Antibodies targeting cd276 antigen and other modulators of cd276 antigen and uses thereof

Abstract

The present invention relates to antibodies or other antigen binding proteins targeting the CD276 antigen also known as B7-H3. The present invention provides improved antibody collections that bind to novel positions within the CD276 antigen and are particularly useful as therapeutic agents in the treatment of CD276 positive cancers. Furthermore, the present invention provides antibody conjugates and bispecific antibodies developed based on the novel anti-CD276 antibodies of the present invention. Furthermore, the present invention discloses the therapeutic use of antibodies and other modulators in the treatment of CD276 positive cancers. Finally, nucleic acid constructs encoding the molecules of the present invention, recombinant cells expressing them, and specific uses and methods are provided.

Description

[0001] This application is a divisional application of Chinese patent application No. 202080081062.3, filed on November 18, 2020, entitled "Antibody targeting CD276 antigen and other modulators of CD276 antigen and their uses". Technical Field

[0002] This invention relates to antibodies or other antigen-binding proteins targeting the CD276 antigen (also known as B7-H3). The invention provides an improved collection of antibodies that bind at novel sites within the CD276 antigen and are specifically used as therapeutic agents in the treatment of CD276-positive cancers. Furthermore, the invention provides antibody-drug conjugates and bispecific antibodies developed based on novel anti-CD276 antibodies of the invention. Additionally, the invention discloses the therapeutic use of antibodies and other modulators in the treatment of CD276-positive cancers. Finally, nucleic acid constructs encoding the molecules of the invention, recombinant cells expressing them, and specific uses and methods are provided. Background Technology

[0003] Humanized monoclonal antibodies (mAbs) such as rituximab and Herceptin, used to treat B-cell lymphoma and Her2 / neu-positive breast cancer, respectively, have greatly improved treatment options for these disease entities. At the heart of the therapeutic activity of these mAbs is their ability to stimulate effector cells carrying the Fc receptor (FcR), which leads to target cell lysis (“antibody-dependent cell cytotoxicity”, ADCC). To increase efficacy, promising strategies include efforts to enhance the ability of a given antitumor mAb to stimulate FcR-mediated ADCC by modulating the amino acid sequence, for example, by using SDIE to modify the glycosylation pattern of the 1 or CH2 domain[1]. Another approach to improve the recruitment of NK and other potential effector cells is to fuse antibodies against cytokines such as IL2 or IL-15. These immunocytokines have shown promising activity against pediatric neuroblastoma[2] and are currently being evaluated in clinical trials in patients with malignant melanoma. A new class of immunocytokines containing a modified IL-15 moiety has recently been described, conferring target-cell-limited activity (MIC protein)[3].

[0004] The most promising approach to optimizing antitumor antibodies appears to be antibody-mediated T cell stimulation, which has a higher effector potential compared to NK cells[4]. Indeed, antibody-based strategies that allow T cell recruitment of anticancer cells have achieved remarkable success: (i) monoclonal antibodies (mAbs) that block inhibitory “checkpoint” molecules (such as CTLA4 or PD-1 / PD-L1) on T cells have induced long-lasting remissions even in patients with high tumor burden. However, durable responses have been achieved only in a small number of patients, and the side effects caused by off-target activation of T cells are quite significant[5]. (ii) T cells transfected with chimeric receptor (CAR T cells) containing antibody portions targeting B-cell-associated CD19 molecules and CD3-associated signaling domains have eliminated large numbers of malignant cells in patients with B-cell-derived malignancies [6, 7] and (iii) Bonatumab is a bispecific antibody (bsAb) with CD19xCD3 specificity, in the form of a so-called bispecific T cell connector (BiTE), which received breakthrough recognition in B-cell-derived acute lymphoblastic leukemia in 2014 [8-10].

[0005] However, in initial clinical studies, the activity of CAR T cells and bispecific antibodies against solid tumors appeared to be minimal [11-12], likely due to limited pathways for T cells to reach the solid tumor site. Reports indicate that adequate influx of effector cells is a critical prerequisite for most immunotherapeutic strategies against solid tumors, suggesting that even large numbers of tumor-specific T cells cannot exert sufficient antitumor activity unless a pro-inflammatory environment is created at the tumor site [13, 14]. Therefore, optimal target antigens are expressed not only on tumor cells but also on tumor blood vessels to allow adequate influx of immune cells into the damaged vascular endothelium and subsequently destroy tumor cells.

[0006] CD276 belongs to the immunoglobulin class. CD276, also known as B7-H3 or B7RP-2, is a 77 kDa glycosylated class 1 transmembrane protein involved in the immune stimulation signaling cascade. It is part of the B7 molecular family and consists of an N-terminal signal peptide, extracellular V- and C-Ig-like domains, a transmembrane region, and a 45-amino acid-long C-terminus. The B7 family and its receptor CD28 family are responsible for the binding of antigens to T cells. This occurs through active co-stimulation and passive co-inhibition. B7-H3 was first described in 2001 by Chapoval, AI et al. (Nat Immunol, 2001. 2(3): pp. 269-74) as a co-stimulatory molecule involved in T cell responses and IFN-γ production. In this case, B7-H3 acts as an inhibitor of T cell stimulation while simultaneously stimulating the production of effector cytokines such as IL-4 and IFN-γ (Prasad, DV et al., J Immunol, pp. 2500-6). In addition, it is involved in stimulating primary CD8 cytotoxic T cells (CTLs), which have been shown to reduce tumor growth rates. Two isotypes are known: human and mouse, which differ in the number of V- and C-Ig-like domains. The human protein (4 Ig B7H3) consists of 4 tandem Ig-like domains, while the mouse protein consists of only 2 domains (2 Ig B7H3). Unlike other members of the B7 family, CD276 (B7-H3) RNA is present in almost all human tissues. However, protein translation is tightly regulated by microRNAs, leading to overexpression in many human cancers such as prostate cancer, non-small cell lung cancer, gastric cancer, neuroblastoma, and ovarian cancer

[25] . Studies in prostate cancer have shown that CD276 expression is associated with tumor spread, tumor recurrence, and overall survival. CD276 is one of the very few target antigens that exhibit this dual expression on tumor and vascular cells, and its own immunohistochemical studies have confirmed that expression is quite specific for malignant tumors compared to normal tissues. In addition to dual expression, CD276 is also known as an immune checkpoint molecule that inhibits T cell function. More information about CD276 is available from the UniProt database, November 2019 edition, accession number: Q5ZPR3. The amino acid sequences of four human isotypes are provided herein as SEQ ID NO: 41-44.

[0007] WO 2008 / 116219 discloses a monoclonal anti-CD276 antibody 8H9, which binds to the antigen in the 4G domain and induces antibody-dependent cell cytotoxicity (ADCC).

[0008] In summary, there is a need to allow for other therapeutic options that effectively target tumor masses, which can be achieved through tumor-associated antigens present on the blood vessels of the targeted tumor. Therefore, this invention seeks to identify antibodies that can be used to treat cancer. Summary of the Invention

[0009] Generally, the main aspects of the present invention can be described in a brief manner as follows:

[0010] In a first aspect, the present invention relates to isolated antigen-binding proteins (ABPs) that specifically bind to B7-H3 protein or variants thereof, wherein the isolated ABPs are capable of inducing antibody-dependent cell-mediated cytotoxicity against cells expressing B7-H3.

[0011] In a second aspect, the present invention relates to a bispecific antigen-binding protein (ABP) comprising a first antigen-binding domain capable of binding to the B7-H3 antigen or a variant thereof and a second antigen-binding domain capable of binding to the human differentiation cluster 3 (CD3) antigen.

[0012] In a third aspect, the present invention relates to isolated nucleic acids comprising encoding any of the ABPs in the first aspect, or antigen-binding fragments or monomers of the ABPs, such as heavy or light chains, or sequences encoding bispecific ABPs according to the second aspect.

[0013] In a fourth aspect, the present invention relates to nucleic acid constructs (NACs) comprising the nucleic acid of the third aspect and one or more additional sequence features, said sequence features allowing the expression of the encoded ABP or bispecific ABP, or a component of said ABP or bispecific ABP (e.g., antibody heavy or light chain) in cells.

[0014] In a fifth aspect, the present invention relates to a recombinant host cell comprising a nucleic acid molecule or NAC according to the third or fourth aspect.

[0015] In a sixth aspect, the present invention relates to a pharmaceutical composition comprising: (i) an ABP or bispecific ABP of the first or second aspect, or (ii) a nucleic acid or NAC of the third or fourth aspect, or (iii) a recombinant host cell and a pharmaceutically acceptable carrier, stabilizer and / or excipient according to the fifth aspect.

[0016] In a seventh aspect, the present invention relates to a component used as a pharmaceutical, wherein the component is selected from the following list: (i) ABP or bispecific ABP of the first or second aspect, or (ii) nucleic acid or NAC of the third or fourth aspect, or (iii) recombinant host cell according to the fifth aspect and (iv) pharmaceutical composition according to the sixth aspect.

[0017] In an eighth aspect, the present invention relates to a method for enhancing a cell-mediated immune response against human cells expressing human B7-H3, the method comprising, in the presence of immune cells such as T cells or natural killer (NK) cells, contacting the cells with (i) an ABP or bispecific ABP according to the first or second aspect, or (ii) a nucleic acid or NAC according to the third or fourth aspect, or (iii) a recombinant host cell according to the fifth aspect and (iv) a pharmaceutical composition according to the sixth aspect, thereby enhancing a cell-mediated immune response against the human cells, preferably cytotoxic.

[0018] In a ninth aspect, the present invention relates to a method for preventing and / or treating proliferative diseases in a subject, the method comprising administering to the subject a therapeutically effective amount of the component described in the seventh aspect; and wherein the proliferative disease is characterized by the expression of B7-H3 in cells associated with the proliferative disease. Detailed Implementation

[0019] The elements of the invention will be described below. These elements are listed with particular embodiments; however, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The embodiments and preferred embodiments described differently should not be construed as limiting the invention to only the explicitly described embodiments. This description should be understood to support and cover embodiments that combine two or more explicitly described embodiments or combine one or more explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, unless the context otherwise indicates, it should be considered that any permutation and combination of all the descriptive elements disclosed in this application are disclosed in the description of this application.

[0020] This invention provides peptides and proteins in some aspects and embodiments that comprise an antigen-binding domain of an anti-CD276 antibody as disclosed herein. The peptides and proteins advantageously and specifically recognize and bind to CD276 (also known as B7-H3) with high affinity. The peptides and proteins advantageously and specifically recognize and bind soluble CD276, and also specifically recognize and bind CD276 expressed on the cell surface. CD276 is expressed or overexpressed in a variety of human tumors, including pediatric solid tumors and adult cancers. Examples of cancers expressing or overexpressing CD276 include, but are not limited to, neuroblastoma, Ewing's sarcoma, rhabdomyosarcoma, and prostate cancer, ovarian cancer, colorectal cancer, and lung cancer. CD276 is also expressed in tumor angiogenesis and is a marker of tumor endothelial cells. Without being bound by any particular theory or mechanism, it is believed that by specifically recognizing and binding to CD276, the peptides and proteins of this invention can advantageously target CD276-expressing cancer cells and / or tumor angiogenesis. In embodiments of this invention, the peptides and proteins of this invention can elicit an antigen-specific response to CD276. Therefore, without being bound by specific theories or mechanisms, it is believed that by specifically recognizing and binding to CD276, the peptides and proteins of the present invention can provide one or more of the following: detecting cancer cells and / or tumor blood vessels expressing CD276, targeting and disrupting cancer cells and / or tumor blood vessels expressing CD276, reducing or eliminating cancer cells and / or tumor blood vessels, promoting the penetration of immune cells and / or effector molecules into tumor sites and / or tumor blood vessels, and enhancing / expanding anticancer and / or antitumor vascular responses.

[0021] In a first aspect, the present invention relates to isolated antigen-binding proteins (ABPs) that specifically bind to B7-H3 protein or variants thereof, wherein the isolated ABPs are capable of inducing antibody-dependent cell-mediated cytotoxicity against cells expressing B7-H3.

[0022] Antigen-binding protein targeting CD276

[0023] As used herein, “antigen-binding protein” (“ABP”) refers to a protein that specifically binds to a target antigen, such as a protein that binds to one or more epitopes displayed or present on the target antigen. The antigen of the ABP of this invention is CD276 or its orthologs (or paralogs) or other variants; and the ABP may optionally bind one or more domains of said CD276 or variants (e.g., an epitope may be displayed or present thereon by one or more extracellular domains of said CD276 or variants). Typically, antigen-binding proteins are antibodies (or fragments thereof), but other forms of antigen-binding proteins are also contemplated in this invention. For example, the ABP may be another (non-antibody) receptor protein derived from a small, robust non-immunoglobulin “scaffold,” such as those equipped with binding functional groups using combinatorial protein design methods (Gebauer & Skerra, 2009; Curr Opin Chem Biol, 13:245).Specific examples of such non-antibody ABPs include: affinity molecules based on the Z domain of protein A (Nygren, 2008; FEBS J 275:2668); Affilin based on γ-B crystals and / or ubiquitin (Ebersbach et al., 2007; J Mo Biol, 372:172); Affimer based on serum cystatin (Johnson et al., 2012; Anal Chem 84:6553); Affitin based on Sac7d from *Sacchariformis suis* (Krehenbrink et al., 2008; J Mol Biol 383:1058); Alphabody based on a triple-helix coil (Desmet et al., 2014; Nature Comms 5:5237); Antiticalin based on lipocalcinin (Skerra, 2008; FEBS J 275:2677); and Avimer based on the A domain of various membrane receptors. (Silverman et al., 2005; NatBiotechnol 23:1556); DARPins based on ankyrin repeat motifs (Strumpp et al., 2008; DrugDiscov Today, 13:695); Fynomer based on the SH3 domain of Fyn (Grabulovski et al., 2007; JBiol Chem 282:3196); Kunitz domain peptides based on the Kunitz domain of various protease inhibitors (Nixon et al., Curr opin Drug Discov Devel, 9:261); and Centyrin and Monobody based on the type III domain of fibronectin (Diem et al., 2014; Protein Eng Des Sel 27:419 doi: 10.1093 / protein / gzu016; Koide & Koide, 2007; Methods Mol Biol 352:95). In the context of the ABP of this invention, ABP specifically binds to human CD276, preferably isotype 1.

[0024] The term "epitope" includes any epitope that can be bound by antigen-binding proteins such as antibodies. An epitope is a region of an antigen that is bound by an antigen-binding protein targeting the antigen, and when the antigen is a protein, it includes (e.g., by an antigen-binding protein of the protein) a specific amino acid that binds to the antigen-binding protein. Epitope epitopes can include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and can have specific three-dimensional structural features and / or specific charge features. Typically, antigen-binding proteins specific to a particular target antigen will preferentially recognize epitopes on the target antigen in complex mixtures of proteins and / or macromolecules.

