102] The Fc region may contain one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to the wild-type Fc region without changing the desired functionality. For example, the present disclosure includes antibodies that include one or more modifications in the Fc region such that the binding interaction between Fc and FcRn may be modified (e.g., enhanced or attenuated). Also, non-limiting examples of Fc modifications include, for example, the amino acid modification of Leu234Ala / Leu235Ala (or LALA) in the IgG1 Fc region that changes antibody-dependent cellular cytotoxicity (ADCC) or other effector functions, and the proline (P) mutation of serine (S) at position 228 in the amino acid sequence of the human IgG4 Fc region. The S228P mutation belongs to IgG4 stabilizing mutations that help prevent half-antibody formation by stabilizing the disulfide in the core hinge of the IgG4 molecule and reducing Fab arm exchange. It is generally known that an increase in binding to FcγRIIIa generally results in an increase in ADCC. Similarly, a decrease in binding to FcγRIIb (inhibitory receptor) may also be beneficial in some situations. Amino acid substitutions utilized in the subject antibodies include those listed in U.S. Patent Nos. 8,188,321 (particularly FIG. 41) and 8,084,582, and U.S. Patent Application Publication Nos. 20060235208 and 20070148170, all of which are hereby expressly incorporated by reference in their entirety. Specific variants utilized 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 both Fc domains of the antibodies disclosed herein.

[0103] The variable regions are operably linked to the IgG constant regions in the heavy and light chains, respectively. In some embodiments, the variable regions are directly linked to the IgG constant regions. In some other embodiments, the variable regions are linked to the IgG constant regions via a linker that may comprise only one amino acid. Peptide linkers for constructing polypeptide sequences are well established in the art. For example, linkers of (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n are included, where n is an integer of at least 1 (generally 3 to 4 or 5). Alternatively, linkers derived from immunoglobulin heavy or light chains (e.g., sequences derived from the hinge region), and any peptide sequence having sufficient length and flexibility to allow recombinant binding of the variable region to the constant region and to allow each region to perform its biological function are included.

[0104] Unless otherwise specified, the numbering of the constant regions of the antibodies herein is based on the EU numbering, similar to Kabat. Thus, in the context of IgG, the "CH" domains are as follows: "CH1" refers to positions 118 - 215 according to the EU index, similar to Kabat; "hinge" refers to positions 216 - 230 according to the EU index, similar to Kabat; "CH2" refers to positions 231 - 340 according to the EU index, similar to Kabat; "CH3" refers to positions 341 - 447 according to the EU index, similar to Kabat.

[0105] Functionality of anti - B7H3 antibodies The antibodies or antigen - binding portions thereof disclosed herein are characterized by certain functional features or properties. In some embodiments, the antibodies (including both chimeric and humanized antibodies) have one or more of the following properties. (a) Specifically binds to human 4IgB7H3 - expressing cells with high affinity (e.g., less than 2 nM as measured by FACS). (b) Binds to human 2IgB7H3 with a significantly lower affinity compared to the affinity for human 4IgB7H3, and the difference between the antibody that binds to 2IgB7H3 and the antibody that binds to 4IgB7H3 is significant. (c) It binds specifically and with higher affinity (e.g., less than 5 nM as measured by FACS) to cynomolgus monkey B7H3 than the reference antibody. And (d) It shows good internalization ability against B7H3-expressing cancer cells.

[0106] A nucleic acid molecule encoding the antibody of the present disclosure In some embodiments, the present disclosure is directed to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the heavy chain variable region and / or the light chain variable region of the isolated antibody disclosed herein. In some embodiments, the isolated nucleic acid molecule disclosed herein comprises the nucleic acid sequence set forth in SEQ ID NO: 16. In some embodiments, the isolated nucleic acid molecule disclosed herein comprises the nucleic acid sequence set forth in SEQ ID NO: 17.

[0107] The 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 further described below), the cDNAs encoding the light and heavy chains of the antibodies produced by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), the nucleic acids encoding such antibodies can be recovered from the gene libraries.

[0108] An isolated nucleic acid encoding the VH region can be converted into a full-length heavy chain gene by operably linking the nucleic acid encoding VH to another DNA molecule encoding the heavy chain constant regions (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 encompassing such 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.

[0109] An isolated nucleic acid encoding a VL region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding the VL to another DNA molecule encoding a light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see, e.g., Kabat et al. supra), and DNA fragments encompassing such 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.

[0110] Once DNA fragments encoding VH and VL segments are obtained, such DNA fragments can be further manipulated by standard recombinant DNA techniques to convert, for example, variable region genes into full-length antibody chain genes, Fab fragment genes or scFv genes. In such manipulations, 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. The term "operably linked" as used in this context is intended to mean that the two DNA fragments are linked such that the amino acid sequences encoded by the two DNA fragments are maintained in-frame.

[0111] In some embodiments, the present disclosure is directed to vectors comprising the nucleic acid sequences disclosed herein. The vector may be any vector suitable in the context of the present disclosure and includes chromosomal vectors, episomal vectors, and synthetic nucleic acid vectors (nucleic acid sequences comprising a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculoviruses, yeast plasmids, vectors obtained 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, which may be, for example, a linear expression element (as described, for example, in Sykes and Johnston, Nat Biotech 17, 355-59 (1997)), a small nucleic acid vector (as described, for example, in U.S. Patent No. 6,077,835 and / or International Publication No. 00 / 70087), a plasmid vector, such as pBR322, pUC19 / 18 or pUC118 / 119, the minimal size nucleic acid vector of "midge" (as described, for example, in Schakowski et al., Mol Ther 3, 793-800 (2001)) or a precipitated nucleic acid vector construct, such as a CaP04 precipitate construct (as described, for example, in International Publication No. 200046147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986), Wigler et al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981)). Such nucleic acid vectors and their use are well known in the art (see, for example, U.S. Patent No. 5,589,466 and U.S. Patent No. 5,973,972).

[0112] In one embodiment, the vector is suitable for antibody expression in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503 - 5509 (1989)), pET vectors (Novagen, Madison WI, etc.). Further or alternatively, the vector can be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system can be utilized. Suitable vectors include, for example, vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH (a review is described in F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987) and Grant et al., Methods in Enzymol 153, 516 - 544 (1987)).

[0113] Further or alternatively, the vector can be a vector suitable for expression in mammalian cells, for example, a vector containing glutamine synthetase as a selectable marker, such as the vector described in Bebbington (1992) Biotechnology (NY) 10: 169 - 175.

[0114] Also, the nucleic acid and / or vector can include a nucleic acid sequence encoding a secretion / targeting sequence into the periplasmic space or cell culture medium that can target a polypeptide, for example, a nascent polypeptide chain. Such sequences are known in the art and include a secretion leader or signal peptide.

[0115] The vector can contain or be associated with any suitable promoter, enhancer, and other expression-promoting elements. Examples of such elements include strong expression promoters (e.g., the human CMV IE promoter / enhancer, as well as the RSV promoter, SV40 promoter, SL3-3 promoter, MMTV promoter, and HIV LTR promoter), effective poly(A) termination sequences, origins of replication of plasmid products in Escherichia coli, antibiotic resistance genes as selectable markers, and / or convenient cloning sites (e.g., polylinkers). Additionally, the nucleic acid can include an inducible promoter, such as CMV IE, relative to a constitutive promoter.

[0116] In some embodiments, the disclosure relates to a host cell comprising the vector specified above herein.

[0117] Accordingly, the disclosure also relates to recombinant eukaryotic or prokaryotic host cells that produce the antibodies of the disclosure, such as transfectomas. The antibodies can be expressed in the recombinant eukaryotic or prokaryotic host cells to produce the antibodies of the disclosure.

[0118] Examples of host cells include yeast, bacteria, plant cells, and mammalian cells. Mammalian host cells for expressing the antibodies of the present disclosure include Chinese Hamster Ovary (CHO) cells (described in Urlaub and Chasin, (1980) Proc. Natl. Acad. ScL USA 77:4216-4220 and used, for example, with a DHFR selectable marker as described in R. J. Kaufman and P. A. Sharp (1982) J. MoI. Biol. 159:601-621, including dhfr CHO cells), COS cells, and SP2 cells. In particular, for use with NSO myeloma cells, another expression system is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841.Also, monkey kidney CV1 strain transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney strain (293 or 293 cells subcloned and grown by suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216); mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251); monkey kidney cells (CV1 ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL2); dog kidney cells (MDCK, ATCC CCL34); buffalo rat liver cells (BRL 3A, ATCC CRL1442); human lung cells (W138, ATCC CCL75); human liver cells (HepG2, HB8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., 1982, Annals N.Y. Acad. Sci. 383:44-68); MRC5 cells; FS4 cells; mouse myeloma cells, for example, NSO (for example, RCB0213, 1992, Bio / Technology 10:169) and SP2 / 0 cells (for example, SP2 / 0-Ag14 cells, ATCC CRL1581); rat myeloma cells, for example, YB2 / 0 cells (for example, YB2 / 3HL.P2.G11.16Ag.20 cells, ATCC CRL1662); PER.C6 cells; and human hepatoma strain (HepG2) are also included. CHO cells are one of the cell lines that can be used in this specification, and CHO-K1, DUK-B11, CHO-DP12, CHO-DG44 (Somatic Cell and Molecular Genetics 12:555 (1986)), and Lec13 are exemplary host cell lines. In the case of CHO-K1, DUK-B11, DG44, or CHO-DP12 host cells, they can be modified to lack the ability to fucosylate the proteins expressed therein.In some embodiments, the host cells of the present specification are selected from CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6 or NSO cells or lymphocyte cells.

[0119] Prokaryotes suitable for this purpose include eubacteria, such as gram-negative or gram-positive organisms, such as the family Enterobacteriaceae, such as Escherichia, such as Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescens, and Shigella, as well as Bacilli, such as Bacillus subtilis and B. licheniformis, Pseudomonas, such as Pseudomonas aeruginosa, and Streptomyces.