[0025] According to the present invention, preferred epitopes that can be targeted by antibodies and their variants include those epitopes located at any of the following amino acid positions in human CD276. These positions are the amino acids involved in the protein-protein interaction between the ABP of the present invention and the human CD276 protein. Therefore, an epitope is a region having at least 8 and preferably no more than 20 consecutive amino acids of the human CD276 amino acid sequence provided herein, and includes the following positions:

[0026] Epitopes in the IgC domain of human CD276 according to the present invention include epitopes including Q179. Epitopes in the IgV domain of human CD276 include any one, any two, or all of A115, G43, and / or F120. Other epitopes include R127 or G130.

[0027] An antigen-binding protein is "specific" when it binds to one antigen (e.g., CD276; such as human CD276, its orthologs, and other variants) more preferentially (e.g., more strongly or more broadly) than to a second antigen. In the context of ABP, the term "specific binding" (or "specifically binding," etc.) as used herein means that the ABP will preferentially bind to the desired antigen (e.g., CD276, particularly the IgC domain of CD276) rather than to other proteins (or other molecules), such as to CD276, compared to one or more other immunoglobulin (Ig) superfamily genes. Therefore, preferably, the binding affinity of ABP to an antigen (e.g., CD276) is at least 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10000, 105, or even at least 106 times, with the most preferred value being at least 2, compared to the binding affinity of ABP to other targets (e.g., unrelated proteins, such as mouse or human Fc domains, or streptavidin).

[0028] The terms "IgV domain" (or "Ig-like V domain") and "IgC domain" (or "Ig-like C domain") used in this article refer broadly to domains of members of the Ig superfamily. These domains correspond to structural units with a unique folding pattern called Ig-like folds. Ig-like folds consist of a sandwich of two antiparallel β-chains, with a conserved disulfide bond between the two layers in most, but not all, domains. The IgC domains of Ig, TCR, and MHC molecules share the same sequence pattern, referred to as C1 set domains in the Ig superfamily. Other IgC domains belong to the IgC2 set domains. IgV domains also share a sequence pattern, referred to as V set domains.

[0029] As used in this article, "orthologous" refers to a variant that originates from the same ancestral gene but exists in another organism due to a speciation event. Orthologous CD276 is generally expected to retain the same function as human CD276 (or have the same function as humans).

[0030] The term “paralog” as used in this article refers to a variant of a gene that originates from the same ancestral gene through a replication event in the same organism. Paralogs of CD276 are generally expected to be immunoglobulin superfamily proteins, particularly those with at least 70%, 80%, 85%, or 90% sequence identity with CD276 (if any such paralog exists in humans).

[0031] In the context of proteins, the term "variant" as used herein refers to any natural or non-natural form of such a protein that, compared to a reference protein, contains one or more amino acid mutations but has significant amino acid sequence identity with the reference protein, for example, at least 70% or 75% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, and most preferably at least 95%, 96%, 97%, 98%, or 99% amino acid sequence identity. Preferably, the variant of the protein has and / or retains at least one function / activity that is the same as, substantially the same as, or similar to that of the reference protein. Variants of CD276 can include orthologs of human CD276 and natural variants. Variants of CD276 can also correspond to human CD276 having one or more amino acid residues inserted into or deleted from the amino acid sequence, such as those CD276 variants naturally present in the population or those obtained through genetic manipulation, such as specifically engineering amino acid changes into one or more domains (e.g., extracellular domains) of the variant. Variants of CD276 include fusion proteins of CD276 (e.g., human CD276 fused to a heterologous polypeptide chain such as an Fc immunoglobulin domain or tag), and / or CD276 conjugated to another chemical motif such as an effector group or a labeling group. In some embodiments, variants of CD276 comprise fragments of CD276, such as polypeptides consisting of one or more IgC or IgV domains (or regions or subdomains thereof) of CD276 without one or any other (or any other) domains.

[0032] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more amino acid molecules, determined by alignment and comparison of sequences. "Identity percentage" represents the percentage of identical residues among amino acids or nucleotides in the compared molecules, calculated based on the size of the smallest molecule being compared. For these calculations, the gaps in the alignment (if any) are preferably resolved by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or peptides include those described in *Computational Molecular Biology* (edited by Lesk, AM), 1988, New York: Oxford University Press; *Biocomputing Informatics and Genome Projects* (edited by Smith, DW), 1993, New York: Academic Press; *Computer Analysis of Sequence Data, Part I* (eds. by Griffin, AM and Griffin, HG), 1994, New Jersey: Humana Press; von Heinje, G., 1987, *Sequence Analysis in Molecular Biology*, New York: Academic Press; *Sequence Analysis Primer* (edited by Gribskov, M. and Devereux, J.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0033] When calculating the percentage of identity, the sequences being compared are typically aligned in a way that yields the best match between the sequences. An example of a computer program that can be used to determine the percentage of identity is the GCG package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI). The computer algorithm GAP is used to align two polypeptides or polynucleotides whose sequence identity percentage is to be determined. The sequences are aligned to achieve the best match of their respective amino acids or nucleotides (“match span”, determined by the algorithm). A vacancy opening penalty (which is calculated as 3 times the average diagonal, where the “average diagonal” is the average of the diagonals of the comparison matrix used; the “diagonal” is a fraction or number assigned to each perfect amino acid match by a specific comparison matrix) and a vacancy expansion penalty (which is typically 1 / 10 of the vacancy opening penalty), along with comparison matrices such as PAM 250 or BLOSUM 62, are used in conjunction with the algorithm.

[0034] The standard comparison matrix (see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919 for the BLOSUM 62 comparison matrix) can also be used by the algorithm.

[0035] Examples of parameters that can be used to determine the percentage of identity between peptide or nucleotide sequences using the GAP procedure are as follows: (i) Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453; (ii) Comparison matrix: BLOSUM62, from Henikoff et al., 1992, supra; (iii) Vacancy penalty: 12 (but no penalty for end gaps); (iv) Vacancy length penalty: 4; (v) Similarity threshold: 0.

[0036] The preferred method for determining the similarity between a protein or nucleic acid and human CD276, its paralogs, orthologs, or other variants is the method provided by Blast search supported by Uniprot (e.g., http: / / www.uniprot.org / uniprot / Q5ZPR3); particularly for amino acid identity, those using the following parameters: program: blastp; matrix: blosum62; threshold: 10; filtered: false; empty: true; maximum reported hits: 250.

[0037] Some alignment protocols used to align two amino acid sequences may result in only a small region of matching between the two sequences, and this small aligned region may have very high sequence identity, even if there is no obvious relationship between the two full-length sequences. Therefore, if needed, the chosen alignment method (GAP procedure) can be adjusted to produce alignments spanning at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, or other numbers of consecutive amino acids across the target polypeptide or its region.

[0038] In a particular embodiment of the invention, CD276 is human CD276, preferably a protein comprising an amino acid sequence selected from SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44 (especially SEQ ID NO. 41), or a protein having no more than two, four, six, eight or ten amino acid substitutions, insertions or deletions compared to these sequences, such as no more than one, two or three.

[0039] In the context of variants of CD276, the present invention includes those embodiments in which variants of CD276 comprise a protein having an amino acid sequence having at least 80%, 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO: 41 (in particular, at least 92% or 95% sequence identity).

[0040] The ABP of the present invention includes one or more complementary determination regions.

[0041] In a particular embodiment, the ABP of the present invention may preferably include at least one complementarity-determining region (CDR), such as a complementarity-determining region from an antibody (particularly from a human antibody), and in a particular embodiment, the ABP may include an amino acid sequence having at least 80%, 85%, 90% or 95% sequence identity (preferably at least 90%) compared to the CDR sequence shown in Table 1 herein, or having no more than three or two, preferably no more than one, amino acid substitution, deletion or insertion CDR.

[0042] The term “complementarity-determining region” (or “CDR” or “hypervariant region”) used in this article refers to one or more hypervariable or complementarity-determining regions (CDRs) present in the variable regions of the light or heavy chains of an antibody. See, for example: “IMGT”, Lefranc et al., 20003, Dev Comp Immunol 27:55; Honegger & Plückthun, 2001, J Mol Biol 309:657; Abhinandan & Martin, 2008, Mol Immunol 45:3832; Kabat et al. (1987): Sequences of Proteins of Immunological Interest National Institutes of Health, Bethesda, Md. These expressions include hypervariable regions as defined by Kabat et al. (1983), Sequences of Proteins of Immunological Interest, US Dept of Health and Human Services, or hypervariable loops in the three-dimensional structure of the antibody (Chothia and Lesk, 1987; J Mol Biol 196:901). CDRs in each chain are closely packed together via framework regions and, together with CDRs in the other chain, contribute to the formation of antigen-binding sites. Within the CDRs are selected amino acids that have been described as selection-determining regions (SDRs), representing key contact residues used by the CDR in antibody-antigen interactions (Kashmiri, 2005; Methods 36:25).

[0043] As described above, in certain embodiments of the present invention, the ABP may include at least one complementarity-determining region (CDR). In some such embodiments, the ABP of the present invention includes at least one complementarity-determining region 3 (CDR3), for example, having at least 80%, 85%, 90%, or 95% (preferably at least 90%) sequence identity with an amino acid sequence selected from those heavy chain and light chain CDR3 sequences shown in Table 1 (e.g., sequences selected from SEQ ID Nos: 3, 7, 11, 15, 19, 23, 27, 31, 35, and 39), or having no more than three or two, preferably no more than one, amino acid substitution, deletion, or insertion complementarity-determining region 3.

[0044] Optionally, or in addition to the CDR3 sequence, the ABP of the present invention may contain at least one CDR1 and / or at least one CDR2 (e.g., CDR2 from an antibody, particularly from a human antibody). Preferably, the ABP of the present invention contains at least one such CDR3, as well as at least one such CDR1 and at least one such CDR2, more preferably wherein each such CDR has at least 80%, 85%, 90%, or 95% (preferably at least 90%) amino acid sequence identity compared to the sequences of the corresponding (heavy chain or light chain) CDR1, CDR2, and CDR3 selected from Table 1, or has no more than three or two, preferably no more than one, amino acid substitution, deletion, or insertion.

[0045] In a particular embodiment, the ABP of the present invention may be an antibody or its antigen-binding fragment.

[0046] As used herein, the term "antibody" can be understood in the broadest sense as any immunoglobulin (Ig) capable of binding to its epitope. An antibody like this is a type of ABP. A full-length "antibody" or "immunoglobulin" is typically a heterotetrameric glycoprotein of about 150 kDa, composed of two identical light chains and two identical heavy chains. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has uniformly spaced intrachain disulfide bonds. Each heavy chain has an amino-terminal variable domain (VH) followed by three carboxyl-terminal constant domains (CH). Each light chain has a variable N-terminal domain (VL) and a single C-terminal constant domain (CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to cells or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Other forms of antibodies include heavy chain antibodies, which consist of only two heavy chains and lack the two light chains normally present in antibodies. Heavy chain antibodies include hcIgG (IgG-like) antibodies of camelids such as dromedary camels, camels, llamas, and alpacas, and IgNAR antibodies of cartilaginous fish (e.g., sharks). Other forms of antibodies include single-domain antibodies (sdAbs, which developer Ablynx calls Nanobody), which are antibody fragments composed of a single monomeric variable antibody domain. Single-domain antibodies are typically produced from heavy chain antibodies but can also be derived from conventional antibodies.

[0047] Antibodies (or fragments thereof) may include, for example, chimeric, humanized, (fully) human, or hybrid antibodies with dual or multiple antigen or epitope specificity, antibody fragments and antibody subfractions, such as Fab, Fab', or F(ab')2 fragments, single-chain antibodies (scFv), etc. (described below), including hybrid fragments of any immunoglobulin or hybrid fragments of any natural, synthetic, or genetically engineered protein that act like antibodies by binding to a specific antigen to form a complex.

[0048] Therefore, in some embodiments, the ABP of the present invention may comprise an antibody heavy chain or an antigen-binding fragment thereof, and / or an antibody light chain or an antigen-binding fragment thereof.

[0049] In other embodiments, the ABP of the present invention may comprise a variable region of the antibody heavy chain or an antigen-binding fragment thereof, and / or a variable region of the antibody light chain or an antigen-binding fragment thereof, and in other embodiments, the ABP of the present invention may comprise variable regions CDR1, CDR2 and CDR3 of the antibody heavy chain, and / or variable regions CDR1, CDR2 and CDR3 of the antibody light chain.

[0050] In a specific embodiment of the invention, when the ABP comprises an antibody heavy chain sequence and / or an antibody light chain sequence, or an antigen-binding fragment thereof; compared to the CDR3 sequences selected from those heavy chain CDR3 sequences shown in Table 1 (e.g., sequences selected from SEQ ID No: 3, 11, 19, 27 and 35), the antibody heavy chain sequence or fragment thereof may contain at least 80%, 85%, 90%; or 95% (preferably at least 90%) sequence identity, or have no more than three or two, preferably no more than one amino acid substitution, deletion or insertion CDR3, and / or wherein compared to the CDR3 sequences selected from those light chain CDR3 sequences shown in Table 1 (e.g., sequences selected from SEQ ID No: 7, 15, 23, 31 and 39), the antibody light chain sequence or fragment thereof may contain at least 80%, 85%, 90%; or 95% (preferably at least 90%) sequence identity, or have no more than three or two, preferably no more than one amino acid substitution, deletion or insertion CDR3.

[0051] In other embodiments of the invention, when the ABP comprises an antibody heavy chain or an antigen-binding fragment thereof, the antibody heavy chain sequence or fragment thereof may further comprise having at least 80%, 85%, or 90%; or 95% (preferably at least 90%) sequence identity, or having no more than three or two, preferably no more than one, amino acid substitution, deletion, or insertion, compared to sequences selected from SEQ ID No: 1, 9, 17, 25, and 33; and / or having at least 80%, 85%, or 90%; or 95% (preferably at least 90%) sequence identity, or having no more than three or two, preferably no more than one, amino acid substitution, deletion, or insertion, compared to sequences selected from SEQ ID No: 2, 10, 18, 26, and 34 (e.g., the CDR2 sequence disclosed in Table 1).

[0052] In other embodiments of the invention, the ABP of the invention comprises an antibody light chain or an antigen-binding fragment thereof, wherein the antibody light chain sequence or fragment thereof further comprises having at least 80%, 85%, or 90%; or 95% (preferably at least 90%) sequence identity, or having no more than three or two, preferably no more than one amino acid substitution, deletion, or insertion, compared to sequences selected from SEQ ID Nos: 5, 13, 21, 29, and 37; and / or having at least 80%, 85%, or 90%; or 95% (preferably at least 90%) sequence identity, or having no more than three or two, preferably no more than one amino acid substitution, deletion, or insertion, compared to sequences selected from SEQ ID Nos: 6, 14, 22, 30, and 38 (e.g., the light chain CDR2 sequence disclosed in Table 1).