[0120] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for vectors encoding bispecific antibodies. Saccharomyces cerevisiae or baker's yeast is most commonly used among lower eukaryotic host microorganisms. However, numerous other genera, species, and strains are generally available and useful herein. For example, Schizosaccharomyces pombe; Kluyveromyces hosts such as 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, etc.; Yarrowia (European Patent No. 402,226); Pichia pastoris (European Patent No. 183,070); Candida; Trichoderma reesia (European Patent No. 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as Aspergillus nidulans and Aspergillus niger, etc.

[0121] In some embodiments, the host cell can contain a first nucleic acid construct and a second nucleic acid construct stably integrated into the cell genome. In another embodiment, the present disclosure provides cells containing a non-integrated nucleic acid containing the first nucleic acid construct and the second nucleic acid construct specified above, such as a plasmid, cosmid, phagemid, or linear expression element.

[0122] In still further aspects, the present disclosure relates to a transgenic non-human animal or plant containing a nucleic acid encoding one or more sets of human heavy and light chains, and this animal or plant produces the antibody of the present disclosure.

[0123] In a further aspect, the present disclosure relates to hybridomas that produce the antibodies of the present disclosure as defined herein. In still further aspects, the present disclosure relates to a transgenic non-human animal or plant containing a nucleic acid encoding one or more sets of human heavy and light chains, and this animal or plant produces the antibody of the present disclosure.

[0124] In one aspect, the present disclosure is (i) a nucleic acid sequence encoding an antibody or an antigen-binding portion thereof, or a VH or VL domain of an antibody according to any one of the embodiments disclosed herein, (ii) a nucleic acid sequence encoding the amino acid sequence of a heavy or light chain, (iii) a nucleic acid sequence encoding the amino acid sequence of a constant region, or (iv) a combination of two or more of the above and relates to an expression vector.

[0125] In one aspect, the present disclosure relates to a nucleic acid construct encoding one or more amino acid sequences presented in the sequence listing.

[0126] In one aspect, the present disclosure relates to a method for producing an antibody according to any one of the embodiments disclosed herein, the method comprising culturing a host cell comprising one or more expression vectors disclosed herein, expressing an antibody, and purifying the antibody from the culture medium. In one aspect, the present disclosure relates to a host cell comprising the expression vector as defined above. In one embodiment, the host cell is a recombinant eukaryotic, recombinant prokaryotic or recombinant microbial host cell.

[0127] Pharmaceutical composition In one aspect, the present disclosure is directed to a pharmaceutical composition comprising at least one antibody or antigen-binding portion thereof disclosed herein and a pharmaceutically acceptable carrier.

[0128] Components of the composition The pharmaceutical composition may contain one or more additional pharmaceutically active ingredients, for example, another antibody or drug. Also, the pharmaceutical compositions of the present disclosure can be administered in combination therapy, for example, together with another immunostimulant, anti-cancer agent, anti-viral agent or vaccine, to achieve an additive or synergistic effect. Examples of pharmaceutically acceptable carriers can include pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, non-aqueous media, antibacterial substances, isotonic substances, buffers, antioxidants, anesthetics, suspension / dispersing substances, chelating substances, diluents, adjuvants, excipients or non-toxic auxiliary substances, and various combinations of components known in the art.

[0129] Suitable components may include, for example, antioxidants, extenders, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickening substances, coloring substances, emulsifiers or stabilizers, such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercapto glycerol, thioglycolic acid, mercapto sorbitol, butyl methyl anisole, butylated hydroxytoluene, and / or propylgalacte. According to the present disclosure, a solvent containing the antibody or antigen-binding fragment of the present disclosure may contain one or more antioxidants, such as methionine, that reduce the oxidation of the antibody or its antigen-binding fragment. By oxidation-reduction, a decrease in binding affinity can be prevented or reduced, thereby enhancing antibody stability and extending the shelf life. Thus, in some embodiments, the present disclosure provides a composition comprising one or more antibodies or antigen-binding fragments thereof and one or more antioxidants, such as methionine. In the present disclosure, various methods are further provided for mixing the antibody or its antigen-binding fragment with one or more antioxidants, such as methionine, so as to prevent the oxidation of the antibody or its antigen-binding fragment, extend the shelf life, and / or enhance the activity.

[0130] For further illustration, pharmaceutically acceptable carriers may include, for example, aqueous media such as sodium chloride injection, Ringer's injection, isotonic glucose injection, sterile water injection or glucose and lactated Ringer's injection; non-aqueous media such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil or peanut oil; antibacterial substances at bacteriostatic or fungistatic concentrations; isotonic substances such as sodium chloride or glucose; buffer solutions such as phosphate or citrate buffer solutions; antioxidants such as sodium bisulfite; local anesthetics such as procaine hydrochloride; suspending and dispersing substances such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose or polyvinylpyrrolidone; emulsifying substances such as polysorbate 80 (TWEEN-80); sequestering or chelating substances such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. The antibacterial substances used as carriers may be added to the pharmaceutical composition in a multi-dose container, and examples include phenol or cresol, mercury agents, benzyl alcohol, chlorobutanol, methyl p-hydroxybenzoate and propyl p-hydroxybenzoate, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, physiological saline, glucose, glycerol or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying substances, pH buffering substances, stabilizers, solubility enhancers, or substances such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrin.

[0131] Administration, Formulation, and Dosage The pharmaceutical compositions of the present disclosure may be administered to a subject in need thereof in vivo by various routes, including oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal routes, or alternatively by implantation or inhalation, but are not limited thereto. The compositions of the subject may be formulated into preparations in solid, semi-solid, liquid or gaseous form, including tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols, but are not limited thereto. Appropriate formulations and routes of administration may be selected according to the intended application and treatment regimen.

[0132] Formulations suitable for enteral administration include hard or soft gelatin capsules, pills, tablets including coated tablets, elixirs, suspensions, syrups or inhalants, and controlled release forms thereof.

[0133] Formulations 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 incorporated (e.g., within liposomes or other microparticles). In addition, such liquids may contain other pharmaceutically acceptable components, such as antioxidants, buffers, preservatives, stabilizers, bacteriostatic agents, suspending substances, thickening substances, and solutes that render the formulation isotonic with the blood (or other relevant body fluid) of the intended recipient. Examples of excipients include, for example, water, alcohol, polyols, glycerol, vegetable oils, etc. Examples of isotonic carriers suitable for use in such formulations include sodium chloride injection solution, Ringer's solution, or lactated Ringer's injection solution. Similarly, specific dosing regimens, including dose, timing, and repetition, depend on the specific individual and the individual's medical history, as well as empirical considerations, such as pharmacokinetics (e.g., half-life, clearance rate, etc.).

[0134] The dosing frequency may be determined and adjusted throughout the course of therapy and is based on the reduction of the number of proliferating or tumorigenic cells, the maintenance of such reduction of tumorous cells, the reduction of tumorous cell proliferation or the delay of metastasis occurrence. In some embodiments, the dosage may be adjusted or attenuated to manage potential side effects and / or toxicities. Alternatively, a sustained continuous release formulation of the subject therapeutic composition may be appropriate.

[0135] It is understood by those skilled in the art that the appropriate dosage may vary depending on the patient. Determination of the optimal dosage generally involves balancing the level of therapeutic benefit against any risks or adverse side effects. The dosage level selected depends on a variety of factors including the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of treatment, other drugs, compounds, and / or materials being co-administered, the severity of the symptoms, and the species, sex, age, weight, symptoms, general health, and prior medical history of the patient, but is not limited thereto. The dosage is generally selected to achieve at the site of action a local concentration that achieves the desired effect without substantially causing adverse effects or harmful side effects, but the amount of the compound and the route of administration are ultimately at the discretion of the physician, veterinarian or clinician.

[0136] Generally, the antibodies or antigen-binding portions thereof of the present disclosure can be administered in a variety of ranges. Such ranges include from about 5 μg / kg body weight to about 100 mg / kg body weight per single dose; from about 50 μg / kg body weight to about 5 mg / kg body weight per single dose; from about 100 μg / kg body weight to about 10 mg / kg body weight per single dose. Other ranges include from about 100 μg / kg body weight to about 20 mg / kg body weight and from about 0.5 mg / kg body weight to about 20 mg / kg body weight per single 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 single dose.

[0137] In any case, the antibody or antigen-binding portion thereof of the present disclosure is preferably administered to a subject in need thereof at any time. The dosing frequency can be determined by those skilled in the art, such as a treating physician, based on considerations such as the symptoms to be treated, the age of the subject to be treated, the severity of the symptoms to be treated, and the overall health status of the subject to be treated.

[0138] In certain preferred embodiments, the course of treatment comprising the antibody or antigen-binding portion thereof of the present disclosure includes multiple doses of a selected pharmaceutical product over a period of weeks or months. More particularly, the antibody or antigen-binding portion thereof of the present disclosure can be administered at a frequency of once per day, two days, four days, one week, ten days, two weeks, three weeks, one month, six weeks, two months, ten weeks, three months or less. In this regard, it is understood that the dosage can be changed or the interval adjusted based on the patient's response and clinical practice.

[0139] Also, the dosage and regimen of the disclosed therapeutic composition in an individual who has received one or more administrations can be determined empirically. For example, an individual can be given the therapeutic composition produced as described herein at an escalating dosage. In selected embodiments, the dosage can be gradually increased or decreased or attenuated based on empirical determination or findings of side effects or toxicity, respectively. To evaluate the effectiveness of the selected composition, markers of a particular disease, disorder or condition can be followed as previously described. In cancer, such markers include direct measurement of tumor size by palpation or visual findings, indirect measurement of tumor size by x-ray or other imaging techniques; improvement when evaluated by direct tumor biopsy and microscopic examination of tumor samples; measurement of indirect tumor markers (e.g., PSA for prostate cancer) or tumorigenic antigens identified according to the methods described herein, reduction of pain or paralysis; improvement of speech, vision, respiration or other disorders associated with the tumor; improvement of appetite; or improvement of quality of life or extension of survival when measured by an approved test. It will be apparent to those skilled in the art that the dosage varies depending on the individual, the type of neoplastic condition, the stage of the neoplastic condition, whether the neoplastic condition has started to metastasize to other sites in the individual, and past treatments and combination therapies used.

[0140] Formulations suitable for parenteral administration (e.g., intravenous injection) contain the antibodies or antigen-binding portions thereof 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 includes 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 includes 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.

[0141] Applications of the present disclosure In some aspects, the present disclosure provides a method of preventing or treating a disease, disorder or condition associated with B7H3 in a subject, comprising administering to the subject (e.g., a human) in need of treatment a therapeutically effective amount of the antibody disclosed herein. The disease or condition can be cancer, an autoimmune disease or an infectious disease.

[0142] Regardless of whether it is malignant or benign and whether it is primary or secondary, various cancers associated with B7H3 can be treated or prevented by the methods provided by the present disclosure. The cancer can be a solid cancer or a hematological malignancy.Examples of such cancers include lung cancer, such as bronchogenic carcinoma (e.g., squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondroma (non-cancerous), and sarcoma (cancerous); heart cancer, such as myxoma, fibroma, and rhabdomyoma; bone cancer, such as osteochondroma, chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma, giant cell tumor, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, Ewing's tumor (Ewing's sarcoma), and reticulum cell sarcoma; brain cancer, such as glioma (e.g., glioblastoma multiforme), anaplastic astrocytoma, astrocytoma, oligodendroglioma, medulloblastoma, chordoma, schwannoma, 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, epidermoid carcinoma, adenocarcinoma, leiomyosarcoma, gastric adenocarcinoma, enterolipoma, enteroneurofibroma, enterofibroma, colonic polyp, and colorectal cancer; liver cancer, such as hepatocellular adenoma, hemangioma, hepatocellular carcinoma, fibrolamellar carcinoma, cholangiocarcinoma, hepatoblastoma, and angiosarcoma; kidney cancer, such as renal adenocarcinoma, renal cell carcinoma, adrenal tumor, and transitional cell carcinoma of the renal pelvis; bladder cancer; blood cancers, such as acute lymphocytic (lymphoblastic) leukemia, acute myeloid (myeloblastic, myelocytic, myeloblastic, myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., Sezary syndrome and hairy cell leukemia), chronic myelogenous (myeloid, myelocytic, granulocytic) leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, mycosis fungoides, and myeloproliferative disorders (including myeloproliferative disorders such as polycythemia vera, myelofibrosis, thrombocythemia, and chronic myelogenous leukemia); skin cancer, such as basal cell carcinoma, squamous cell carcinoma, melanoma, Kaposi's sarcoma, and Paget's disease; head and neck cancer; eye-related cancers, such as retinoblastoma and intraocular melanoma; cancers of the male genital 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 genital system, such as uterine cancer (endometrial cancer), cervical cancer (cervical carcinoma), ovarian cancer (ovarian carcinoma), vulvar cancer, vaginal cancer, fallopian tube cancer, and cystic teratoma; thyroid cancer (including papillary, follicular, undifferentiated, or medullary cancer); pheochromocytoma (adrenal); non-cancerous parathyroid proliferation; pancreatic cancer; and blood cancers, such as leukemia, myeloma, non-Hodgkin's lymphoma, and Hodgkin's lymphoma.In certain embodiments, the cancer is selected from a neurological tumor, melanoma, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, stomach cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer, and ovarian cancer.

[0143] In some embodiments, examples of cancers include low grade / follicular non-Hodgkin's 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 lesion NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenström macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myelogenous leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as B-cell cancers including B-cell lymphoma, including vascular proliferative abnormalities associated with phakomatosis, edema (e.g., edema associated with brain tumors), B-cell proliferative disorders, and Majocchi syndrome, but are not limited thereto.More specific examples include, but are not limited to, recurrent 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, nodular marginal zone lymphoma, hairy cell leukemia, plasmacytoma and / or multiple myeloma, low-grade / follicular lymphoma, intermediate-grade / follicular NHL, mantle cell lymphoma, follicular center lymphoma (follicular), intermediate-grade diffuse NHL, diffuse large B-cell lymphoma, intermediate-grade NHL (including intermediate-grade frontline NHL and intermediate-grade recurrent NHL), recurrent 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 lesion NHL, Burkitt lymphoma, precursor (peripheral) large granular lymphocytic leukemia, mycosis fungoides and / or Sézary syndrome, cutaneous (cutaneous) lymphoma, anaplastic large cell lymphoma, angiocentric lymphoma.

[0144] In some embodiments, examples of cancers include, but are not limited to, B cell proliferative disorders, including but not limited to lymphoma (e.g., B cell non-Hodgkin lymphoma (NHL)) and lymphocytic leukemia. Such lymphomas and lymphocytic leukemias include, for example, a) follicular lymphoma, b) small non-cleaved 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), nodular 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) lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, h) acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B cell prolymphocytic leukemia, i) plasma cell tumors, multiple myeloma, plasmacytoma, and / or j) Hodgkin disease.

[0145] In some other embodiments, the disease or condition is an autoimmune disease. Examples of autoimmune diseases that can be treated by the antibodies or antigen-binding portions thereof disclosed herein include, among others, autoimmune encephalomyelitis, lupus erythematosus, and rheumatoid arthritis. Also, the antibodies or antigen-binding portions thereof can be used to treat or prevent infectious diseases, inflammatory diseases (e.g., allergic asthma), and chronic graft-versus-host disease.

[0146] Combination The antibodies can be used in combination with various anti-cancer agents, cytotoxic agents, chemotherapeutic agents, or cellular immunotherapies. For example, the antibodies of the present disclosure can be administered in combination with anti-PD-1 / PD-L1 antibodies. Also, the antibodies of the present disclosure can be administered in combination with CAR-T therapy.

[0147] Cell immunotherapy

[0148]

[0149] Chemotherapy In some embodiments, the antibodies disclosed herein can be used in combination with cell immunotherapy, also known as adoptive cell therapy. As is generally known, cell immunotherapy is a form of treatment that uses cells of the human immune system to remove cancer. Such techniques may include directly isolating unique immune cells and simply increasing their numbers (e.g., by activating and expanding a patient's immune cells outside the body and injecting them into the patient), or may include genetically modifying immune cells (by gene therapy) to enhance their anti-cancer ability. Cell immunotherapy can be deployed in various ways, including but not limited to tumor-infiltrating lymphocyte (TIL) therapy, modified T cell receptor (TCR-T) therapy, chimeric antigen receptor (CAR) T cell therapy, CAR NK cell therapy, and natural killer (NK) cell therapy. In some embodiments, the antibody can be used in combination with additional anti-tumor therapies, such as tumor-infiltrating lymphocyte (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. Administration of the additional anti-tumor therapy can be performed before, after, or simultaneously with administration of the antibody or pharmaceutical composition disclosed herein.The term "anticancer substance" or "antiproliferative substance" means any substance that can be used in the treatment of cell proliferative disorders, such as cancer, and includes, but is not limited to, cytotoxic substances, cell division inhibitors, antiangiogenic substances, dealkylating substances, chemotherapeutic substances, radiation therapy and radiation therapy substances, targeted anticancer substances, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiation therapy, and antimetastatic substances and immunotherapy substances. In the selected embodiments considered above, it is understood that such anticancer substances may include conjugates and may be associated with the disclosed site-specific antibodies prior to administration. More specifically, in certain embodiments, the selected anticancer substance is linked to the mismatched cysteine of the modified antibody to produce the modified conjugate described herein. Accordingly, such modified conjugates are expressly contemplated as being within the scope of the present disclosure. In other embodiments, the disclosed anticancer substances are administered in combination with site-specific conjugates containing the various therapeutic substances described above.

[0150] As used herein, the term "cytotoxic substance" means a substance that is toxic to cells, reduces or inhibits cell function, and / or causes cell destruction. In certain embodiments, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic substances include bacteria (e.g., diphtheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcal enterotoxin A), fungi (e.g., α sarcin, restrictocin), plants (e.g., abrin, ricin, modeccin, viscumin, yamabushitoxin, saporin, gelonin, momoridin, trichosanthin, barley toxin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, chlorambu officinalis inhibitor, gelonin, mitegellin, restrictocin, phenomycin, neomycin, and trichothecene) or low molecular weight toxins or enzymatically active toxins of animals (e.g., cytotoxic Rnase, including fragments and / or variants thereof, such as extracellular pancreatic Rnase; Dnase I), but are not limited thereto.

[0151] For the purposes of the present disclosure, a "chemotherapeutic substance" includes a compound (e.g., a cytotoxic or cytostatic agent) that non-specifically reduces or inhibits the growth, proliferation, and / or survival of cancer cells. Such chemical substances often act on intracellular processes necessary for cell growth or division and are thus generally particularly effective against cancer cells that rapidly grow and divide. For example, vincristine inhibits cells from entering mitosis by depolymerizing microtubules. Generally, a chemotherapeutic substance can include any chemical substance designed to inhibit or designed to inhibit cancer cells or cells that have the potential to become cancerous or generate tumorigenic progeny (e.g., TIC). Such substances are often administered in combination and are often most effective in regimens such as CHOP or FOLFIRI.