[0053] In other embodiments of the invention, the ABP of the present invention may contain an antibody variable chain sequence having at least 80%, 85%, 90%; or 95% (preferably at least 90%) sequence identity, compared to sequences selected from SEQ ID NO. 4, 8, 12, 16, 20, 24, 28, 32, 36 and 40 (e.g., the VH or VL sequences disclosed in Table 1).

[0054] In a specific embodiment of the invention, the ABP of the invention comprises an antigen-binding fragment of an antibody, wherein the antigen-binding fragment comprises CDR1, DR2, and CDR3. In some such embodiments, CDR1 is selected from those disclosed in Table 1, CDR2 is selected from those disclosed in Table 1, and CDR3 is selected from those disclosed in Table 1 (e.g., CDR1, DR2, and CDR3 are selected from CDR1, CDR2, and CDR3 sequences having amino acid sequences of SEQ ID No. 1, 2, 3 or 5, 6, 7 or 9, 10, 11 or 13, 14, 15 or 17, 18, 19 or 21, 22, 23 or 25, 26, 27 or 29, 30, 31 or 33, 34, 35 or 37, 38, 39, respectively); and in each case, independently, optionally having no more than three or two, preferably no more than one, amino acid substitution, insertion, or deletion compared to these sequences.

[0055] In other specific embodiments of the present invention, the ABP of the present invention may comprise antibody heavy chain variable regions CDR1, DR2, and CDR3, and / or antibody light chain variable regions CDR1, CDR2, and CDR3, wherein CDR1 has the amino acid sequence of heavy chain or light chain CDR1 shown in Table 1 (e.g., having an amino acid sequence selected from SEQ ID No. 1, 5, 9, 13, 17, 21, 25, 29, 33, and 37), and wherein CDR2 has the amino acid sequence of heavy chain or light chain CDR2 shown in Table 1 (e.g., having an amino acid sequence selected from SEQ ID No. 2, 6, 10, 14, 18, 22, 26, 30, 34, and 38), and wherein CDR3 has the amino acid sequence of heavy chain or light chain CDR3 shown in Table 1 (e.g., having an amino acid sequence selected from SEQ ID No. 1, 5, 9, 13, 17, 21, 25, 29, 33, and 37). (Amino acid sequences of 3, 7, 11, 15, 19, 23, 27, 31, 35 and 39); in each case independently, optionally having no more than three or two, preferably no more than one, amino acid substitution, insertion or deletion compared to these sequences.

[0056] In this preferred embodiment, the ABP can be an antibody or its antigen-binding fragment, comprising at least one, preferably two, antibody heavy chain sequences and at least one, preferably at least two antibody light chain sequences, wherein the at least one, preferably two, antibody heavy chain sequences and at least one, preferably two, antibody light chain sequences contain CDR1 to CDR3 sequences selected from any combination of heavy chain CDRs shown in Table A and / or any combination of light chain CDRs shown in Table A; in each case, independently, optionally, it has no more than three or two, preferably no more than one, amino acid substitution, insertion, or deletion compared to these sequences. Particularly preferred are antibodies with the heavy chain and light chain sequence combinations shown in the first row of Table A below.

[0057] Table A: Preferred combinations of heavy chain CDRs and light chain CDRs

[0058]

[0059] In a preferred embodiment, the present invention relates to an isolated antigen-binding protein (ABP) that specifically binds to CD276 protein or a variant thereof, wherein the isolated ABP is capable of inducing antibody-dependent cell-mediated cytotoxicity in cells expressing CD276 protein; and the ABP has at least one antigen-binding domain, wherein:

[0060] (A) comprising the antibody heavy chain CDR1 sequence shown in SEQ ID NO: 1, the antibody heavy chain CDR2 sequence shown in SEQ ID NO: 2, and the antibody heavy chain CDR3 sequence shown in SEQ ID NO: 3; and the antibody light chain CDR1 sequence shown in SEQ ID NO: 5, the antibody light chain CDR2 sequence shown in SEQ ID NO: 6, and the antibody light chain CDR3 sequence shown in SEQ ID NO: 7; and independently, optionally having no more than three or two, preferably no more than one, amino acid substitution, insertion, or deletion compared to these sequences [such ABP is or is derived from an antibody internally named 7C4 disclosed herein]; or

[0061] (B) comprising the antibody heavy chain CDR1 sequence shown in SEQ ID NO: 9, the antibody heavy chain CDR2 sequence shown in SEQ ID NO: 10, and the antibody heavy chain CDR3 sequence shown in SEQ ID NO: 11; and the antibody light chain CDR1 sequence shown in SEQ ID NO: 13, the antibody light chain CDR2 sequence shown in SEQ ID NO: 14, and the antibody light chain CDR3 sequence shown in SEQ ID NO: 15; and independently, optionally having no more than three or two, preferably no more than one, amino acid substitution, insertion, or deletion compared to these sequences [such ABP is or is derived from an antibody internally named 11A7 disclosed herein]; or

[0062] (C) comprising the antibody heavy chain CDR1 sequence shown in SEQ ID NO: 17, the antibody heavy chain CDR2 sequence shown in SEQ ID NO: 18, and the antibody heavy chain CDR3 sequence shown in SEQ ID NO: 19; and the antibody light chain CDR1 sequence shown in SEQ ID NO: 21, the antibody light chain CDR2 sequence shown in SEQ ID NO: 22, and the antibody light chain CDR3 sequence shown in SEQ ID NO: 23; and independently, optionally having no more than three or two, preferably no more than one, amino acid substitution, insertion, or deletion compared to these sequences [such ABP is or is derived from an antibody internally named 8D9 disclosed herein]; or

[0063] (D) comprising the antibody heavy chain CDR1 sequence shown in SEQ ID NO: 25, the antibody heavy chain CDR2 sequence shown in SEQ ID NO: 26, and the antibody heavy chain CDR3 sequence shown in SEQ ID NO: 27; and the antibody light chain CDR1 sequence shown in SEQ ID NO: 29, the antibody light chain CDR2 sequence shown in SEQ ID NO: 30, and the antibody light chain CDR3 sequence shown in SEQ ID NO: 31; and independently, optionally having no more than three or two, preferably no more than one, amino acid substitution, insertion, or deletion compared to these sequences [such ABP is or is derived from an antibody internally named 10A7 disclosed herein]; or

[0064] (E) comprising the antibody heavy chain CDR1 sequence shown in SEQ ID NO: 33, the antibody heavy chain CDR2 sequence shown in SEQ ID NO: 34, and the antibody heavy chain CDR3 sequence shown in SEQ ID NO: 35; and the antibody light chain CDR1 sequence shown in SEQ ID NO: 36, the antibody light chain CDR2 sequence shown in SEQ ID NO: 37, and the antibody light chain CDR3 sequence shown in SEQ ID NO: 38; and in each case, independently, optionally having no more than three or two, preferably no more than one, amino acid substitution, insertion, or deletion compared to these sequences [such ABP is or is derived from an antibody internally named 8H8 disclosed herein].

[0065] In other preferred embodiments of the invention, the ABP may be an antibody or its antigen-binding fragment, comprising at least one, preferably at least two, antibody heavy chain sequences and at least one, preferably at least two, antibody light chain sequences, wherein, independently in each case, each antibody heavy chain sequence and antibody light chain sequence comprises a variable region sequence from the heavy chain and light chain variable domain combinations shown in Table B, optionally having no more than ten, nine, eight, seven, six, five, four, preferably no more than three, two, or one amino acid substitution, insertion, or deletion compared to these sequences. Particularly preferred are antibodies with the heavy chain and light chain sequence combinations shown in the first row of Table B below.

[0066] Table B: Preferred combinations of variable domains for heavy and light chains

[0067]

[0068] In all preferred embodiments of the ABP of the present invention, the ABP is isolated and / or substantially pure.

[0069] In the context of proteins such as ABPs (examples of which may be antibodies), the term "isolated" as used herein refers to a protein purified from a protein or polypeptide or other contaminants that may interfere with its therapeutic, diagnostic, preventative, research, or other uses. The isolated ABP according to the invention may be recombinant, synthetic, or modified (non-natural) ABP. In the context of nucleic acids or cells, the term "isolated" as used herein refers to nucleic acids or cells purified from DNA, RNA, proteins or polypeptides or other contaminants (e.g., other cells) that may interfere with their therapeutic, diagnostic, preventative, research, or other uses, or it refers to recombinant, synthetic, or modified (non-natural) nucleic acids. Preferably, the isolated ABP or nucleic acid or cell is substantially pure. In this context, a "recombinant" protein or nucleic acid is a protein or nucleic acid produced using recombinant technology. Methods and techniques for producing recombinant nucleic acids and proteins are known in the art.

[0070] In the context of proteins such as ABPs (examples of which may be antibodies), the term "isolated" as used herein refers to a protein purified from a protein or polypeptide or other contaminants that may interfere with its therapeutic, diagnostic, preventative, research, or other uses. The isolated ABP according to the invention may be recombinant, synthetic, or modified (non-natural) ABP. In the context of nucleic acids or cells, the term "isolated" as used herein refers to nucleic acids or cells purified from DNA, RNA, proteins or polypeptides or other contaminants (e.g., other cells) that may interfere with their therapeutic, diagnostic, preventative, research, or other uses, or it refers to recombinant, synthetic, or modified (non-natural) nucleic acids. Preferably, the isolated ABP or nucleic acid or cell is substantially pure. In this context, a "recombinant" protein or nucleic acid is a protein or nucleic acid produced using recombinant technology. Methods and techniques for producing recombinant nucleic acids and proteins are known in the art.

[0071] In some embodiments, the ABP of the present invention may (e.g., one or more epitopes represented by one or more domains of CD276) be combined with CD276 or its paralogs, orthologs, or other variants (e.g., any CD276 or variants described herein), wherein the KD is less than 20 nM, for example less than about 10 nM, 5 nM, or 2 nM (particularly less than about 1 nM). In a preferred embodiment, the ABP of the present invention will be combined with CD276 (e.g., the epitope of said CD276) or a variant thereof, wherein the KD is less than 100 pM. In a preferred embodiment, the ABP of the present invention will be combined with said CD276 or a variant thereof, wherein the KD is about (+ / - 5) 10 pM. In some embodiments, the binding of the ABP of the present invention, such as the antibody of the present invention, to human cell lines expressing said CD276 or its variants can occur at an EC50 of less than about 10 µg / mL, 5 µg / mL, 2 µg / mL, 1 µg / mL, 0.5 µg / mL, or 0.2 µg / mL, preferably less than 2 µg / mL. In some embodiments, the binding of the ABP of the present invention, such as the antibody of the present invention, to rhesus monkey cell lines expressing orthologs or variants of said CD276 can occur at an EC50 of less than about 10 µg / mL, 5 µg / mL, 2 µg / mL, 1 µg / mL, 0.5 µg / mL, 0.2 µg / mL, 0.1 µg / mL, less than 50 ng / mL, or less than 25 ng / mL, preferably less than 10 ng / mL, more preferably less than 5 ng / mL, and most preferably about 4 ng / mL.

[0072] In other embodiments, the ABP of the present invention may: (i) bind to CD276 or a variant of CD276, wherein the KD is less than 20 nM, for example less than about 10 nM, 5 nM or 2 nM (in particular, less than about 1 nM), less than 100 pM, or about 10 pM; and / or bind to human cell lines expressing CD276 or a variant of CD276, wherein the EC50 is less than 10 ng / mL.

[0073] As used herein, the term "KD" refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods established in the art, such as plasmonic resonance (BIAcore®), ELISA, and KINEXA. A preferred method for determining the KD of an antibody is by using surface plasmonic resonance, preferably using a biosensor such as the BIAcore® system or by ELISA. As used herein, "Ka" (or "K-assoc") refers generally to the binding rate of a specific antibody-antigen interaction, while the term "Kd" (or "K-diss") refers to the dissociation rate of a specific antibody-antigen interaction.

[0074] In one embodiment, the ABP of the present invention is a polyclonal antibody (mixture), or the antigen-binding fragment is a fragment of a polyclonal antibody (mixture).

[0075] In another preferred embodiment of all ABPs of the present invention, the ABP is an antibody or an antigen-binding fragment thereof, the antibody is a monoclonal antibody, or the antigen-binding fragment is a fragment of a monoclonal antibody.

[0076] As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody derived from a group of antibodies with substantially identical amino acid sequences. Monoclonal antibodies are typically highly specific. Furthermore, unlike conventional (polyclonal) antibody formulations, which often comprise different antibodies targeting different determinants (e.g., epitopes), each mAb typically targets a single determinant on an antigen. In addition to their specificity, mAbs have the advantage of being synthesized from cell cultures (hybridomas, recombinant cells, etc.) uncontaminated with other immunoglobulins. The mAbs used in this article include, for example, chimeric, humanized, or human antibodies or antibody fragments.

[0077] The monoclonal antibodies according to the invention can be prepared by methods well known to those skilled in the art. For example, mice, rats, or rabbits can be immunized with a target antigen and an adjuvant. Spleen cells are collected from animals that have received multiple immunizations at intervals as pools, and test bleeding is performed to assess serum antibody titers. Spleen cells are prepared for immediate use in fusion experiments or stored in liquid nitrogen for future fusions. Fusion experiments are then performed according to the procedure in Stewart & Fuller, J. Immunol. Methods 1989, 123:45-53. mAb secretors are screened from the supernatant of wells containing growing hybrids by, for example, enzyme-linked immunosorbent assay (ELISA). Clones of ELISA-positive cultures are sorted by limiting dilution or fluorescence-activated cell sorting, typically producing hybridomas established from single colonies. The ability of an antibody (including antibody fragments or subfractions) to bind a specific antigen can be determined by binding assays known in the art, for example, using the target antigen as a binding chaperone.

[0078] In other preferred embodiments, the ABP of the present invention is an antibody or an antigen-binding fragment thereof, wherein the antibody is a human antibody, a humanized antibody or a chimeric-human antibody, or wherein the antigen-binding fragment is a fragment of a human antibody, a humanized antibody or a chimeric-human antibody.

[0079] Human antibodies can also be obtained via in vitro methods. Suitable examples include, but are not limited to, phage display (CAT, Morphosys, Dyax, Biosite / Medarex, Xoma, Yumab, Symptomogen, Alexion, Affimed, etc.). In phage display, a polynucleotide encoding a single Fab or Fv antibody fragment is expressed on the surface of the phage particle (see, for example, Hoogenboom et al., J. Mol. Biol., 227: 381 (1991); Marks et al., J Mol. Biol 222: 581 (1991); U.S. Patent No. 5,885,793). Phages are “screened” to identify those antibody fragments that have affinity for the target. Thus, some such processes mimic immune selection by displaying a library of antibody fragments on the surface of filamentous phages and then selecting phages by their binding to the target. In some of these processes, high-affinity functional neutralizing antibody fragments are isolated. Therefore, a complete human antibody gene library can be established by cloning naturally rearranged human V genes from peripheral blood lymphocytes (see, for example, Mullinax et al., Proc Natl Acad Sci (USA), 87: 8095-8099 (1990)) or by generating fully synthetic or semi-synthetic phage display libraries with human antibody sequences (see Knappik et al., 2000; J Mol Biol 296:57; de Kruif et al., 1995; J Mol Biol 248:97).

[0080] Alternatively, the antibodies described herein can be prepared using XenoMouse® technology. These mice are capable of producing human immunoglobulin molecules and antibodies, but are deficient in producing mouse immunoglobulin molecules and antibodies. In particular, preferred embodiments for the transgenic production of mice and antibodies are disclosed in U.S. Patent Application Serial No. 08 / 759620, filed December 3, 1996, and International Patent Application No. WO 98 / 24893, published June 11, 1998, and International Patent Application No. WO 00 / 76310, published December 21, 2000. See also Mendez et al., Nature Genetics, 15:146-156 (1997). Using this technology, fully human monoclonal antibodies against a variety of antigens have been produced. Essentially, XenoMouse® lines of mice are immunized with a target antigen (e.g., CD276), lymphocytes (e.g., B-cells) are recovered from hyperimmunized mice, and the recovered lymphocytes are fused with myeloid cell lines to prepare immortalized hybridoma cell lines. These hybridoma cell lines are screened and selected to identify those that produce antibodies specific to the target antigen. Other "humanized" mice are also commercially available, such as Medarex-HuMab mice, Kymab-Kymouse, Regeneron-Velocimmune mice, Kirin-TC mice, Trianni-Trianni mice, OmniAb-OmniMouse, Harbour Antibodies-H2L2 mice, and Merus-MeMo mice. Other "humanized" species are also available: rats: OmniAb-OmniRat, OMT-UniRat; chickens: OmniAb-OmniChicken.

[0081] According to the present invention, the term "humanized antibody" refers to an immunoglobulin chain or fragment thereof (e.g., Fab, Fab', F(ab')2, Fv, or other antigen-binding sequence of the antibody) containing a minimal sequence derived from a non-human immunoglobulin (but usually still at least partially derived from a non-human immunoglobulin). For the most part, humanized antibodies are human immunoglobulins (receptor antibodies), wherein the CDR residues of the receptor antibody are replaced by CDR residues from a non-human immunoglobulin (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and ability. Thus, at least a portion of the frame sequence of the antibody or fragment thereof may be a human common frame sequence. In some cases, the Fv frame residues of the human immunoglobulin need to be replaced by corresponding non-human residues to increase specificity or affinity. Furthermore, humanized antibodies may contain residues not found in either the receptor antibody or the input CDR or frame sequence. These modifications are made to further improve and maximize antibody performance. Typically, humanized antibodies will contain substantially all of at least two variable domains, wherein all or substantially all of the CDR regions correspond to those of non-human immunoglobulins, and all or substantially all of the frame regions are those of human immunoglobulin common sequences. Humanized antibodies preferably also contain at least a portion of an immunoglobulin constant region, typically a constant region of a human immunoglobulin, which can be modified (e.g., by mutation or glycoengineering) to optimize one or more properties of that region and / or improve (e.g., the function of a therapeutic antibody), such as increasing or decreasing Fc effector function or increasing serum half-life. Exemplary such Fc modifications (e.g., Fc engineering or Fc enhancement) are described elsewhere herein.

[0082] According to the invention, the term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain genes have been established, said light chain and / or heavy chain genes typically being genetically engineered to consist of variable and constant regions of immunoglobulins that are identical or homologous to corresponding sequences in different species, such as mice or humans. Alternatively, the variable region gene is derived from a particular antibody species or subclass, while the remainder of the chain is derived from another antibody species or subclass, either of the same or different species. It also includes fragments of such antibodies. For example, a typical therapeutic chimeric antibody is a heterozygous protein consisting of a variable domain or antigen-binding domain from a mouse antibody and a constant domain or effector domain from a human antibody, although other mammalian species may also be used.

[0083] In a particular embodiment of this invention, the ABP comprises an antigen-binding domain of an antibody, wherein the antigen-binding domain is of a human antibody. Preferably, the ABP comprises an antigen-binding domain of an antibody or an antigen-binding fragment thereof, which is a human antigen-binding domain; (ii) the antibody is a monoclonal antibody, and wherein the antigen-binding fragment is a fragment of a monoclonal antibody; and (iii) the antibody is a human antibody or a humanized antibody, or wherein the antigen-binding fragment is a fragment of a human antibody, a humanized antibody, or a chimeric-human antibody.

[0084] Human antibodies typically consist of two light chains: a κ light chain and a λ light chain, each containing a variable region and a constant region. Heavy chains are typically divided into μ, δ, γ, α, or ε chains, which define antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. Human IgG has multiple isotypes, including but not limited to IgG1, IgG2, IgG3, and IgG4. Human IgM isotypes include IgM and IgM2. Human IgA isotypes include IgA1 and IgA2. In humans, the IgA and IgD isotypes contain four heavy chains and four light chains; the IgG and IgE isotypes contain two heavy chains and two light chains; and the IgM isotype contains ten or twelve heavy chains and ten or twelve light chains. The antibodies according to the present invention can be IgG, IgE, IgD, IgA, or IgM immunoglobulins.

[0085] In some embodiments, the ABP of the present invention is an IgG antibody or a fragment thereof. In some embodiments, the ABP of the present invention is an IgE antibody or a fragment thereof. In some embodiments, the ABP of the present invention is an IgD antibody or a fragment thereof. In some embodiments, the ABP of the present invention is an IgA antibody or a fragment thereof. In some embodiments, the ABP of the present invention is an IgM antibody or a fragment thereof. Preferably, the ABP of the present invention is, comprises, or is derived from IgG immunoglobulin or a fragment thereof; for example, human, human-derived IgG immunoglobulin, or rabbit or rat-derived IgG, and / or IgG2 immunoglobulin or a fragment thereof. When the ABP of the present invention is, comprises, or is derived from rat-derived IgG, then preferably, the ABP is, comprises, or is derived from rat IgG2a or IgG2b immunoglobulin. When the ABP of the present invention is, comprises, or is derived from human-derived IgG, then preferably, the ABP of the present invention is, comprises, or is derived from human IgG1, IgG2, or IgG4, and most preferably, the ABP of the present invention is, comprises, or is derived from human IgG1 or IgG2.

[0086] Therefore, in a specific embodiment of the present invention, ABP is an antibody, wherein the antibody is an IgG, IgE, IgD, IgA or IgM immunoglobulin; preferably an IgG immunoglobulin.

[0087] The ABP of the present invention, which contains at least a portion of the immunoglobulin constant region (typically the human immunoglobulin constant region), can modify such (e.g., human) immunoglobulin constant region, for example by glycoengineering or mutation, to optimize one or more properties of the region and / or improve the function of (e.g., therapeutic) antibodies, such as increasing or decreasing Fc effector function or increasing serum half-life.

[0088] The ABPs of this invention, particularly those used in this method, include antibodies that induce antibody-dependent cytotoxicity (ADCC) in cells expressing CD276. ADCC of anti-CD276 antibodies can be improved by using antibodies with low levels of fucose or lacking fucose. Fucose-deficient antibodies are associated with enhanced ADCC (antibody-dependent cytotoxicity) activity, particularly at low doses (Shields et al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al., 2003, J. Biol. Chem. 278:3466).

[0089] Methods for preparing fucose-free antibodies or antibodies with reduced fucose levels involve growth in rat myeloma YB2 / 0 cells (ATCC CRL 1662). YB2 / 0 cells express low levels of FUT8 mRNA, which encodes an enzyme (α,1,6-fucotransferase) required for the fucosylation of a polypeptide.

[0090] Alternatively, during the expression of such an antibody, inhibitors targeting enzymes involved in glycan modification can be used, including: tunicamycin, which selectively inhibits the formation of GlcNAc-PP-Dol (GlcNAc-PP-Dol is the first step in the formation of a core oligosaccharide, which is a precursor to an N-glycosidic linked glycan chain); kifunensine and von Willebrand extract as inhibitors of glycosidase I; bromocondulitol as an inhibitor of glycosidase II; 1-deoxynojirimycin and 1,4-dioxy-1,4-imino-D-mannitol as inhibitors of glycosidase I; squalene as an inhibitor of glycosidase II; and so on. Examples of glycosyltransferase-specific inhibitors include deoxy derivatives of substrates that are anti-N-acetylglucosamine transferase V (GnTV). It is also known that 1-deoxynojirimycin inhibits the synthesis of complex sugar chains and increases the ratio of high-mannose and heterozygous sugar chains (Glycobiology series 2 - Destiny of Sugar Chain in Cell, edited by Katsutaka Nagai, Senichiro Hakomori and Akira Kobata, 1993).

[0091] Based on these data, several cell lines have been genetically engineered to produce antibodies with or without low levels of fucose (Mori et al., 2004; Yamane-Ohnuki et al., 2004), enabling the engineering of glycosylation patterns of IgG to select therapeutic monoclonal antibodies that exhibit specific features of Fc-γ-R binding, which can be used for a variety of conditions.

[0092] Umana et al. and Davis et al. stated that engineered lgG1 antibodies containing increased amounts of bimeric complex oligosaccharides (bimeric A / -acetylglucosamine, GlcNAC) allowed for stronger ADCC compared to their parental counterparts (Umana et al., 1999; Davies et al., 2001). Secondly, the absence of fucose on the N-linked oligosaccharide in human lgG1 has been shown to improve FCGRIII binding and ADCC.

[0093] GLYCART BIOTECHNOLOGY AG (Zurich, CH) has expressed N-acetyl-glucosamine transferase III (GnTIII), which catalyzes the addition of bipartite GlcNac residues to N-linked oligosaccharides in the Chinese hamster ovary (CHO) cell line and has shown that the resulting lgG1 antibody has a larger ADCC (WO 99 / 54342; WO 03 / 011878; WO 2005 / 044859).

[0094] WO20070166306 relates to the modification of an antibody against CD19 containing 60% N-acetylglucosamine dimeric oligosaccharides and 10% unfucosylated N-acetylglucosamine dimeric oligosaccharides, said dimeric oligosaccharides being produced in mammalian human 293T embryonic kidney cells transfected with (i) cDNA of an anti-CD19 antibody and (ii) cDNA of a GnTIII enzyme.

[0095] Compared to the same lgG1 produced in wild-type CHO cells, recombinant human lgG1 produced in YB2 / 0 cells (Shinkawa et al., 2003; Siberil et al., 2006) or in CHO-Lec13 (Shields et al., 2002) exhibited enhanced ability to induce cytotoxicity, showing low or no fucose content. Conversely, no correlation was observed between galactose and ADCC, and the content of bimeric GlcNAC had only a slight effect on ADCC (Shinkawa et al., 2003).

[0096] By removing or replacing the fucose in the Fc moiety of antibodies, KYOWA HAKKO KOGYO (Tokyo, Japan) has enhanced Fc binding and improved ADCC, thereby increasing the potency of MAbs (US 6946292). This improved Fc-γ-RIIIA-dependent effector function of low-fucosylated IgG shows no dependence on the Fc-γ-RIII allelic form (Niwa et al., 2005). Moreover, it has recently been shown that a lower antigen density is required to induce effective ADCC when IgG has a lower fucose content compared to high-fucosylated IgG.

[0097] Laboratoire Frangais du Fractionnement et des (LFB) (France) indicated that the ratio of Fuc / Gal in MAb oligosaccharides should be equal to or less than 0.6 to obtain antibodies with high ADCC (FR 2861080).

[0098] Cardarelli et al., 2019, generated anti-CD19 antibodies in Ms-704PF CHO cells lacking the FUT8 gene, which encodes α1,6-fucosyltransferase. In this study, defucosylation of the antibodies requires engineering of enzyme-deficient cell lines. Amino acid mutations are not considered in this study.

[0099] Herbst et al. produced a humanized lgG1 MAb, MEDI-551, expressed in the fucosyltransferase-deficient producer CHO cell line. Amino acid mutations were not considered in this study (Herbst et al., 2010). S. Siberil et al. used the rat myeloma YB2 / 0 cell line to produce an anti-RhD MAb with low fucosyl content. The MAb produced in wild-type CHO cells exhibited a high fucosyl content (81%), while the same MAb produced in YB2 / 0 cells showed a lower fucosyl content (32%). Amino acid mutations were not considered in this study (Siberil et al., 2006).

[0100] Therefore, the ABP of the present invention can be prepared and / or can have one or more of the features of the above-described glycoengineering (e.g., defucosylation) method / antibody.

[0101] Alternative methods to increase the ADDC activity of the ABP of the present invention include mutations in the Fc portion of such ABP, particularly mutations that increase antibody affinity for the Fc-γ-R receptor.

[0102] Therefore, any ABP of the present invention described above can be produced using different isotypes or mutant isotypes to control the degree of binding to different Fc-γ-R receptors. Antibodies lacking the Fc region (such as the Fab fragment) lack binding to different Fc-γ receptors. The choice of isotype also affects binding to different Fc-γ receptors. The affinity of various human IgG isotypes for three different Fc-γ receptors, Fc-γ-RI, Fc-γ-RII, and Fc-γ-RIII, has been determined (see Ravetch & Kinet, Annu. Rev. Immunol. 9, 457 (1991)). Fc-γ-RI is a high-affinity receptor that binds to IgG in monomeric form, while the latter two are low-affinity receptors that bind to IgG only in polymeric form. Typically, IgG1 and IgG3 exhibit significant binding activity to all three receptors, IgG4 shows significant binding activity to Fc-γ-RIs, and IgG2 exhibits significant binding activity only to one Fc-γ-RII (called IIaLR) (see Parren et al., J. Immunol. 148, 695 (1992). Therefore, human isotype IgG1 is usually chosen because a stronger binding to Fc-γ receptors is required, while IgG2 is usually chosen because of its weaker binding.