[0152] Examples of anti-cancer substances that can be used in combination with the site-specific constructs of the present disclosure (either as components of site-specific conjugates or in an unconjugated state) include alkylating agents, alkyl sulfonates, aziridines, ethyleneimines, and methylamelamine, acetogenins, camptothecin, bryostatin, callystatin, CC-1065, cryptophycin, dolastatin, duocarmycin, erythrobins, pancratistatin, sarcodictyin, spongistatin, nitrogen mustard, antibiotics, enediynes, dynemicin, bisphosphonates, esperamicin, chromoprotein enediyne anti-cancer chromophores, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (ADRIAMYCIN, registered trademark), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, oligomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptozotocin, tubercidin, ubenimex, dinostatin, zorubicin;Antimetabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogs, purine analogs, androgens, anti-adrenal agents, folic acid supplements, for example, folic acid, aceglutamide, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demeclocycline, diaziquone, eflornithine, elliptinium acetate, epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansinoids, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, OR), razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2”-trichlorotriethylamine; trichothecenes (particularly, T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitopropritol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, chlorambucil; GEMZAR (registered trademark) gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE (registered trademark) vinorelbine; novantrone;Teniposide; Edatrexate; Daunomycin; Aminopterin; Xeloda; Ibandronic acid; Irinotecan (Camptosar, CPT-11), topoisomerase inhibitor RFS2000; Difluoromethylornithine; Retinoid; Capecitabine; Combretastatin; Leucovorin; Oxaliplatin; inhibitors of PKC-alpha, Raf, H-Ras, EGFR, and VEGF-A that reduce cell proliferation, and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing, but are not limited thereto. This definition includes antihormonal substances that act to regulate or inhibit the action of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit the aromatase enzyme that regulates estrogen production in the adrenal gland, antiandrogens; and Troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, ribozymes, such as VEGF expression inhibitors and HER2 expression inhibitors; vaccines, Proleukin (PROLEUKIN, registered trademark) rIL-2; Lurtotecan (LURTOTECAN, registered trademark) topoisomerase 1 inhibitor; Abarelix (ABARELIX, registered trademark) rmRH; Vinorelbine and Esperamicin, and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing are also included.;

[0153] Combined with radiotherapy In addition, the present disclosure provides a combination of an antibody and radiotherapy (i.e., any mechanism for locally inducing DNA damage in tumor cells, such as gamma irradiation, X-rays, UV irradiation, microwaves, electron emission, etc.). Also contemplated are combination therapies that use the specific delivery of a radioisotope to tumor cells, and the disclosed antibodies may be used in association with a target anticancer agent or other targeting means. Typically, radiotherapy is administered by pulse over a period of about 1 to about 2 weeks. Radiotherapy may be administered to a subject with head and neck cancer for about 6 to 7 weeks. Radiotherapy may be administered as a single dose or as multiple sequential doses.;

[0154] Pharmaceutical packs and pharmaceutical kits Also provided are a pharmaceutical pack and a pharmaceutical kit comprising one or more containers containing an antibody or an antigen-binding portion thereof in a dose of one or two or more doses. In certain embodiments, a unit dose is provided, which unit dose comprises, for example, a predetermined amount of a composition comprising an antibody or an antigen-binding portion thereof, with or without one or more additional substances. In other embodiments, such unit doses are supplied by pre-filled disposable syringes for injection. In still other embodiments, the composition contained in the unit dose may contain physiological saline, sucrose, etc.; a buffer, for example, phosphoric acid, etc., and / or may be formulated within a stable and effective pH range. Alternatively, in certain embodiments, the composition may be provided as a lyophilized powder that can be reconstituted upon addition of a suitable liquid, such as sterile water or physiological saline. In certain preferred embodiments, the composition contains one or more substances that inhibit aggregation of the protein, including but not limited to sucrose and arginine. By affixing or associating any label on or with the container, it is indicated that the enclosed composition is used for the treatment of optimal tumor pathologies.

[0155] Also, the present disclosure provides a kit for generating a single-dose or multi-dose administration unit of an antibody and one or more optional anti-cancer substances. The kit includes 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 may be formed from 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 another preferred embodiment, the container includes a sterile port (e.g., the container may be an intravenous injection solution bag or a vial with a stopper that can be punctured by a subcutaneous injection needle). Such a kit generally contains a pharmaceutically acceptable formulation of the antibody in a suitable container and may contain one or more anti-cancer substances in the same or different containers. The kit may also contain other pharmaceutically acceptable formulations for either diagnostic or combination therapy. For example, in addition to the antibody or its antigen-binding portion of the present disclosure, such a kit may contain any one or more of a variety of anti-cancer substances, such as chemotherapeutic or radiotherapeutic drugs; anti-angiogenic substances; anti-metastatic substances; targeted anti-cancer substances; cytotoxic substances; and / or other anti-cancer substances.

[0156] More specifically, the kit may have a single container containing the disclosed antibody or its antigen-binding portion, with or without further components, or may have separate containers for each desired substance. Alternatively, the antibody and any anti-cancer substances of the kit may be separately maintained in separate containers prior to administration to the patient. The kit may also include a second / third container as a means for containing a sterile pharmaceutically acceptable buffer or other diluent, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and glucose solution.

[0157] When providing the components of the kit in one or more solutions, the solution is preferably an aqueous solution, and sterilized water or physiological saline is particularly preferred. However, the components of the kit can be provided as dry powders. When providing the reagent or component as a dry powder, the powder can be reconstituted by adding a suitable solvent. It is also envisioned that the solvent can be provided in a separate container.

[0158] As briefly shown above, the kit can also contain an antibody or an antigen-binding portion thereof and means for administering any components to a patient, such as one or more needles, an I.V. bag or syringe, or an eye dropper, pipette, or other instrument that can inject or introduce the formulation into an animal or apply it to an affected part of the body. Further, the kits of the present disclosure typically include means for containing vials, etc., and other components that have strict restrictions regarding commercial sale, such as blow-molded plastic containers that house and maintain syringes or desired vials and other instruments.

[0159] Summary of the Sequence Listing A sequence listing containing a number of amino acid sequences and nucleotide sequences is attached to this application. In the following Tables A, B, and C, a summary of the sequences of the W301088 antibody exemplified herein is presented, which includes W301088-1.145.16, its chimeric version W301088-1.145.16-xIgG1KV320, and its humanized version W301088-1.145.16-z3-p1-uIgG1KV320. W301088-1.145.16-xIgG1KV320 has the same variable region as the parental mouse clone of W301088-1.145.16. [Table 2] [Table 3] [Table 4]

[0160] [Examples] The present disclosure described generally as such will be more readily understood by reference to the following examples, which are provided for illustrative purposes only and are not intended to limit the present disclosure. These examples are not intended to represent that all the following experiments are, nor are they merely the experiments that have been conducted.

Example

[0161] Preparation of Antigen, Reference Antibody, and Cell Line 1.1 Preparation of Commercially Available Materials

Table 5

[0162] 1.2 Generation of Antigen The amino acid sequence encoding the extracellular domain of human 4IgB7H3 (UniprotID: Q5ZPR3, amino acids 29 - 461) was first codon-optimized for mammalian cell expression and then synthesized by Sangon Biotech (Shanghai, China). This DNA segment was subcloned into a pcDNA3.3 expression vector with 6xHis at the C-terminus and then transfected into Expi293F cells (Invitrogen, A14635). Five days after 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 3.5 kDa). The protein concentration was determined on a NanoDrop device by absorbance at 280 nm. Electrophoresis of 2 μg of the purified protein was performed on an SDS-PAGE gel (Invitrogen) with and without reducing reagent. The purity of the purified protein was quantified by HPLC-SEC using a TSKgel G3000SWXL size exclusion column (Tosoh, 008541). The purified protein was stored at -80 °C. Human 2IgB7H3 and cynomolgus monkey B7H3 were purchased from ACRO (Cat. B73-H52E2) and ACRO (Cat. B73-C52Ha), respectively.

[0163] 1.3 Generation of reference antibody (BMK) The anti-B7H3 antibody enoblituzumab (MacroGenics, US Patent Application Publication No. 20120294796: Seq 117, 119) was used as the reference antibody. The nucleic acid sequence encoding the variable domain of this antibody was first codon-optimized for mammalian cell expression and then synthesized by Sangon Biotech (Shanghai, China). This DNA segment was then subcloned into a modified pcDNA3.4 expression vector having the constant region of human IgG1. Plasmids containing the VH gene and the VL gene were co-transfected into Expi293F cells (Invitrogen, A14635). Five days after 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 on a NanoDrop device by absorbance at 280 nm. Electrophoresis of 2 μg of the purified antibody was performed on an SDS-PAGE gel (Invitrogen) with or without reducing reagent. 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.

[0164] 1.4 Establishment of stable cell lines / cell pools A human 4IgB7H3-expressing cell line was generated. Briefly, CHOK1 cells were transfected with a pcDNA3.3 expression vector containing full-length 4IgB7H3 using the Lipofectamine 2000 transfection kit (Invitrogen, 11668027) according to the manufacturer's protocol. 48 h after transfection, the cells were subcultured in a selection medium (F12-K supplemented with 10% FBS and 15 μg / mL blasticidin) in a T75 flask. After 2 or 3 passages of selection, a human 4IgB7H3-expressing stable cell line (W3XX088-CHOK1.hPro1.2A5) was obtained by blasticidin selection and limiting dilution, and the expression level was determined by FACS using an anti-B7H3 antibody.

[0165] A cynomolgus monkey B7H3-expressing cell pool was generated. Briefly, FlipinCHO cells were transfected with a pcDNA5 / pOG44 expression vector containing full-length cynomolgus monkey B7H3 using the Lipofectamine 2000 transfection kit (Invitrogen, 11668027) according to the manufacturer's protocol. 48 h after transfection, the cells were subcultured in a selection medium (F12 supplemented with 10% FBS and 600 μg / mL hygromycin B) in a T75 flask. After 2 or 3 passages of selection, a high-expression stable cell pool (WBP3XX088-FlpinCHO.cPro1.pool) was obtained by hygromycin B selection and BD FACSMelody™ cell sorting.