[0103] The association between increased Fc-γ-R binding and mutant Fc has been confirmed using targeted cytotoxic cell assays (Shields et al., 2001, J. Biol. Chem. 276:6591-6604; Presta et al., 2002, BiochemSoc. Trans. 30:487-490). Methods to increase ADCC activity through specific Fc region mutations include Fc variants selected from those containing at least one amino acid substitution at the following positions: 234, 235, 239, 240, 241, 243, 244, 245, 247, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313, 325, 327, 328, 329, 330, and 332, wherein the residues in the Fc region are numbered according to Kabat's EU index (Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987).

[0104] In certain specific embodiments, the Fc variant includes at least one substitution selected from the following: L234D, L234E, L234N, L234Q, L234T, L234H, L234Y, L234I, L234V, L234F, L235D, L235S, L235N, L235Q, L235T, L235H, L235Y, L235I, L235V, L235F, S239D, S239E, S239N, S239Q, S239F, S239T, S239H, S239Y, V240I, V240A, V240T, V240M, F241W , F241L, F241Y, F241E, F241R, F243W, F243L, F243Y, F243R, F243Q, P244H, P245A, P247V, P247G, V262I, V262A, V262T, V262E, V263I, V263A . D265H, D265T, V266I, V266A, V266T, V266M, S267Q, S267L, E269H, E269Y, E269F, E269R, Y296E, Y296Q, Y296D, Y296N, Y296S, Y296T, Y296L, Y296I, Y296H, N297S, N297D, N297E, A298H, T299I, T299L, T299A, T299S, T299V, T299H, T299F, T299E, W313F, N325Q, N325L, N325I, N325D, N 325E, N325A, N325T, N325V, N325H, A327N, A327L, L328M, L328D, L328E, L328N, L328Q, L328F, L328I, L328V, L328T, L328H, L328A, P329F, A330L, A330Y, A330V, A330I, A330F, A330R, A330H, I332D, I332E, I332N, I332Q, I332T, I332H, I332Y, and I332A, where the residues in the Fc region are numbered according to the EU index of Kabat.

[0105] The Fc variants can also be selected from V264L, V264I, F241W, F241L, F243W, F243L, F241L / F243L / V262I / V264I, F241W / F243W, F241W / F243W / V262A / V264A, F241L / V262I, F243L / V264I, F243L / V262I / V264W, F241Y / F243Y / V262T / V264T, F241E / F243R / V262E / V264R, F241E / F243Q / V262T / V264E, F241R / F243Q / V262T / V264R, F241E / F243Y / V262T / V264R, L328M, L328E, L328F, I332E, L3238M / I332E, P244H, P245A, P247V, W313F, P244H / P245A / P247V, P247G, V264I / I332E, F241E / F243R / V262E / V264R / I332E, F241E / F243Q / V262T / 264E / I332E, F241R / F243Q / V262T / V264R / I332E, F241E / F243Y / V262T / V264R / I332E, S298A / I332E, S239E / I332E, S239Q / I332E, S239E, D265G, D265N, S239E / D265G, S239E / D265N, S239E / D265Q, Y296E, Y296Q, T299I, A327N, S267Q / A327S, S267L / A327S, A327L, P329F, A330L, A330Y, I332D, N297S, N297D, N297S / I332E, N297D / I332E, N297E / I332E, D265Y / N297D / I332E, D265Y / N297D / T299L / I332E, D265F / N297E / I332E, L328I / I332E, L328Q / I332E, I332N, I332Q, V264T, V264F, V240I, V263I, V266I, T299A, T299S, T299V, N325Q, N325L, N325I, S239D, S239N, S239F, S239D / I332D, S239D / I332E, S239D / I332N, S239D / I332Q, S239E / I332D, S239E / I332N, S239E / I332Q, S239N / I332D, S239N / I332E, S239N / I332N,S239N / I332Q, S239Q / I332D, S239Q / I332N, S239Q / I332Q, Y296D, Y296N, F241Y / F243Y / V262T / V264T / N297D / I332E, A330Y / I332E, V264I / A330Y / I332 E, A330L / I332E, V264I / A330L / I332E, L234D, L234E, L234N, L234Q, L234T, L234H, L234Y, L234I, L234V, L234F, L235D, L235S, L235N, L235Q, L235T, L23 5H, L235Y, L235I, L235V, L235F, S239T, S239H, S239Y, V240A, V240T, V240M, V263A, V263T, V263M, V264M, V264Y, V266A, V266T, V266M, E269H, E269Y, E 269F, E269R, Y296S, Y296T, Y296L, Y296I, A298H, T299H, A330V, A330I, A330F, A330R, A330H, N325D, N325E, N325A, N325T, N325V, N325H, L328D / I332E L328E / I332E, L328N / I332E, L328Q / I332E, L328V / I332E, L328T / I332E, L328H / I332E, L328I / I332E, L328A, I332T, I332H, I332Y, I332A, S239E / V264 I / I332E, S239Q / V264I / I332E, S239E / V264I / A330Y / I332E, S239E / V264I / S298A / A330Y / I332E, S239D / N297D / I332E, S239E / N297D / I332E, S239D / D26 5V / N297D / I332E, S239D / D265I / N297D / I332E, S239D / D265L / N297D / I332E, S239D / D265F / N297D / I332E, S239D / D265Y / N297D / I332E, S239D / D265H / N 297D / I332E, S239D / D265T / N297D / I332E, V264E / N297D / I332E, Y296D / N297D / I332E, Y296E / N297D / I332E, Y296N / N297D / I332E, Y296Q / N297D / I332EY296H / N297D / I332E, Y296T / N297D / I332E, N297D / T299V / I332E, N297D / T299I / I332E, N297D / T299L / I332E, N297D / T299F / I332E、N297D / T299H / I332E、N297D / T299E / I332E、N297D / A330Y / I332E、N297D / S298A / A330Y / I332E、S239D / A330Y / I332 E, S239N / A330Y / I332E, S239D / A330L / I332E, S239N / A330L / I332E, V264I / S298A / I332E, S239D / S298A / I332E, S239N / S298A / I332E, S239D / V264I / I332E, S239D / V264I / S298A / I332E, and S239D / 264I / A330L / I332E, where the residues in the Fc region are numbered according to the EU index of Kabat. See also WO2004029207, which is incorporated herein by reference.

[0106] In certain embodiments, mutations can be made at, near, or adjacent to sites in the hinge region in all isotypes (e.g., replacing residues 234, 235, 236, and / or 237 with another residue) to reduce affinity for Fc-γ receptors, particularly Fc-γ-RI receptors (see, for example, US6624821). Optionally, positions 234, 236, and / or 237 are replaced with alanine, and position 235 is replaced with glutamic acid (see, for example, US5624821). Position 236 is deleted in the human IgG2 isotype. Exemplary fragments of amino acids 234, 235, and 237 of human IgG2 are Ala Ala Gly, Val Ala Ala, AlaAla Ala, Val Glu Ala, and Ala Glu Ala. Preferred combinations of mutants are L234A, L235E, and G237A, or preferred combinations of human isotype IgG1 are L234A, L235A, and G237A. A particularly preferred ABP of the present invention is an antibody having one of these three mutations in human isotype IgG and the Fc region. Other substitutions that reduce binding to the Fc-γ receptor are the E233P mutation (particularly in mouse IgG1) and D265A (particularly in mouse IgG2a). Other examples of mutations and combinations of mutations that reduce Fc and / or C1q binding are E318A / K320A / R322A (particularly in mouse IgG1) and L235A / E318A / K320A / K322A (particularly in mouse IgG2a). Similarly, residue 241 (Ser) in human IgG4 can be substituted with, for example, proline to interrupt Fc binding.

[0107] Additional mutations can be made in the constant region to modulate effector activity. For example, mutations can be made in the IgG1 or IgG2a constant region at A330S, P331S, or both. For IgG4, mutations can be made at E233P, F234V, and L235A (with G236 deletion), or any combination thereof. IgG4 can also have one or both of the following mutations: S228P and L235E. The use of interrupted constant region sequences to modulate effector function is further described, for example, in WO2006118959 and WO2006036291.

[0108] Additional mutations can be made in the constant region of human IgG to modulate effector activity (see, for example, WO200603291). These include the following substitutions for human IgG1: (i) A327G, A330S, P331S; (ii) E233P, L234V, L235A, G236 deletion; (iii) E233P, L234V, L235A; (iv) E233P, L234V, L235A, G236 deletion, A327G, A330S, P331S; and (v) E233P, L234V, L235A, A327G, A330S, P331S; or in particular, (vi) L234A, L235E, G237A, A330S, and P331S (e.g., for human IgG1), wherein the residue numbers in the Fc region are the numbers of the Kabat EU index. See also WO2004029207, which is incorporated herein by reference.

[0109] The affinity of antibodies against FcRs can be altered by mutating residues in certain heavy chain constant regions. For example, disrupting glycosylation sites on human IgG1 can reduce FcR binding, thereby decreasing the effector function of the antibody (see, for example, WO2006036291). The tripeptide sequences NXS and NXT (where X is any amino acid except proline) are enzyme recognition sites for N-residue glycosylation. Disruption of any tripeptide amino acid, particularly in the CH2 region of IgG, will prevent glycosylation at this site. For example, the N297 mutation in human IgG1 prevents glycosylation and reduces FcR binding to antibodies.

[0110] A preferred enhancement of Fc receptor binding can be achieved by introducing a mutant of the Fc domain of human IgG1 (often referred to as “SDIE” in this paper, which represents the mutation S239D / I332E).

[0111] While activation of ADCC and CDC is generally desirable for therapeutic antibodies, in some cases, the ABP of the present invention, which cannot activate effector functions, is preferred (e.g., the ABP of the present invention with an unknown modulator). For these purposes, IgG4 is typically used, but it has become less popular in recent years due to the unique ability of this subclass to perform Fab arm exchange, where heavy chains can be exchanged between IgG4s in vivo. Therefore, Fc engineering methods can also be used to identify key interaction sites between the Fc domain and the Fc-γ receptor and C1q, and then mutate these sites, for example, in the Fc of the ABP of the present invention, to reduce or eliminate binding. By alanine scanning, Duncan and Winter (1998; Nature 332:738) first isolated the binding site of C1q with the region covering the hinge and upper CH2 of the Fc domain. Researchers at Genmab identified mutants K322A, L234A, and L235A, whose combination was sufficient to almost completely eliminate the binding of Fc-γ-R and C1q (Hezareh et al., 2001; J Virol 75:12161). Similarly, MedImmune later identified a set of three mutations, L234F / L235E / P331S (designated TM), which had very similar effects (Oganesyan et al., 2008; Acta Crystallographica 64:700). Another approach is glycosylation modification on the asparagine 297 of the Fc domain, which is known to be required for optimal FcR interaction. Loss of binding to FcRs has been observed in the N297 point mutation (Tao et al., 1989; J Immunol 143:2595), enzymatically deglycosylated Fc domains (Mimura et al., 2001; J Biol Chem 276:45539), recombinant expression of antibodies in the presence of glycosylation inhibitors (Walker et al., 1989; Biochem J 259:347), and expression of Fc domains in bacteria (Mazor et al., 2007; Nat Biotechnol 25:563). Therefore, this invention also includes embodiments of ABP in which such techniques or mutations have been used to reduce effector function.

[0112] Due to FcRn-mediated recycling, IgG naturally persists for a long time in serum (such as human serum), thus its typical half-life is approximately 21 days. Nevertheless, much effort has been made to design pH-dependent interactions between the Fc domain and FcRn to enhance affinity at pH 6.0 while maintaining minimal binding at pH 7.4. Researchers at PDL BioPharma discovered the mutant T250Q / M428L, which resulted in an approximately 2-fold increase in the IgG half-life in rhesus monkeys (Hinto et al., 2004; J Biol Chem 279:6213), and researchers at MedImmune have discovered the mutant M252Y / S254T / T256E (referred to as YTE), which resulted in an approximately 4-fold increase in the IgG half-life in cynomolgus monkeys (Dall'Acqua et al., 2006; J Biol Chem 281:23514). The ABP of this invention can also be polyethylene glycol-modified. PEGylation, i.e., chemical coupling with the synthetic polymer polyethylene glycol (PEG), has emerged as a recognized technique for developing biological agents with prolonged action, with approximately 10 clinically approved protein and peptide drugs to date (Jevsevar et al., 2010; Biotechnol J 5:113). The ABP of this invention can also be modified with PAS protein, a biological alternative to PEGylation for prolonging the plasma half-life of active pharmaceutical proteins (Schlapschy et al., 2013; Protein Eng Des Sel 26:489; XL-protein GmbH, Germany). Therefore, this invention also includes embodiments in which this technique or mutation has been used to prolong the plasma half-life, particularly of ABP in human plasma.

[0113] Antibody fragments include “Fab fragments,” which consist of a constant domain and a variable domain for each heavy and light chain, bound together by an adjacent constant region of the light chain and the first constant domain (CH1) of the heavy chain. These can be formed from conventional antibodies by protease digestion (e.g., with papain), but similar Fab fragments can also be prepared through genetic engineering. Fab fragments include Fab', Fab, and “Fab-SH” (which is a Fab fragment containing at least one free thiol group).

[0114] The difference between Fab' fragments and Fab fragments is that they contain additional residues at the carboxyl terminus of the first constant domain of the heavy chain, including one or more cysteine ​​residues from the antibody hinge region. Fab' fragments include "Fab'-SH" (which is a Fab' fragment containing at least one free thiol group).

[0115] Furthermore, the antibody fragment includes an F(ab')2 fragment containing two light chains and two heavy chains. The heavy chains contain a constant region (“hinge region”) between the CH1 and CH2 domains, allowing interchain disulfide bonds to form between the two heavy chains. Therefore, the F(ab')2 fragment consists of two Fab' fragments linked together by disulfide bonds between the two heavy chains. The F(ab')2 fragment can be prepared by conventional antibody cleavage using an enzyme that performs cleavage below the hinge region (e.g., pepsin) or through genetic engineering.

[0116] The “Fv region” contains variable regions from the heavy and light chains, but lacks a constant region. A “single-chain antibody” or “scFv” is an Fv molecule in which the variable regions of the heavy and light chains are linked by a flexible linker to form a single polypeptide chain that forms the antigen-binding region.

[0117] The “Fc region” contains two heavy chain segments, each containing the antibody’s CH2 and CH3 domains. These two heavy chain segments are linked together by two or more disulfide bonds and by hydrophobic interactions through the CH3 domain.

[0118] Therefore, in some embodiments, the ABP of the present invention is selected from the following antibody fragments: Fab', Fab, Fab'-SH, Fab-SH, Fv, scFv and F(ab')2.

[0119] In embodiments of ABPs that are immunoglobulin fragments such as antibody fragments, it is preferred to be those fragments capable of binding to the extracellular domain (e.g., an epitope shown), such as any epitope or other binding features described herein, of CD276 or its paralogs, orthologs or other variants; and more preferably, the fragment is a regulator (e.g., an inhibitor or antagonist) of the expression, function, activity and / or ability of CD276 or its paralogs, orthologs or other variants.

[0120] In a preferred embodiment, the ABP of the present invention is an antibody, wherein at least a portion of the frame sequence of the antibody or a fragment thereof is a human common frame sequence, for example, a frame sequence containing human germline encoded.

[0121] In some embodiments, the ABP of the present invention is modified or engineered to increase antibody-dependent cell cytotoxicity (ADCC). As those skilled in the art will now understand, such an ABP of the present invention has particular efficacy in treating diseases or conditions associated with cellular resistance to immune cells such as CTLs (e.g., CD276-positive cancers); because the ADCC mechanism (cell-mediated immune defense, in which effector cells of the immune system actively lyse target cells whose membrane surface antigens have been bound by specific antibodies) will enhance cells resistant to immune cells such as CTLs, thus leading to increased attachment and / or lysis of these cells by effector cells of the immune system.

[0122] As used in this article, “treatment” is synonymous with treating a disease, disorder, or symptom, including alleviating the symptoms of a disease, disorder, or symptom, inhibiting the development of a disease, disorder, or symptom, causing the disease, disorder, or symptom to subside, and / or curing a disease, disorder, or symptom.

[0123] Various techniques for modifying or engineering the ABP of the present invention to enhance ADCC are known (Satoh et al., 2006; Expert Opin Biol Ther 6:1161; WO2009 / 135181). Therefore, this embodiment includes embodiments in which the ABP of the present invention can be defucosylated (GlycArt Biotechnology), for example, wherein the antibody is produced in CHO cells with the endogenous FUT8 gene knocked out; or the ABP can be a “glycoengineered antibody” (SeattleGenetics), for example, wherein the addition of a fucose analogue to CHO cells expressing the antibody results in a significant reduction in fucosylation. Other defucosylation methods that can be applied to the ABP of the present invention are described elsewhere herein.

[0124] Other techniques for modifying or engineering the ABP of this invention to enhance ADCC include mutations in the Fc portion of the ABP (e.g., described in more detail elsewhere herein), particularly in the case of one or more such mutations in human Fc residues 234, 235, 236 and / or 237, and / or residues 330, 331; wherein such numbering of the Fc residues is the numbering of Kabat’s EU index (Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987).

[0125] Therefore, in some embodiments, the ABP of the present invention is modified or engineered to enhance antibody-dependent cell-mediated cytotoxicity (ADCC), preferably wherein the ABP is defucosylated and / or the Fc of the ABP is mutated (e.g., wherein the Fc is mutated using one or more of the following residue changes: L234A, L235E, G237A, A330S, and / or P331S). In alternative embodiments, the ABP of the present invention is modified or engineered to reduce antibody-dependent cell-mediated cytotoxicity (ADCC).

[0126] In certain other embodiments, the ABP of the present invention is modified to prolong its serum half-life, particularly in human serum. For example, the ABP of the present invention may be polyethylene glycol-modified and / or PAS protein-modified, or have an Fc region modified with T250Q / M428L or M252Y / S254T / T256E.

[0127] In some embodiments, the ABP of the present invention binds to one or more epitopes displayed by the extracellular domain of (a) a variant of CD276 or CD27611; or binds to two or more epitopes displayed by the extracellular domain of (b) a variant of CD276 or CD27611. Preferably, one or more said epitopes are displayed between amino acid residues 23 and 241 of SEQ ID NO: 371 (ECD of human CD276 protein), for example, between amino acid residues 23 and 136 of SEQ ID NO: 371 (Ig-like V-domain of human CD276 protein).

[0128] The ABP of the present invention can be monospecific (i.e., it has an antigen-binding domain that binds to only one antigen) or multispecific (i.e., it has two or more different antigen-binding domains that bind to different antigens). For example, “bispecific,” “dual-specific,” or “bifunctional” ABPs or antibodies are hybrid ABPs or antibodies having two different antigen-binding sites, respectively. A bispecific antigen-binding protein and antibody is a multispecific antigen-binding protein antibody and can be prepared by a variety of methods, including but not limited to hybridoma fusion or ligation of Fab' fragments (see, for example, Songsivilai and Lachmann, 1990; Kostelny et al., 1992). The two binding sites of the bispecific antigen-binding protein or antibody will bind to two different epitopes, which may be located on the same or different protein targets.

[0129] In some such implementations, the ABP can be a bispecific, trispecific, or tetraspecific antibody. In particular, the bispecific antibody is selected from: bispecific T-cell adaptor (BiTE) antibody, biparental heavy targeting molecule (DART), CrossMAb antibody, DutaMab™ antibody, DuoBody antibody; Triomab, TandAb, bispecific NanoBody, tandem scFv, diabody, single-chain diabody, HSA body, (scFv)2 HSA antibody, scFv-IgG antibody, Dock and Lock bispecific antibody, DVD-IgG antibody, TBTI DVD-IgG, IgG-fynomer, tetravalent bispecific tandem IgG antibody, dual-targeting domain antibody, chemically linked bispecific (Fab')2 molecule, cross-linked mAb, dual-effect Fab IgG (DAF-IgG), orthoFab-IgG, bispecific CovX-Body, bispecific hexavalent trimer, and ART-Ig.

[0130] Therefore, in some embodiments, the ABP of the present invention is a multispecific antibody comprising at least two antigen-binding domains, wherein each antigen-binding domain specifically binds to a different antigen epitope.

[0131] Therefore, in some embodiments, when expressed on the surface of mammalian cells, the ABP of the present invention binds (e.g., via one or more first antigen-binding domains) to the extracellular domains of CD276, paralogs, orthologs, and other variants, and further includes one or more additional antigen-binding domains that bind to antigens other than said CD276 or variants. In some embodiments of the ABP of the present invention, such additional antigens may be another immunoglobulin superfamily gene; and / or such additional antigens may be antigens present on mammalian T cells. Antigens present on mammalian T cells that can bind via such additional antigen-binding domains include CD3, CD40, OX-40, ICOS, and 4-1BB. In some embodiments of the ABP of the present invention, such additional antigens may also be albumin, such as human albumin. It may also be another component of blood or serum to which binding of the ABP would prolong the serum half-life of the ABP, for example, a half-life similar to that when bound to albumin.

[0132] Preferred variants of the ABP of the present invention relate to bispecific compounds comprising an antigen-binding region of the antibody of the present invention and a second antigen-binding region against CD3, such as the scFv construct known as UCHT1. A preferred construct of the present invention comprises two antigen-binding domains and two anti-CD3 binding domains of the 7C4 antibody specified herein, such as the UCHT1 scFv construct described above.

[0133] In other embodiments, the ABP of the present invention may include two or more antigen-binding regions, preferably two, three or four antigen-binding regions.

[0134] In other embodiments, the ABP of the present invention may comprise a chimeric antigen receptor (CAR), and preferably comprises an extracellular antigen-binding region, a membrane anchor such as a transmembrane domain, and an intracellular region such as an intracellular signaling region.

[0135] In a preferred embodiment, the ABP of the present invention may include at least one antibody constant domain, particularly wherein at least one antigen constant domain is a CH1, CH2 or CH3 domain or a combination thereof.

[0136] In other such embodiments, the ABP of the present invention, having an antibody constant domain, comprises a mutated Fc region, for example, to enhance the interaction of the Fc region with the Fc receptor (Fc receptor on immune effector cells) (e.g., Saxena & Wu, 2016; Front Immunol 7:580). Examples and embodiments thereof are described elsewhere herein.

[0137] In other embodiments, the ABP of the present invention may include effector groups and / or labeling groups.

[0138] The term "effect group" refers to any group, particularly one coupled to another molecule such as an antigen-binding protein, which is used as a cytotoxic agent. Examples of suitable effector groups are radioisotopes or radionuclides. Other suitable effector groups include toxins, therapeutic groups, or chemotherapeutic groups. Examples of suitable effector groups include calicheamicin, auristatin, geldanamycin, α-amanitine, pyrrolobenzodiazepine, and maytansine.

[0139] The term "tag" or "tag group" refers to any detectable tag. Typically, there are many types of tags, depending on the analysis used to detect them: a) isotopic tags, which can be radioactive or heavy isotopes; b) magnetic tags (e.g., magnetic powder); c) redox active moieties; d) fluorescent dyes; enzyme groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); e) biotinylated groups; and f) predetermined polypeptide epitopes recognized by secondary reporter sequences (e.g., leucine zipper pairs, binding sites of secondary antibodies, metal-binding domains, epitope tags, etc.).

[0140] In some implementations, the effector or labeling group binds to another molecule (e.g., ABP) via spacer arms of various lengths to reduce potential steric hindrance.

[0141] The preferred embodiments of the invention then relate to ABP modified to include immune cytokine functional groups. Interleukin-15 (IL-15) or modified IL-15 is preferred. However, the use of other immune cytokines in antibody fusion constructs is well documented, including antibody-cytokine fusion proteins containing IL-2, IL-12, IL-21, TNFα, and interferon α, β, and γ.

[0142] The preferred IL15 molecule used as the fusion protein for the ABP of the present invention is a molecule with reduced affinity for IL-15Rα compared to wild-type IL-15. The modified IL-15 polypeptide used in the present invention preferably contains at least one amino acid substitution at one or more positions of the amino acid sequence corresponding to human wild-type IL-15 (derived from UniProt, accession number P40933-1), specifically positions 92, 93, 94, 95, 96, 97, 98, 99, 100, 112, 113, 114, 115, and / or 116. Therefore, preferably, the ABP of the present invention may contain one or two antigen-binding domains disclosed herein and, for example, be fused to the antibody constant region, one or more of the above-described modified IL-15 polypeptides. Such a construct is disclosed in EP 3265478, which is incorporated herein by reference in its entirety.

[0143] In a second aspect, the present invention relates to a bispecific antigen-binding protein (ABP) comprising a first antigen-binding domain capable of binding to the CD276 (B7-H3) antigen or a variant thereof and a second antigen-binding domain capable of binding to the human differentiation cluster 3 (CD3) antigen.

[0144] A "bispecific" or "bifunctional" ABP is an ABP with two distinct epitope / antigen-binding domains (or "sites"), thus exhibiting binding specificity for two different target epitopes. These two epitopes can be epitopes of the same antigen or, as preferred in this invention, epitopes of different antigens such as the different antigens CD276 and CD3.

[0145] A “bispecific ABP” can be an ABP that binds an antigen or epitope to one of two or more binding arms defined by a first pair of heavy and light chains or a main chain and a shorter / smaller chain, and causes different antigens or epitopes to bind to a second arm defined by a second pair of heavy and light chains or a main chain and a smaller chain. Embodiments of such a bispecific ABP have two distinct antigen-binding arms in both the specificity and the CDR sequence. Typically, a bispecific ABP is monovalent for each antigen it binds to, meaning it binds to the corresponding antigen or epitope using only one arm. However, bispecific antibodies can also dimerize or multiply, which is preferred in the context of this invention. For example, in the form of a dimerized IgGsc as described herein, the antibody can have two binding sites for each antigen ( Figure 1B (Third-side construct). The bispecific antibody can be a hybrid ABP, which may have a first binding region defined by a first light chain variable region and a first heavy chain variable region, and a second binding region defined by a second light chain variable region and a second heavy chain variable region. The present invention envisions that one of these binding regions may be defined by a heavy chain / light chain pair. In the context of the present invention, a bispecific ABP may have a first binding site defined by variable regions of the main chain and the smaller chain, and a distinct second binding site defined by a variable region of an scFv fragment contained in the ABP main chain.

[0146] Methods for preparing bispecific ABPs are known in the art, such as the chemical conjugation of two different monoclonal antibodies, or the chemical conjugation of two antibody fragments, such as two Fab fragments. Alternatively, bispecific ABPs can be prepared via quadroma techniques, i.e., by fusing hybridomas that generate parental antibodies. Due to the random sorting of the H and L chains, a potential mixture of ten different antibody structures is generated, of which only one possesses the desired binding specificity.

[0147] The bispecific ABP of this invention can be used as a monoclonal antibody (mAb) targeting each target. In some embodiments, the antibody is chimeric, humanized, or fully human. The bispecific antibody can be, for example, a bispecific tandem single-chain Fv, a bispecific Fab2, or a bispecific diabody.

[0148] Based on the domains included in the ABP of this invention, the bispecific ABP of this invention may comprise a Fab fragment, which typically includes a hinge region, a CH2 domain, and a single-chain Fv fragment. This bispecific antibody is called a “Fabsc”-ABP and was first described in International Patent Application WO 2013 / 092001. More specifically, the “Fabsc” form of ABP used herein generally refers to the bispecific ABP of this invention having a Fab fragment, which typically includes a hinge region located at the C-terminus of the Fab fragment connected to the N-terminus of the CH2 domain, which in turn is connected to the N-terminus of the scFv fragment. This “Fabsc” does not contain or substantially does not contain a CH3 domain. In this context, “does not contain” or “substantially does not contain” means that the ABP does not contain a full-length CH3 domain. This preferably means that the ABP contains 10 or fewer, preferably 5 or fewer, preferably 3 or even fewer amino acids of the CH3 domain.

[0149] According to the disclosure by Coloma and Morrison (Nat Biotechnol 15:159-63, 1997), the bispecific ABP of the present invention may also have a CH3 domain, typically arranged at the C-terminus of the CH2 domain. This molecule is also referred to herein as an “IgGsc” form ABP, and denotes the bispecific ABP of the present invention having a Fab fragment, which typically includes a hinge region located at the C-terminus of the Fab fragment, typically connected to the N-terminus of the CH2 domain, which in turn typically connects to the N-terminus of the CH3 domain, which in turn typically connects to the N-terminus of the scFv fragment. Figure 1B An illustrative example of an IgGsc format ABP is shown. This bispecific ABP is preferred in the context of this invention.

[0150] Alternatively, the “Fabsc” or “IgGsc” ABP of the present invention may also have an “Fc-weakened” CH2 domain (including a hinge region). This “Fc-weakening” is achieved by deleting and / or substituting (mutating) at least one selected amino acid residue in the CH2 domain capable of mediating binding to the Fc receptor. In an illustrative embodiment, the at least one amino acid residue in the hinge region or CH2 domain capable of mediating binding to the Fc receptor and which is deleted or mutated is selected from sequence positions 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 265, 297, 327, and 330 (sequence positions numbered according to the EU index). In illustrative examples, this Fc-decreased ABP may contain at least one mutation selected from the following: deletion of amino acid 228, deletion of amino acid 229, deletion of amino acid 230, deletion of amino acid 231, deletion of amino acid 232, deletion of amino acid 233, substitution of Glu233→Pro, substitution of Leu234→Val, deletion of amino acid 234, substitution of Leu235→Ala, deletion of amino acid 235, deletion of amino acid 236, deletion of amino acid 237, deletion of amino acid 238, substitution of Asp265→Gly, substitution of Asn297→Gln, substitution of Ala327→Gln, and substitution of Ala330→Ser (sequence positions are numbered according to the EU-index, see, for example, Figures 10 and 1P of International Patent Application WO 2013 / 092001). In the case of bispecific antibodies that activate T cells (e.g., against tumor cells), Fc attenuation may be desirable to prevent the antibody from binding to cells carrying the Fc receptor, which could lead to undesirable off-target activation of T cells.