Example

[0166] Content of WBP301088 antibody 2.1 Immunization of animals Four 6- to 8-week-old Balb / c mice were immunized with 200 - 400 μg of human 4IgB7H3-encoding DNA plasmid per animal. The adjuvant mixture included Adju-Phos, CpG-ODN, and GM-CSF. The animals were given subcutaneous, intramuscular, and hydrodynamic tail vein injections once a week. Blood was collected from the mice after 3 injections (first bleeding) and 5 injections (second bleeding). Serum titers were measured by ELISA and FACS.

[0167] The serum antibody titer against the 4IgB7H3 antigen was measured using an ELISA assay. Plates (Nunc) were coated overnight at 4°C with 100 μL of 0.5 μg / mL his-tag, and then blocked for 1 hour at ambient temperature with blocking buffer (2% BSA in PBS). The plates were then washed and incubated for 1 hour at ambient temperature with 1 μg / mL antigen. After washing, mouse sera were serially diluted 1:3 starting from a 1:100 dilution with blocking buffer and incubated for 2 hours at ambient temperature. The plates were then washed and incubated for 1 hour with secondary antibody goat anti-mouse IgG-Fc-HRP (Bethyl, A90-231P). After washing, TMB substrate was added and the reaction was stopped with 2M HCl. Absorbance at 450 nm was read using a microplate reader (Molecular Device). Serum titers were determined by twice the background.

Table 6

[0168] The serum antibody titer against the 4IgB7H3 antigen was measured using a FACS assay. Briefly, modified B7H3-expressing cells were seeded at a density of 1 × 10 5Cells were seeded in a 96-well U-bottom plate at the cell / well level, centrifuged at 1500 rpm for 4 minutes at 4°C, and then the supernatant was removed. Mouse serum, starting from a 1:100 dilution with 1×PBS / 1% BSA and serially diluted in a 1:3 stepwise manner, was added to resuspend the cells, and the cells were incubated at 4°C for 1 hour. The cells were washed twice with 180 μL of 1×PBS / 1% BSA. Goat anti-mouse IgG-Fc Alexa 647 (Jackson, 109-605-098) as the secondary antibody was added to resuspend the cells, incubated in the dark at 4°C for 30 minutes, and then washed with 180 μL of 1×PBS / 1% BSA. Finally, the cells were resuspended in 100 μL of 1×PBS / 1% BSA, and the fluorescence intensity was measured by FACS (BD Canto II) and analyzed using FlowJo Version software. The serum titer was determined by a two-fold background.

Table 7

[0169] When the serum titer was sufficiently high, after inoculating the final booster of 400 μg of DNA by hydrodynamic tail vein injection and 4×10 6 of MCF-7 cells by plantar injection and subcutaneous injection, fusion was carried out. Cell fusion was performed using lymph nodes and spleens.

[0170] 2.2 Hybridoma content The lymph nodes and spleens of immunized mice were homogenized and filtered to remove blood clots and cell debris. Sp2 / 0 myeloma cells in the logarithmic growth phase were collected and centrifuged. According to the general electrofusion procedure, B cells were fused with Sp2 / 0 myeloma cells at a ratio of 1:1 in an electrofusion solution. The fused cells were suspended in DMEM medium supplemented with 20% FBS and 1×HAT, and then transferred into a 96-well plate (CORNING). The fused cells were cultured in an incubator set at 37°C, 5% CO 2 for 10 - 14 days.

[0171] 2.3 Antibody screening First Screening: A cell-based ELISA assay was used as the first screening to examine the binding of hybridoma supernatants to human 4IgB7H3. Briefly, CHOK1 cell lines transfected with human 4IgB7H3 were seeded in 96-well plates (CORNING) at a density of 5×10 3 cells / well, and then maintained in an incubator set at 37 °C and 5% CO 2 for 2 days. The plates were then washed and incubated with the hybridoma supernatants for 1 hour at ambient temperature. After washing the plates, they were incubated with the secondary antibody, goat anti-mouse IgG-Fc-HRP (Bethyl, A90-231P), for 1 hour. After washing, TMB substrate was added and the interaction was stopped with 2M HCl. The absorbance at 450 nm was read using a microplate reader (Molecular Device).

[0172] Second 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 using a dye. After a 15-minute incubation and washing at ambient temperature, the three cell types were mixed at an equivalent of 1×10 6 cells / mL. The three-cell mixture was transferred into a 96-well U-bottom plate (BD) at a density of 1×10 5 cells / well. Then, the hybridoma supernatants were transferred to the plates 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 for 0.5 hour in the dark at 4 °C. The cells were then washed, resuspended in 1×PBS / 1% BSA, and analyzed by flow cytometry.

[0173] To examine the binding of hybridoma supernatants to human 2IgB7H3, a conventional ELISA assay was used. Plates were pre-coated with human 2IgB7H3 (0.2 μg / mL) at 100 μL / well for 1 hour at ambient temperature. After blocking for 1 hour with 1×PBS / 2% BSA at 200 μL / well, the plates were washed three times with 1×PBST. Hybridoma supernatants were added to the plates at a volume of 100 μL / well and incubated for 2 hours at ambient temperature. After three plate washes with 1×PBST, goat anti-mouse IgG-Fc HRP (Bethyl, A90-231P) at 100 μL / well was added to the plates and incubated for 1 hour at ambient temperature. After six washes, color development was induced by dispensing 100 μL / well of TMB substrate for 5 - 10 minutes, and then the reaction was stopped by adding 100 μL / well of 2M HCl. Absorbance at 450 nm was read using a microplate reader.

[0174] The selected positive clones screened by FACS were further confirmed by SPR (surface plasmon resonance). SPR enables label-free real-time detection of biomolecular interactions. SPR occurs when polarized light is incident on the interface between two media of a conductive surface. This generates a charge density wave called a plasmon, which reduces the intensity of the reflected light at a specific angle known as the resonance angle in proportion to the mass on the sensor surface.

[0175] The binding of supernatants to human 4IgB7H3 was ranked using a Biacore 8K. An activator was prepared immediately before injection by mixing 400 mM of EDC and 100 mM of NHS (GE). The CM5 sensor chip was activated with this activator for 420 seconds. Next, goat anti-mouse Fc IgG (30 μg / mL in 10 mM NaAc, pH 4.5) was injected into the channel at a flow rate of 10 μL / min for 420 seconds. The chip was inactivated with 1 M ethanolamine-HCl. The supernatant was injected into the channel at a flow rate of 10 μL / min for 30 seconds. A 200 nM analyte of human 4IgB7H3 was injected into the channel at a flow rate of 30 μL / min during a 300-second dissociation phase after a 120-second association phase. Glycine (10 mM, pH 1.5) was injected as a regeneration buffer after the dissociation phase. Sensorgrams were subtracted for the reference channel and the buffer channel. The data were then fit to a 1:1 Langmuir binding model.

[0176] The pHrodo iFL dye can conjugate to biomolecules such as antibodies and become highly fluorescent only when found in an acidic environment, such as that of cell lysosomes and cell endosomes. This enables the detection of conjugated biomolecules in live cell assays. AffiniPure F(ab')2 fragments of goat anti-human IgG Fcγ and anti-mouse IgG Fcγ were labeled with the pHrodo iFL Red STP ester amine-reactive dye and purified using a zeba spin desalting column according to the manufacturer's instructions. MCF-7 cells (2×10 4 cells / well) expressing human full-length B7H3 were seeded into 96-well clear-bottom black plates pre-coated with 8 μg / mL of poly-D-lysine. The cells were grown overnight in an incubator set at 37 °C, 5% CO 2 2. The next day, the WBP301088-1 positive strain supernatant was added to the corresponding wells in a volume of 100 μL. The cells were incubated at 37 °C, 5% CO 2Incubated for 0.5 h in an incubator set at [condition]. Human and mouse IgG1 isotype antibodies were used as negative controls. 50 nM pHrodo iFL Red / F(ab') 2 Goat anti-mouse or anti-human IgG Fcγ conjugate was added at a volume of 100 μL / well (diluted in complete culture medium). Then, the plate was incubated in the dark at 37 °C, 5% CO 2 for 5 h in an incubator set at [condition]. After incubation, the supernatant was discarded. The plate was washed with complete culture 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 maintained at ambient temperature for 15 min. 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 using a Perkin Elmer Operetta CLS high-content analysis system.

[0177] In the primary screening, a total of 8460 wells were examined, 279 hybridomas were selected and expanded in 24-well plates. In the secondary screening, the supernatants of the 24-well plates were used to confirm strong binding to human 4IgB7H3 and cynomolgus B7H3, weak binding to human 2IgB7H3, and strong internalization activity against MCF-7. Based on the secondary screening results, the WBP301088-1.145 hybridoma was selected for subcloning.

[0178] 2.4 Subcloning of antibodies The selected positive hybridoma cells were used for subcloning. Cells in the logarithmic growth phase were counted and added to 1.5 mL of semi-solid HAT medium. The cells were gently mixed for 5 - 10 s using a vortex oscillator and then seeded into 6-well plates (CORNING). The plates were incubated at 37 °C, 5% CO 2It was maintained for 7 - 8 days in an incubator set at [conditions]. Each visible single colony was selected into a 96 - well plate (CORNING) with DMEM medium supplemented with 10% FBS. After 2 - 3 days, the cell supernatant was collected and screened using the protocols described above for antibody screening by cell - based ELISA (human 4IgB7H3) and conventional ELISA (human 2IgB7H3). After sub - cloning confirmation screening for human 4IgB7H3 and cynomolgus monkey B7H3 by FACS, the culture supernatant of the selected single - positive clones was collected, and the antibodies were purified for further characterization. WBP301088 - 1.145.16 was obtained after sub - cloning screening and confirmation screening.