[0151] According to the disclosure by Coloma and Morrison (Nat Biotechnol 15:159-63, 1997), the most preferred IgGsc form of the bispecific ABP of the present invention may also have a CH3 domain, typically arranged at the C-terminus of the CH2 domain. This molecule is also referred to herein as the “IgGsc” form of ABP, and denotes the bispecific ABP of the present invention having a Fab fragment, which typically includes a hinge region located at the C-terminus of the Fab fragment, typically connected to the N-terminus of the CH2 domain, which in turn typically connects to the N-terminus of the CH3 domain, which in turn typically connects to the N-terminus of the scFv fragment. Figure 1B An illustrative example of an IgGsc format ABP is shown. This bispecific ABP is preferred in the context of this invention.

[0152] The common feature of the antibody forms Fabsc and IgGsc is that the N-terminal targeting portion is composed of either a "physiological" Fab- or Fab2 region, thereby avoiding the use of a single-chain portion in that part of the molecule. If these forms are used for target-cell-restricted T-cell activation, Fc receptor (FcR) binding attenuation can be employed (if desired or necessary) to prevent FcR-mediated activation. This can be achieved, for example, by introducing defined and well-known mutations into the CH2 domain of the molecules described above, as well as in International Patent Application WO 2013 / 092001 and Armour et al., Eur J Immunol 1999; 292613. Therefore, the IgGsc ABP of the present invention may also have a CH2 domain (including a hinge region), wherein at least one amino acid residue of the hinge region or the CH2 domain capable of mediating Fc receptor binding is deleted or mutated. As described above, the residues in CH2 and the hinge region can be selected from sequence positions 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 265, 297, 327, and 330, respectively (sequence positions are numbered according to the EU-index). However, due to the presence of the CH3 domain in the IgGsc molecule, two separate molecules will (spontaneously) homodimerize through the CH3 domain to form a tetravalent molecule (see again for this). Figure 1B Therefore, the absence or mutation of cysteine ​​residues at sequence positions 226 and / or 229 in the hinge region is not required. Thus, this tetrameric IgGsc ABP of the present invention may have cysteine ​​residues at sequence positions 226 and / or 229 in one of the corresponding hinge domains, consistent with the Kabat number [EU-index].

[0153] Consistent with the above disclosure of bispecific ABPs containing a set of CDR regions mediating CD276 binding and / or binding to leukemia cancer cells, the ABP of the present invention may contain a second binding site that specifically binds to a receptor on immune cells such as T cells or NK cells. This receptor, present on the immune cell, may be a receptor capable of activating or stimulating an immune response in the immune cell. The induced immune response may preferably be a cytotoxic immune response. Such suitable receptors may be, for example, CD3, antigen-specific T cell receptor (TCR), CD28, CD16, NKG2D, Ox40, 4-1 BB, CD2, CD5, programmed cell death protein 1 (PD-1), and CD95. Particularly preferred are ABPs in which the second binding site binds to CD3, TCR, or CD16. Most preferred are ABPs in which the second binding site specifically binds to CD3, TCR, or CD16. A preferred ABP contains a second binding site that corresponds to the antigen-binding site of the anti-CD3 antibody OKT3. The amino acid sequences of the heavy chain variable region and light chain variable region of antibody OKT3 are also described, for example, in Arakawa et al., J. Biochem. 120, 657-662 (1996) and International Patent Application WO 2015 / 158868 (see SEQ ID NOS: 17 and 18 in the sequence listing of WO 2015 / 158868). Another preferred ABP contains a second binding site corresponding to the antigen binding site of the anti-CD3 antibody UCHT1. The VH and VL sequences of the humanized UCHT1 antibody are described in International Patent Application WO 2013 / 092001. Other examples of CD3-binding ABPs that can be used in this invention include ABPs described in European Patent 2155783B1 or European Patent EP2155788B1, which are capable of binding to epitopes of the CD3ε chain in humans, common marmosets, woolly-crowned tamarins, or squirrel monkeys.

[0154] Therefore, the bispecific ABP of the present invention can be a bispecific ABP such as an IgGsc molecule, which comprises the Fab fragment and the scFv fragment described herein. In this molecule, a first binding site can bind CD276 and can be contained in the Fab fragment described herein (or in the context of a bivalent CD276 F(ab)2 in the form of IgGsc), and a second binding site (which can bind an immune receptor) can be contained in the scFv fragment, such as an scFv fragment that specifically binds CD3. Alternatively, the first binding site for CD276 is contained in a single-stranded Fv fragment, and the second binding site (which can bind CD3) is contained in the Fab fragment.

[0155] In some embodiments, the bispecific ABP of the present invention, upon binding, for example, to CD3, does not itself activate immune cells, such as T cells. Instead, it can crosslink and activate the receptor only when both binding sites, such as the CD276-specific binding site and the CD3-specific binding site, bind to the receptor on T cells and CD276 on target cancer cells, triggering effector cells to kill specific target cells. Standard functional assays can be established to assess the ability of the bispecific ABP of the present invention to kill target cells by lymphocytes in the presence or absence of the lymphocytes, to assess / or screen the ability of the ABP to bind to its receptors.

[0156] In some embodiments of the present invention, the bispecific ABP includes a second antigen-binding domain, an anti-CD3 antibody scFv, such as UCHT1 or a variant thereof.

[0157] In a third aspect, the present invention relates to isolated nucleic acids comprising encoding any of the ABPs in the first aspect, or antigen-binding fragments or monomers of the ABPs, such as heavy or light chains, or sequences encoding bispecific ABPs according to the second aspect.

[0158] For example, the component encoded by the nucleic acid of the present invention may be all or part of one chain of the antibody of the present invention; or the component may be the scFv of the ABP. The component encoded by such a nucleic acid may be all or part of one or another chain of the antibody of the present invention; for example, the component encoded by such a nucleic acid may be the ABD of the present invention. The nucleic acid of the present invention may also encode fragments, derivatives, mutants, or variants of the ABP of the present invention, and / or represent polynucleotide components suitable and / or sufficient to be used as hybridization probes, polymerase chain reaction (PCR) primers, or sequencing primers for identifying, analyzing, mutating, or amplifying polynucleotides encoding polypeptides, antisense or repressive nucleic acids (such as RNAi / siRNA / shRNA or gRNA molecules) for inhibiting polynucleotide expression, and the aforementioned complementary sequences.

[0159] In a particular embodiment of the invention, the nucleic acid of the invention comprises a nucleic acid or a functional fragment thereof having a sequence encoding a heavy or light chain CDR, a combination of heavy and / or light chain CDR1, CDR2 and CDR3, or a heavy or light chain variable domain (as shown in Table 1 in each case). In other embodiments, the nucleic acid of the invention comprises a nucleic acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%; or 95% (preferably at least 75%) sequence uniformity with any of the CDRs (preferably CDR3) disclosed herein in Table 1 (or having no more than fifty, forty, thirty, twenty, fifteen, ten or five, preferably no more than three, two or one substitution, insertion or deletion).

[0160] The nucleic acid according to the invention can be genomic, mRNA, cDNA, or synthetically derived DNA or RNA, or combinations thereof, optionally linked to a non-naturally linked polynucleotide. In some embodiments, such a nucleic acid may contain one or more (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 20, particularly 1 to about 5, or preferably all cases of a particular nucleotide in the sequence) non-natural (e.g., synthetic) nucleotides; and / or such a nucleic acid may contain (e.g., coupled to) another chemical moiety, such as a labeling group or effector group; for example, the labeling groups or effector groups described elsewhere herein.

[0161] In one embodiment, the nucleic acid of the present invention may be isolated or substantially pure. In another embodiment, the nucleic acid of the present invention may be recombinant, synthetic and / or modified, or any other non-natural form. For example, relative to natural products, such as human nucleic acid, the nucleic acid of the present invention may contain at least one nucleic acid substitution (or deletion) modification (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more such modifications, particularly 1 to about 5 such modifications, preferably 2 or 3 such modifications).

[0162] Nucleic acids can be of any suitable length, for example, approximately 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000, or more than 5000 nucleotides. For example: Preferably, the siRNA nucleic acid can be about 15 to about 25 base pairs in length (preferably about 19 to about 21 base pairs); preferably, the shRNA nucleic acid can include a stem of 20 to 30 base pairs, a loop of at least 4 nucleotides, and a dinucleotide overhang at the 3' end; preferably, the microRNA can be about 22 base pairs in length; preferably, the mRNA or DNA sequence (e.g., heavy chain or light chain or IgG antibody) encoding ABP or its components of the present invention can be about 500 and 1500 nucleotides, respectively. More preferably, the nucleic acid encoding the mammalian antibody light chain can be about 630 to about 650 nucleotides, and the nucleic acid encoding the mammalian antibody heavy chain can be about 1300 to about 1650 nucleotides. The nucleic acid can include one or more additional sequences, such as regulatory sequences and / or portions of larger nucleic acids. The nucleic acid can be single-stranded or double-stranded and can contain RNA and / or DNA nucleotides, and their artificial variants (e.g., peptide nucleic acids).

[0163] Nucleic acid encoding antibody polypeptides (e.g., heavy or light chain, variable domains only or full length) can be isolated from B cells of mice, rats, llamas, alpacas, chickens, or rabbits immunized with CD276 antibody or fragments thereof, such as one or more domains (or nucleotides encoding and capable of expressing CD276 antigen or fragments thereof). The nucleic acid can be isolated by conventional procedures such as PCR.

[0164] The change can be introduced by mutating the sequence of the nucleic acid of the present invention. Depending on its properties and position in the codon, such a change can lead to a change in the amino acid sequence of the polypeptide it encodes (e.g., an antigen-binding protein). Any technique known in the art can be used to introduce the mutation.

[0165] In one embodiment, one or more specific amino acid residues may be altered using, for example, a site-directed mutation formation protocol. In another embodiment, one or more randomly selected residues may be altered using, for example, a random mutation formation protocol. However, once prepared, the mutant polypeptide can be expressed and screened for desired properties. Mutations can be introduced into nucleic acids without significantly altering the biological activity of the encoded polypeptide. For example, nucleotide substitutions can be performed, resulting in the replacement of non-essential amino acid residues with amino acids.

[0166] Other changes that can be made to the nucleic acid sequence of the present invention (e.g., by mutation) may not alter the amino acid sequence of the encoded polypeptide, but may alter its stability and / or the effectiveness of expression of the encoded polypeptide. For example, through codon optimization, the expression of a given polypeptide sequence can be improved by utilizing more common codons for a given amino acid found in the species in which the nucleotide to be expressed. Methods of codon optimization and alternative methods (e.g., optimization of CpG and G / C content) are described, for example, by Hass et al., 1996 (Current Biology 6:315); WO1996 / 09378; WO2006 / 015789 and WO 2002 / 098443).

[0167] In a fourth aspect, the present invention relates to nucleic acid constructs (NACs) comprising the nucleic acid of the third aspect and one or more additional sequence features, said sequence features allowing the expression of the encoded ABP or bispecific ABP, or a component of said ABP or bispecific ABP (e.g., antibody heavy or light chain) in cells.

[0168] Such NACs may include one or more additional features that allow expression of an ABP or a component of said ABP (e.g., ABD) encoded in a cell (e.g., a host cell). Examples of NACs of the present invention include, but are not limited to, plasmid vectors, viral vectors, mRNA, non-attachment mammalian vectors, and expression vectors, such as recombinant expression vectors. The nucleic acid constructs of the present invention may contain the nucleic acids of the present invention in a form suitable for expression in cells, such as host cells (see below). The nucleic acid constructs of the present invention are typically recombinant nucleic acids and / or may be isolated and / or substantially pure. Recombinant nucleic acids are typically non-natural; particularly if they contain portions derived from different species and / or synthesized, in vitro, or mutagenic.

[0169] In some embodiments, the NAC of the present invention comprises one or more constructs, any one of which contains a nucleic acid encoding a heavy antibody chain or a light antibody chain. In some embodiments, the NAC of the present invention comprises two constructs, one containing a nucleic acid encoding a heavy antibody chain and the other containing a nucleic acid encoding a light antibody, such that expression from the construct can produce a complete antibody molecule. In some embodiments, the NAC of the present invention comprises a construct containing both a heavy antibody chain and a light antibody chain, such that a complete antibody molecule can be expressed from a single construct. In other embodiments, the NAC of the present invention may comprise a single construct encoding a single chain sufficient to form the ABP of the present invention; for example, if the encoded ABP is an scFv or a single-domain antibody (e.g., a camel antibody).

[0170] In some embodiments, the NAC of the present invention includes a sequence encoding all or part of the constant region, such that the entire or part of the heavy chain and / or light chain can be expressed.

[0171] The NAC according to the invention may comprise (or consist of) an mRNA molecule comprising an open reading frame encoding the ABP of the invention, and together with, for example, upstream and downstream elements (e.g., 5' and / or 3' UTRs and / or poly-A extensions) such that the ABP is expressed, and preferably enhances the stability of the mRNA and / or the expression of the ABP. The use of mRNA as an NAC to introduce into cells and express polynucleotides in cells has been described, for example, by Zangi et al. in Nat. Biotechnol, Vol. 31, 898-907 (2013), Sahin et al. (2014) Nature Reviews Drug Discovery 13:759, and Thess et al., Mol. Ther., Vol. 23, no. 9, 1456-1464 (2015). Specific UTRs that may be included in the mRNA NAC of the invention include: the 5' UTR of the TOP gene (WO2013 / 143699), and / or histone stem-loops (WO 2013 / 120629). The mRNA NAC of the present invention may also contain one or more chemical modifications (EP 1685844); including a 5' cap, such as m7G(5')ppp, (5'(A,G(5')ppp(5')A or G(5')ppp(5')G and / or at least one nucleotide analogue of a naturally occurring nucleotide, such as thiophosphate, phosphoramide, peptide nucleotide, methylphosphonate, 7-deaza-guanosine, 5-methylcytosine or inosine.

[0172] NACs, such as those based on DNA, retroviruses, and mRNA, can be used in gene therapy to treat or prevent diseases of the immune system (see Treatment Methods below), thereby administering an NAC containing an expressible sequence encoding the ABP of the present invention to a cell or organism (e.g., by transfection). In particular, the use of mRNA therapy to express antibodies is known from WO2008 / 083949.