[0179] 2.5 Antibody isotype determination Isotype determination ELISA was performed using a 96 - well high - binding plate (Nunc). Each well was coated overnight at 4°C with 2 μg / mL of capture antibodies, goat anti - mouse IgG1 (Bethyl, A90 - 205A), goat anti - mouse IgG2a (Bethyl, A90 - 207A), goat anti - mouse IgG2b (Bethyl, A90 - 209A), goat anti - mouse IgG3 (Bethyl, A90 - 211A), 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 immobilized on the plate was measured using a peroxidase - conjugated goat anti - mouse kappa / lambda light chain antibody. The HRP signal was detected by adding TMB substrate, and the reaction was stopped after 12 minutes using 2M HCl. All incubation steps were performed at ambient temperature, and the plate was washed with PBS between steps. The absorbance at 450 nm was read using a microplate reader (Molecular Device).

[0180] The isotype determination results of WBP301088 - 1.145.16 are shown in the following table (Table 4).

Table 8

[0181] 2.6 Generation of Antibodies When a small amount of purified antibody is required, a T75 flask (CORNING) is generally used to scale up hybridoma cell culture. Monoclonal hybridoma cells were seeded into flasks at 5×10 6 ~1×10 7 cells / T75 flask. The cells were continuously grown for about 7 - 10 days until the cell viability reached about 30 - 40%. The culture supernatant was collected and cell debris was removed by centrifugation. The supernatant was sterile filtered and stored for antibody purification.

[0182] 2.7 Sequencing of Hybridomas Total RNA of hybridoma cell samples was first extracted according to the instructions of TaKaRa's MiniBEST Universal RNA Extraction Kit. Then, RNA was converted to cDNA using Clonetech's SMART RACE cDNA Amplification Kit. Next, the VH domain DNA sequence and VL domain DNA sequence were amplified from the cDNA by 30 cycles of PCR, with denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and then extension at 72°C for 30 seconds. Then, the PCR products were subcloned into a TA cloning vector and then sent to Biosune Biotech (Shanghai, China) for sequencing.

[0183] When the monoclonal nature of the hybridoma cell sample was confirmed by sequencing data, the DNA sequences of the VH domain and VL domain were amplified by PCR. The primers were synthesized by Sangon Biotech (Shanghai, China). Then, the DNA segment was subcloned into a pcDNA3.4 expression vector with the constant region of human IgG1 having the LALA mutation and then sequenced by Tsingke Biotechnology (Beijing, China).

[0184] 2.8 Conversion of IgG Once the monoclonality of the hybridoma cell sample was confirmed by sequencing data, the amino acid sequences of the VH and VL domains were codon-optimized for mammalian cell expression and then synthesized by Sangon Biotech (Shanghai, China). Subsequently, the DNA segments were subcloned into the pcDNA3.4 expression vector with the constant region of human IgG1 having the L234A / L235A (LALA) mutation.

[0185] In this method, W301088-1.145.16 was converted to human IgG1 with the LALA mutation format to obtain the chimeric antibody W301088-1.145.16-xIgG1KV320. After protein A purification and buffer exchange, it was characterized by SDS-PAGE and SEC-HPLC. The protein migrated at apparent molecular weights of 50 kDa and 25 kDa on SDS-PAGE under reducing conditions, corresponding to the heavy and light chains of IgG (data not shown). The purity was high purity exceeding 99% (SEC-HPLC).

Table 9

[0186] 2.9 Humanization A local copy of the open-source software ANARCI (Dunbar J, Deane CM. ANARCI: antigen receptor numbering and receptor classification. Bioinformatics, 2015; 32 (5): 298-300) was run to assign Kabat numbering to W301088-1.145.16. The CDRs were identified according to the definition on Dr. Martin's website (Martin, 2018, supra) and are shown in Table 6.

Table 10

Table 11

Table 12

[0187] The VH domain sequence and VL domain sequence of the mouse antibody W301088 - 1.145.16 were respectively aligned against the human germline sequence repertoires of the VH domain and VL domain of IMGT. Human germline sequences of VH / VL domains with the minimum number of amino acid differences within the framework were selected as the humanization templates for the VH / VL domains of W301088 - 1.145.16. IGHV4 - 31*02 mixed with IGHJ6*01 and IGKV4 - 1*01 mixed with IGKJ4*01 were the most homologous human germline sequences to the VH domain sequence and VL domain sequence of W301088 - 1.145.16 respectively. The CDRs of W301088 - 1.145.16 were transplanted into the frameworks of such two human germline templates to construct the germline sequences.

[0188] Several "back - mutation" positions within the framework were empirically selected to convert the amino acids in the germline sequences to these corresponding amino acids in the original mouse sequence. A set of humanized variants was empirically designed to explore various combinations of such selected back - mutation sites. The germline sequences differed from the original mouse W301088 - 1.145.16 sequence at 16 positions in the VL domain and 20 positions in the VH domain. Among a total of 36 different sites, 14 sites were considered for back - mutation: VH: Q001D, S025T, H040F, K043N, G044K, I048M, V067I, V071R, A085E, V089T, Y091F, VL: I021M, P043S, Y087F. A set of humanized variants was empirically designed to examine various combinations of such back - mutations.

[0189] 2.11 Removal of PTM The VH domain sequences and VL domain sequences of the humanized variants were scanned for several types of important post-translational modification (PTM) sites: asparagine deamidation sites (N-G and N-S) in the CDRs, aspartic acid isomerization sites (D-G) in the CDRs, unpaired C sites in the full length, and N-linked glycosylation sites (N-X-S / T, where X can be any amino acid except P) in the full length. By empirically designing point mutations, such important PTM sites in the humanized variants were removed to avoid the potential risks of PTM modifications. Finally, PTM-removed antibodies that did not affect expression, binding, and thermal stability compared to the parental antibody were selected.

[0190] Since the VL domain sequence of the mouse antibody W301088-1.145.16 contains an N-S-S N-linked glycosylation site, three point mutations VL:N027dQ, VL:S027eP, and VL:S027fA (according to Kabat numbering) were designed to remove the important PTM site. These were combined with the humanized variant to rank k off They were further characterized by ranking.

[0191] 2.12 k by SPR off Ranking k of the filtered supernatant offRanking was performed on the Biacore 8K instrument. The CM5 sensor chip was first activated with 400 mM EDC and 100 mM NHS (GE) at a flow rate of 10 μL / min for 420 seconds. Then, 30 μg / mL anti-human Fc IgG (Jackson) in 10 mM NaAc (pH 4.5) was injected into channels 1 - 8 at a flow rate of 10 μL / min for 420 seconds. Next, the chip was deactivated with 1 M ethanolamine-HCl (GE) at a flow rate of 10 μL / min for 420 seconds. The diluted antibody supernatant was injected into channel Fc2 at a flow rate of 10 μL / min. The supernatant of cell culture without plasmid transfection was used as a negative control and injected into channel Fc1 at a flow rate of 10 μL / min. 100 nM human 4IgB7H3 and running buffer were regularly injected into channels Fc1 - Fc2 at a flow rate of 30 μL / min during the 3600-second dissociation phase after the 180-second association phase. After each run, 10 mM glycine (pH 1.5) was injected to regenerate the chip. Sensorgrams were analyzed using the software attached to the Biacore 8K instrument. The sensorgrams of the samples were subtracted by the reference channel and the buffer channel. Then, a 1:1 Langmuir binding model was fitted to the data.

[0192] 2.13 Combinations with humanized variants of PTM removal mutations The best k off The PTM removal point mutation showing the rate was combined with the most promising k off rate of the humanized variant by site-directed mutagenesis to construct the final construct and verify the affinity.

[0193] k off From the rate, the PTM removal mutation of VL:N027dQ was combined with the selected four humanized mutant amino acids S025T, V071R, Y091F, V089T to construct the final lead. The final lead was named W301088-1.145.16-z3-p1-uIgG1KV320.

Table 13

[0194] 2.14 Generation Scale Transient Transfection and Purification Each plasmid at a final concentration of 1 μg / mL was transiently transfected into 20 mL of Expi293F cells at a density of 3.0×10 6 cells / mL and a cell viability of more than 95% using the ExpiFectamine™ Transient Transfection Kit. The cell culture was grown on a humidified platform shaker at a rotation rate of 150 rpm. While maintaining the temperature at 37°C, the CO 2 level was maintained at 8%.

[0195] Five days after the incubation of the cell culture, the supernatant expressing the target antibody was collected and filtered and purified using a GE MabSelect SuRE Protein A column (Cytiva, 175438). The eluted antibody was dialyzed into PBS via a D-Tube Dialyzer Maxi (EMD Millipore, 71508, MWCO 3.5 kDa). The antibody concentration was determined on a NanoDrop device by absorbance at 280 nm. Electrophoresis of 2 μg of the purified antibody sample was performed on an SDS-PAGE gel (Invitrogen NuPAGE™ 4-12% Bis-Tris Protein Gel) with and without reducing reagent. The purity of the purified antibody sample was quantified by HPLC-SEC using a TSKgel G3000SWXL size exclusion column (Tosoh, 008541). The purified antibody was stored at -80°C.

Example

[0196] In Vitro Characterization of the WBP301088 Antibody 3.1 Detection of Purity by SDS-PAGE and SEC-HPLC The sample was mixed with the loading buffer and heated at 75°C for 10 minutes using a dry bath. After the addition of the sample, SDS-PAGE was run at a constant voltage (200 V) for 35 minutes. The gel was stained and destained with eStain™ L1. The SDS-PAGE results were obtained using a BIO-RAD imaging system.

[0197] The results are summarized in Table 10 below. The protein migrates at apparent molecular weights of 50 kDa and 25 kDa on SDS-PAGE under reducing conditions, corresponding to the heavy and light chains of IgG, while under non-reducing conditions, it migrates as a band of approximately 150 kDa as expected (Figure 1).

[0198] 3.2 Detection of purity by SEC-HPLC The purity of each antibody was detected using a TSKgel G3000SWXL column (Tosoh Bioscience, 0008541) with an Agilent 1260 Infinity II system (Agilent Technologics™). An appropriate volume (5 - 100 μL) of the sample was injected into the column and separated for 20 minutes at a flow rate of 1 mL / min. The running buffer was 50 mM sodium phosphate, 150 mM NaCl, pH 7.0. The retention peak was detected by UV light at a wavelength of 280 nm. The purity of each antibody was analyzed by the SEC-HPLC analysis method, and the total peak areas from 4.5 minutes to 10.5 minutes were integrated. The software for calculation and analysis was OpenLab CDS Workstation (v2.3.0.443 or v2.2.0.484).

[0199] The results are summarized in Table 10 below. The purity of the final lead was high, exceeding 99% (SEC-HPLC, Figure 2).

[0200] 3.3 Thermal stability (T m ) The T of each antibody m(Melting temperature) was investigated using a QuantStudio (registered trademark) 7 Flex Real-Time PCR System (Applied Biosystems). 19 μL of the 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 air bubbles. The plate was heated from 26 °C to 95 °C at a rate of 0.9 °C / min, and the generated fluorescence data was collected. The negative differential coefficient of the fluorescence change for various temperatures was calculated, and the maximum value was defined as the melting temperature T m as. When the protein has multiple unfolding transitions, the first T m was reported and named T m 1. Data collection and T m calculation were automatically performed using QuantStudio (registered trademark) Real Time PCR software (v1.3).

[0201] The T m value of protein W301088-1.145.16-z3-p1-uIgG1KV320 was defined as the maximum value of the negative differential coefficient of the fluorescence change for various temperatures and listed in Table 10 below. The data profile is shown in Figure 3. This dashed line indicates the position of the T m 1 value in the fluorescence curve. The T m 1 of W301088-1.145.16-z3-p1-uIgG1KV320 was 70.1 °C, indicating that it has good thermal stability.

[0202] 3.4 Detection of Hydrophobicity by HIC-HPLC The hydrophobic properties of the antibody were detected using a TSKgel butyl-NPR column (Tosoh, 0042168) with an HPLC 1260 Infinity II system (Agilent Technologics™). Each sample was diluted to 0.5 mg / mL with PBS buffer, and 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 4 ) 2 SO 4 , pH 7.0 (buffer D). The running gradient was 0% - 100% buffer D over 3 - 53 minutes. The retention peaks were detected by UV light at wavelengths of 280 nm and 230 nm. The retention time was analyzed by the HIC-HPLC analysis method, and the peak areas of all peaks between 20 - 40 minutes were integrated. The software for calculation and analysis was OpenLab CDS Workstation (v2.6.0.691).

[0203] The HIC retention time of protein W301088-1.145.16-z3-p1-uIgG1KV320 was 23.52 minutes (Table 10 and Figure 4), indicating that this protein is an antibody with good hydrophobicity.

[0204] 3.5 Diffusion interaction parameters (k D ) DLS-k DMeasurements were investigated using a DynaPro Plate Reader III (Wyatt Technology). During the sample preparation process, the appearance of the samples was recorded during thawing, filtration, and concentration. The samples were concentrated to over 20 mg / mL and then diluted to final concentrations of 2.5 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, and 20 mg / mL with PBS buffer. Next, 7.5 μL of the sample solution was added to a 1536-well microplate (Aurora, ABI1-00110A). The plate was sealed with ClearSeal Film (Hampton Research, HR4-521) and centrifuged at 3,000 rpm for 5 minutes to sediment the sample to the bottom of the well. Each sample was examined in duplicate wells. The plate was placed in the corresponding position, and data collection was performed using DYNAMICS operation software (v7.8.1.3). Five acquisition data were collected for each protein sample, and the acquisition time between each was 5 seconds. For each measurement, the diffusion coefficient was determined and plotted against the protein concentration. k D Values were automatically calculated by the software (v7.8.1.3) as listed in Table 10. The data profile is shown in Figure 5. The k D of protein W301088-1.145.16-z3-p1-uIgG1KV320 in PBS is -5.17 mL / g, indicating that this protein is an antibody with high solubility. During the assay process, the appearance of the protein was also recorded, and a small amount of particles were observed after thawing and gentle shaking. When the buffer was exchanged to 20 mM His, 8% sucrose, 0.02% PS80, pH 6.5, no particles were observed.

[0205] The above results of SDS-PAGE, SEC-HPLC, HIC-HPLC, DSF, and DLS are summarized in Table 10.

Table 14

[0206] 3.6 Binding of Human B7H3 (FACS) Using FACS, the binding of antibodies to human B7H3 was detected. In this method, specific molecules expressed on the surface of live cells can be quantitatively analyzed and identified. Unlabeled cells were used as a control to set a threshold before detection, and the percentage change of each group exceeding the fluorescence intensity threshold was analyzed. MCF-7 cells (1×10 5 cells / well) expressing human full-length B7H3 were incubated for 1 hour at 4°C in a refrigerator with various concentrations of antibodies (3.16-fold serial dilutions from 50 nM to 0.0005 nM in 1×DPBS / 1% BSA) at a volume of 100 μL / well. The anti-human B7H3 reference antibody enobrutuzumab (MacroGenics) was used as a positive control. A 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 for 0.5 hour at 4°C in the dark in a refrigerator. The mean fluorescence intensity (MFI) of the cells was measured by a flow cytometer and analyzed by FlowJo. EC 50 values were calculated using GraphPad Prism 7 software by four-parameter nonlinear regression analysis.

[0207] W301088-1.145.16-z3-p1-uIgG1KV320 showed good binding to B7H3-expressing MCF-7 cells, with an EC 50 of 1.08 nM and a maximum MFI of 19,900. This was comparable to chimeric antibodies and better than the reference antibody enobrutuzumab (MacroGenics) (Table 11, Figure 6).

Table 15

[0208] 3.7 Binding of cynomolgus monkey B7H3 (FACS) The binding of the antibody to cynomolgus B7H3 was detected using FACS. Cynomolgus B7H3 was transfected into FlpinCHO cells (W3XX088-FlpinCHO.cProl.pool) (1×10 5 cells / well), and incubated for 1 hour at 4°C in a refrigerator with various concentrations of the antibody (3.16-fold serial dilution from 50 nM to 0.0005 nM in 1×DPBS / 1% BSA) at a volume of 100 μL / well. The anti-human B7H3 reference antibody enoblituzumab (MacroGenics) was used as a positive control. A 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 for 0.5 hour at 4°C in the dark in a refrigerator. The mean fluorescence intensity (MFI) of the cells was measured using a flow cytometer and analyzed using FlowJo. EC 50 values were calculated using GraphPad Prism 7 software by four-parameter non-linear regression analysis.

[0209] W301088-1.145.16-z3-p1-uIgG1KV320 showed good binding to cynomolgus transfected cells, and the EC 50 was 4.68 nM. This was comparable to the chimeric antibody and was better than enoblituzumab (MacroGenics) (Table 12, Figure 7).

Table 16

[0210] 3.8 Binding of human 4IgB7H3 (SPR) The affinity of the antibody for human 4IgB7H3 was determined using SPR. The affinity of the antibody for human 4IgB7H3 was examined by Biacore 8K. An activator was prepared immediately before injection by mixing 400 mM of EDC and 100 mM of NHS (GE). The CM5 sensor chip was activated with this activator for 420 seconds. Next, goat anti-human Fc IgG (30 μg / mL in 10 mM NaAc, pH 4.5) was injected into the channel at a flow rate of 10 μL / min for 420 seconds. The chip was deactivated with 1 M hydrochloric acid ethanolamine. The antibody diluted with running buffer (1×HBS-EP+) was injected into the channel at a flow rate of 10 μL / min for 30 seconds. Analyte human 4IgB7H3 at 7 concentrations (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 2.125 nM, and 1.563 nM) was regularly injected into the channel at a flow rate of 30 μL / min for 3600 seconds during the dissociation phase after 180 seconds of the association phase. Glycine (10 mM, pH 1.5) was injected as the regeneration buffer after the dissociation phase.

[0211] The sensorgrams of the reference channel and the buffer channel were subtracted from the test sensorgram. The experimental data was fitted to a 1:1 binding model. The molecular weight of human 4IgB7H3 used for the calculation was 47.8 kDa.

[0212] The experimental data was fitted to a 1:1 binding model attached to the Biacore 8K evaluation software. As shown in Table 13 and Figures 8A - 8C, W301088-1.145.16-z3-p1-uIgG1KV320 showed a high binding affinity for human 4IgB7H3. The K D value was 7.08E-11 M, which was better than enobrutuzumab (MacroGenics).

Table 17

[0213] 3.9 Binding of human 2IgB7H3 (SPR) The affinity of the antibody against human 2IgB7H3 was determined using SPR. The affinity of the antibody against human 2IgB7H3 was examined by Biacore 8K. An activator was prepared immediately before injection by mixing 400 mM of EDC and 100 mM of NHS (GE). The CM5 sensor chip was activated with this activator for 420 seconds. Next, goat anti-human Fc IgG (30 μg / mL in 10 mM NaAc, pH 4.5) was injected into the channel at a flow rate of 10 μL / min for 420 seconds. The chip was deactivated with 1 M hydrochloric acid ethanolamine. The antibody diluted with electrophoresis buffer (1×HBS-EP+) was injected into the channel at a flow rate of 10 μL / min for 30 seconds. Analyte human 2IgB7H3 at 7 concentrations (500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, and 7.813 nM) was regularly injected into the channel at a flow rate of 30 μL / min for 300 seconds during the dissociation phase after 120 seconds of the association phase. Glycine (10 mM, pH 1.5) was injected after the dissociation phase as the regeneration buffer.

[0214] The sensorgrams of the reference channel and the buffer channel were subtracted from the test sensorgram. The experimental data was fitted to a 1:1 binding model. The molecular weight of human 2IgB7H3 used for the calculation was 25.2 kDa.

[0215] The experimental data was fitted to a 1:1 binding model attached to the Biacore 8K evaluation software. As shown in Table 14 and Figures 9A - 9C, W301088-1.145.16-z3-p1-uIgG1KV320 showed a weak binding affinity for human 2IgB7H3 compared to the binding to human 4IgB7H3. The K D value was 4.64E-08 M.

Table 18

[0216] 3.10 Binding of Cynomolgus B7H3 (SPR) The affinity of the antibody against cynomolgus B7H3 was determined using SPR. The affinity of the antibody against cynomolgus B7H3 was examined by Biacore 8K. An activator was prepared immediately before injection by mixing 400 mM of EDC and 100 mM of NHS (GE). The CM5 sensor chip was activated with this activator for 420 seconds. Next, goat anti-human Fc IgG (30 μg / mL in 10 mM NaAc, pH 4.5) was injected into the channel at a flow rate of 10 μL / min for 420 seconds. The chip was deactivated with 1 M hydrochloric acid ethanolamine. The antibody diluted with running buffer (1×HBS-EP+) was injected into the channel at a flow rate of 10 μL / min for 30 seconds. Analyte cynomolgus B7H3 at 9 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 regularly injected into the channel at a flow rate of 30 μL / min for 3600 seconds in the dissociation phase after 180 seconds in the association phase. Glycine (10 mM, pH 1.5) was injected as a regeneration buffer after the dissociation phase.

[0217] The sensorgrams of the reference channel and the 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 to a 1:1 binding model. The molecular weight of cynomolgus B7H3 used in the calculation was 49 kDa.

[0218] The experimental data were fitted to a 1:1 binding model attached to the Biacore 8K evaluation software. As shown in Table 15 and Figures 10A - 10C, W301088-1.145.16-z3-p1-uIgG1KV320 showed high binding affinity for cynomolgus B7H3, which was comparable to the binding to human 4IgB7H3. The K D value was 5.74E-11 M, which was better than enobrutuzumab (MacroGenics).

Table 19

[0219] 3.11 Internal migration inspection (HCS) The internal migration of the antibody was determined by operetta CLS (PerkinElmer), a high-content imaging and analysis system. This enables the rapid and sensitive collection and analysis of sample images. MCF-7 cells (1.5×10 4 cells / well) expressing human full-length B7H3 were seeded into 96-well clear-bottom black plates pre-coated with 8 μg / mL poly-D-lysine. After overnight incubation in an incubator set at 37 °C and 5% CO 2 , the cells were incubated for 2 hours at 4 °C in a refrigerator with various concentrations of the antibody (3.16-fold serial dilution from 50 nM to 0.0005 nM in 1×DPBS / 1% BSA) at a volume of 100 μL / well. The anti-human B7H3 reference antibody enoblituzumab (MacroGenics) was used as a positive control. A 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 for 1 hour at 4 °C in the dark in a refrigerator. Then, the cells were washed once and resuspended in 1×DPBS / 1% BSA at 37 °C for 2 hours. After incubation, the supernatant was discarded, 100 μL / well of Hoechst 33342 (diluted 1:5000 in 1×DPBS) was added, and the cells were incubated for 15 minutes at ambient temperature. After washing the cells with 1×DPBS, 100 μL / well of acid wash buffer (100 mM glycine, 150 mM NaCl, pH 2.5) was added, and the cells were incubated for 5 minutes at 4 °C in a refrigerator. The cells were washed once with 1×DPBS and fixed with 60 μL of 4% PFA. The mean fluorescence intensity (MFI) of the cells was measured and analyzed by operetta CLS. The EC 50 value was calculated using GraphPad Prism 7 software by four-parameter non-linear regression analysis.

[0220] W301088-1.145.16-z3-p1-uIgG1KV320 showed good internalization ability against B7H3-expressing MCF-7 cells, and the EC 50 was 0.51 nM. This was comparable to the chimeric antibody and better than the reference antibody enobrituzumab (MacroGenics) (Table 16, Figure 11).

Table 20

[0221] Those skilled in the art will further understand that the present disclosure can be implemented in other specific forms without departing from its spirit or central characteristics. From the foregoing description of the present disclosure, it should be understood that other variations are considered to be within the scope of the present disclosure in that only exemplary embodiments thereof are disclosed. Therefore, the present disclosure is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the appended claims as indicating the scope and content of the present disclosure.

Deposit Number

[0222] ATCC CRL1651 ATCC CCL10 ATCC CCL70 ATCC CRL-1587 ATCC CCL2 ATCC CCL34 ATCC CRL1442 ATCC CCL75 HB8065 ATCC CCL51 RCB0213 ATCC CRL1581 ATCC CRL1662 ATCC 12,424 ATCC 16,045 ATCC 24,178 ATCC 56,500 ATCC 36,906

Claims

1. An antibody or antigen-binding portion thereof that binds to B7H3, comprising the following CDRs. Heavy chain CDR1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 1, HCDR2 containing the amino acid sequence of SEQ ID NO: 2, and HCDR3 containing the amino acid sequence of SEQ ID NO: 3; and Light chain CDR1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 4 or 7, LCDR2 containing the amino acid sequence of SEQ ID NO: 5, and LCDR3 containing the amino acid sequence of SEQ ID NO: 6;

2. An antibody or antigen-binding moiety according to claim 1, comprising a heavy chain variable region (VH) and a light chain variable region (VL), The aforementioned VH is, (A) The amino acid sequence described in Sequence 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) Amino acid sequences 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: 8 or 10; Including, and / or The aforementioned VL is (A) The amino acid sequence described in Sequence 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) Amino acid sequences 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; The antibody or its antigen-binding portion, including the antibody.

3. The antibody or antigen-binding moiety according to claim 2, wherein VH and / or VL comprises one or more amino acid substitutions in the framework region.

4. The antibody or antigen-binding moiety according to claim 3, wherein at least one of the amino acid substitutions is a reverse mutation in which an amino acid derived from a human germline sequence is replaced by a different amino acid at a corresponding position in the parent antibody.

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

6. The antibody or antigen-binding moiety according to claim 1, wherein B7H3 is a human B7H3 protein, a mouse B7H3 protein, a cynomolgus monkey B7H3 protein, or the extracellular domain thereof.

7. The antibody or antigen-binding moiety according to claim 6, wherein B7H3 is the human 4IgB7H3 protein or its extracellular domain.

8. The antibody or antigen-binding portion thereof according to claim 1, which is a complete antibody, scFv, Fab, F(ab')2, or Fv fragment.

9. The antibody or antigen-binding portion thereof according to claim 1, wherein the antibody includes an immunoglobulin constant region, for example, a human IgG constant region.

10. The antibody or antigen-binding moiety according to claim 9, wherein the immunoglobulin constant region is a human IgG1 constant region which may contain one or more modifications, for example, an L234A / L235A (LALA) mutation.

11. The antibody or antigen-binding portion thereof according to claim 1, which is a chimeric antibody or a humanized antibody.

12. (a) the amino acid sequence of the heavy chain of SEQ ID NO: 12 and the amino acid sequence of the light chain of SEQ ID NO: 13; and / or (b) The amino acid sequence of the heavy chain of SEQ ID NO: 14 and the amino acid sequence of the light chain of SEQ ID NO: 15; The antibody or antigen-binding portion thereof according to claim 1, comprising:

13. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the VH region and / or VL region of the antibody or antigen-binding portion thereof as described in claim 1.

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

17.

15. A vector comprising the isolated nucleic acid molecule described in claim 13.

16. A host cell comprising the isolated nucleic acid molecule described in claim 13 or the vector described in claim 15.

17. A pharmaceutical composition comprising an antibody or an antigen-binding moiety thereof as described in claim 1, and a pharmaceutically acceptable carrier.

18. A method for producing an antibody or its antigen-binding portion, The steps of culturing the host cells described in claim 16 under conditions suitable for the expression of an antibody or its antigen-binding portion, The steps of isolating the antibody or its antigen-binding portion from the host cells. The method, including the method described above.

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

20. An agent for preventing or treating a B7H3-related disorder in a subject, comprising an antibody or antigen-binding portion thereof according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 17.

21. The agent according to claim 20, wherein the disorder associated with B7H3 is selected from cancer, autoimmune diseases, and infectious diseases.

22. The agent according to claim 21, wherein the cancer is selected from breast cancer, neurological tumors, melanoma, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, stomach cancer, kidney cancer, bladder cancer, prostate cancer, ovarian cancer, cervical cancer, glioblastoma, esophageal cancer, renal cell carcinoma, endometrial cancer, skin cancer, testicular cancer, thyroid cancer, urothelial carcinoma, lymphoma, for example, non-Hodgkin lymphoma and diffuse large B-cell lymphoma, leukemia, for example, chronic lymphocytic leukemia, and multiple myeloma.

23. The agent according to claim 21, wherein the antibody or its antigen-binding portion is administered in combination with other substances for use in cellular immunotherapy, chemotherapy, radiotherapy, targeted therapy, and / or cancer immunotherapy.

24. An antibody or antigen-binding moiety according to any one of claims 1 to 12, for use in the treatment or prevention of B7H3-related disorders in a subject.

25. The use of an antibody or antigen-binding portion thereof according to any one of claims 1 to 12 in the manufacture of a pharmaceutical product for diagnosing, treating or preventing a disorder associated with B7H3 in a subject.

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

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