[0173] In a fifth aspect, the present invention relates to recombinant host cells comprising nucleic acid molecules or NACs according to the third or fourth aspect. Preferably, such cells are capable of expressing an ABP (or a component thereof) encoded by said NAC. For example, if the ABP of the present invention comprises two separate polypeptide chains (e.g., the heavy and light chains of IgG), the cell of the present invention may comprise a first NAC encoding (and capable of expressing) the heavy chain of such ABP and a second NAC encoding (and capable of expressing) the light chain of such ABP; or, the cell may comprise a single NAC encoding the chain of such ABP. In these ways, such cells of the present invention are capable of expressing the functionalized (e.g., binding and / or inhibitory) ABP of the present invention. The (host) cells of the present invention may be one of the mammalian, prokaryotic, or eukaryotic host cells as described elsewhere herein, particularly Chinese hamster ovary (CHO) cells.

[0174] In some embodiments of this invention, the (host) cell is a human cell; in particular, it can be a human cell (e.g., autologous human cell) sampled from a specific individual. In such embodiments, this human cell can be proliferated and / or manipulated in vitro to introduce the NAC of the invention. Uses of manipulated human cells from a specific individual can include generating the ABP of the invention, including reintroducing a population of such manipulated human cells into a human subject, for example, for therapeutic purposes. In some such uses, the manipulated human cells can be introduced into the same human individual from which they were first sampled; for example, as autologous human cells.

[0175] The human cells used for this manipulation can be any germ cell or somatic cell type in the body. For example, donor cells can be germ cells or somatic cells selected from fibroblasts, B cells, T cells, dendritic cells, keratinocytes, adipocytes, epithelial cells, epidermal cells, chondrocytes, cumulus cells, nerve cells, glial cells, astrocytes, cardiomyocytes, esophageal cells, muscle cells, melanocytes, hematopoietic cells, macrophages, monocytes, and mononuclear cells. Donor cells can be obtained from any organ or tissue in the body; for example, they can be cells selected from organs such as the liver, stomach, intestines, lungs, pancreas, cornea, skin, gallbladder, ovary, testis, kidney, heart, bladder, and urethra.

[0176] In a sixth aspect, the present invention relates to a pharmaceutical composition comprising: (i) an ABP or bispecific ABP of the first or second aspect, or (ii) a nucleic acid or NAC of the third or fourth aspect, or (iii) a recombinant host cell and a pharmaceutically acceptable carrier, stabilizer and / or excipient according to the fifth aspect.

[0177] For therapeutic use, the ABP, nucleic acid, or NAC (or cell, such as host cell) of the present invention can be formulated into a pharmaceutical composition suitable for administration to animals or humans. The term "pharmaceutical composition" refers to a mixture of substances comprising a therapeutically active substance (such as the ABP of the present invention) for pharmaceutical use.

[0178] For example, the pharmaceutical composition of the present invention may contain 0.1% to 100% by weight of the active ingredient, such as about 0.5% by weight, 1% by weight, 2% by weight, 3% by weight, 4% by weight, 5% by weight, 6% by weight, 8% by weight, 10% by weight, 15% by weight, 20% by weight, 25% by weight, 30% by weight, 40% by weight, 50% by weight, 60% by weight, 70% by weight, 80% by weight, 90% by weight, 95% by weight, 96% by weight, 97% by weight, 98% by weight or 99% by weight, preferably about 1% to about 20% by weight, about 10% by weight to 50% by weight or about 40% by weight to 90% by weight.

[0179] As used herein, the term "pharmaceutically acceptable" excipients, stabilizers, or carriers is intended to include any and all solvents, solubilizers, fillers, stabilizers, binders, absorbents, bases, buffers, lubricants, controlled-release carriers, diluents, emulsifiers, wetting agents, dispersion media, coatings, antibacterial or antifungal agents, isotonic agents, and absorption delay agents, etc., compatible with drug administration. The use of such media and reagents for the active pharmaceutical ingredient is known in the art. Their use in the composition should be considered unless any conventional media or reagent is incompatible with the active compound. Supplements may also be incorporated into the composition.

[0180] The pharmaceutical compositions of the present invention (or those used in the present invention) are generally formulated to be compatible with their intended route of administration. Examples of routes of administration include oral, parenteral, such as intrathecal, intra-arterial, intravenous, intradermal, subcutaneous, oral, transdermal (topical), and transmucosal administration.

[0181] In some embodiments, the pharmaceutical composition containing ABP or NAC is in a unit dose form of 10 mg to 1000 mg. In some embodiments, the pharmaceutical composition containing ABP or NAC is in a unit dose form of 10 mg to 200 mg. In some embodiments, the pharmaceutical composition containing ABP is in a unit dose form of 200 mg to 400 mg. In some embodiments, the pharmaceutical composition containing ABP or NAC is in a unit dose form of 400 mg to 600 mg. In some embodiments, the pharmaceutical composition containing ABP or NAC is in a unit dose form of 600 mg to 800 mg. In some embodiments, the pharmaceutical composition containing ABP or NAC is in a unit dose form of 800 mg to 100 mg.

[0182] Exemplary unit dose forms of pharmaceutical compositions containing ABP or NAC are tablets, capsules (such as powders, granules, microtablets, or micropellets), suspensions, or disposable pre-loaded syringes. In some embodiments, a kit is provided for generating a single-dose administration unit. The kit may contain a first container with the dried active ingredient and a second container with the aqueous formulation. Alternatively, the kit may contain single-lumen and multi-lumen pre-loaded syringes.

[0183] The toxicity and therapeutic efficacy (e.g., effectiveness) of such active ingredients can be determined using standard pharmaceutical procedures in cell culture or laboratory animals, for example, to determine the LD50 (the dose that is lethal to 50% of the population) and ED50 (the therapeutically effective dose to 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Active agents exhibiting a large therapeutic index are preferred. While compounds exhibiting toxic side effects can be used, care should be taken to design delivery systems that target such compounds to sites in the affected tissue to minimize potential damage to uninfected cells, thereby reducing side effects.

[0184] Based on all aspects and embodiments of the medical uses and treatments provided herein, the effective dose for administering at least one dose of ABP or NAC to a subject requiring treatment with ABP or NAC is generally from about 0.01 mg / kg to about 100 mg / kg per dose, for example, from about 1 mg / kg to about 10 mg / kg per dose. In some embodiments, the effective dose for administering at least one dose of ABP or NAC to the subject is from about 0.01 mg / kg to about 0.1 mg / kg per dose, from about 0.1 mg / kg to about 1 mg / kg per dose, from about 1 mg / kg to about 5 mg / kg per dose, from about 5 mg / kg to about 10 mg / kg per dose, from about 10 mg / kg to about 50 mg / kg per dose, or from about 50 mg / kg to about 100 mg / kg per dose.

[0185] For the prevention or treatment of disease, the appropriate dose of ABP or NAC (or a pharmaceutical composition containing it) depends on the type of disease to be treated, the severity and duration of the disease, whether the ABP or NAC and / or pharmaceutical composition is for preventive or therapeutic purposes, prior treatment, the patient's clinical history, age, size / weight, and response to the ABP or NAC and / or pharmaceutical composition, and the judgment of the attending physician. The ABP or NAC and / or pharmaceutical composition is appropriately administered to the patient either once or as a series of treatments. If such an ABP or NAC and / or pharmaceutical composition is administered as a series of treatments, the total number of administrations for a given course of treatment can be approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than approximately 10 treatments. For example, treatment may be administered once daily (or twice, 3, or 4 times a day) for a week, a month, or even several months. In some embodiments, the course of treatment may be continued indefinitely.

[0186] In a seventh aspect, the present invention relates to components used as pharmaceuticals, wherein the components are selected from the following list: (i) ABP or bispecific ABP of the first or second aspect, or (ii) nucleic acids or NAC of the third or fourth aspect, or (iii) recombinant host cells according to the fifth aspect and (iv) pharmaceutical compositions according to the sixth aspect.

[0187] In related aspects, the present invention also relates to methods for treating or preventing diseases, disorders or conditions in mammalian subjects in need, comprising administering to the subject at least once an effective amount of the regulatory compound as described above, or specifically administering to the subject at least once an effective amount of the ABP, NAC, (host) cell or pharmaceutical composition as described above.

[0188] In another related aspect, the present invention also relates to the use of the products of the present invention as described above or the regulatory compounds as described above (especially the ABP of the present invention) in the preparation of a medicine, particularly for the treatment of diseases, disorders or conditions in mammalian subjects, especially wherein the disease, disorder or condition is the disease, disorder or condition described herein.

[0189] The term "treatment" in this invention refers to therapies, such as therapeutic treatments, and preventive or inhibitory measures against diseases (or disorders or conditions). Thus, for example, successful administration of a compound according to the invention prior to the onset of a disease results in treatment of the disease. "Treatment" also includes the administration of a compound according to the invention after the onset of a disease to improve or eradicate the disease (or its symptoms). Administration of the CD276-binding compound according to the invention after the onset of disease and after clinical symptoms may alleviate clinical symptoms and may improve the disease, and also includes treatment of the disease. Those who "require treatment" include objects (e.g., human objects) that already have a disease, disorder, or condition, and those that are susceptible to or suspected of having a disease, disorder, or condition, including those in which the disease, disorder, or condition is to be prevented.

[0190] In specific embodiments of these aspects, the modulating compound is the modulating compound described above, and / or the ABP, NAC, (host) cell, or pharmaceutical composition of the present invention; particularly the ABP of the present invention.

[0191] Such compounds can be, for example, compounds that enhance the killing and / or lysis of natural killer cells (ADCC) and / or CDC against cells expressing CD276 or CD276 variants (e.g., ABP or inhibitory nucleic acids).

[0192] In other aspects described elsewhere in this document, methods for detecting and / or diagnosing diseases, disorders, or conditions in mammalian subjects are provided.

[0193] In one particular implementation, the disease, disorder, or symptom is characterized by a pathological immune response.

[0194] In other specific embodiments, the disease, disorder, or symptom is characterized by the expression of CD276 or a variant of CD276, particularly by the expression of CD276 or a variant of CD276 by cells associated with the disease, disorder, or symptom, such as cancer cells. For example, the disease, disorder, or symptom may be associated with the undesirable presence of CD276-positive cells or CD276 variant-positive cells. Preferably, the disease, disorder, or symptom is characterized by a vascular system comprising cells expressing CD276 or a variant thereof.

[0195] In some alternative embodiments, the disease, disorder, or symptom that can be treated by the subject matter of the present invention is characterized by the expression of CD276; in particular, it is characterized by abnormal expression, such as overexpression (or underexpression), characterization, or activity of CD276 in a given cell or tissue (e.g., those cells or tissues associated with a proliferative disease of the subject) compared to a healthy subject or normal cells.

[0196] In other specific embodiments, the disease, disorder, or symptom is characterized by the expression and / or activity of CD276, particularly by the expression of CD276 mRNA and / or protein in such cells, and / or positivity for such CD276 expression and / or activity.

[0197] In another specific embodiment, the disease, disorder, or symptom is a proliferative disease (or a symptom associated with such disorder or disease), particularly when the product or regulatory compound (e.g., the ABP, nucleic acid, NAC, or recombinant host cell of the present invention, particularly the ABP of the present invention) is present.

[0198] "Proliferative disorders" are diseases characterized by abnormal proliferation of cells. A proliferative disorder does not imply any restriction on the rate of cell growth, but only indicates a loss of normal control affecting growth and cell division. Therefore, in some embodiments, cells in a proliferative disorder may have the same rate of cell division as normal cells, but do not respond to signals that limit such growth. Within the scope of "proliferative disorders," a proliferative disorder is a growth or tumor that is an abnormal growth of tissue or cells. Cancer is understood in the art and includes any of a variety of malignant tumors characterized by cell proliferation, said cells having the ability to invade surrounding tissues and / or metastasize to new colonization sites. Proliferative disorders include cancer, atherosclerosis, rheumatoid arthritis, idiopathic pulmonary fibrosis, and cirrhosis. Non-cancerous proliferative disorders also include cell proliferation in the skin, such as psoriasis and its various clinical forms, Leter's syndrome, pityriasis rubra pilaris, and proliferative variations of keratinization disorders (e.g., actinic keratosis, senile keratosis), scleroderma, etc.

[0199] In a more specific implementation plan, proliferative diseases are cancers or tumors, particularly solid tumors (or conditions associated with such cancers or tumors). Such proliferative diseases include, but are not limited to, head and neck cancer, squamous cell carcinoma, multiple myeloma, solitary plasmacytoma, renal cell carcinoma, retinoblastoma, germ cell tumor, hepatoblastoma, hepatocellular carcinoma, melanoma, renal rhabdomyosarcoma, Ewing sarcoma, chondrosarcoma, any hematologic malignancy (e.g., chronic lymphocytic leukemia, chronic myelomonocytic leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, chronic myeloid leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, chronic myeloid leukemia, myelodysplastic syndrome, hairy cell leukemia, mast cell leukemia, mast cell tumor, follicular lymphoma, diffuse large cell lymphoma). Lymphoma, mantle cell lymphoma, marginal zone lymphoma, Burkitt lymphoma, mycosis fungoides, Seary syndrome, cutaneous T-cell lymphoma, peripheral T-cell lymphoma, chronic myeloproliferative disorders, myelofibrosis, myeloid metaplasia, systemic mastocytosis, and central nervous system tumors (e.g., brain cancer, glioblastoma, non-glial cell brain cancer, meningioma, pituitary adenoma, vestibular schwannoma, primitive neuroectodermal tumor, medulloblastoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, ependymoma, and choroid plexus papilloma), myeloproliferative disorders (e.g., polycythemia vera, thrombocytosis, primary myelofibrosis), soft tissue sarcoma, thyroid cancer, endometrial cancer, carcinoid tumors, or liver cancer.

[0200] In a preferred embodiment, aspects of the invention relate to, for example, the ABP of the invention for detecting / diagnosing, preventing, and / or treating proliferative diseases, including but not limited to cancers (including breast cancer, prostate cancer, gastric cancer, lung cancer, colorectal cancer and / or colon cancer, hepatocellular carcinoma, melanoma), lymphomas (including non-Hodgkin's lymphoma and mycosis fungoides), leukemia, sarcoma, mesothelioma, brain cancer (including glioma), germ cell tumors (including testicular cancer and ovarian cancer), choriocarcinoma, kidney cancer, pancreatic cancer, thyroid cancer, head and neck cancer, endometrial cancer, cervical cancer, bladder cancer, or gastric cancer.

[0201] Therefore, in a preferred embodiment, the ABP, bispecific ABP, or NAC according to the present invention are used for the prevention and / or treatment of cancer, such as cancer characterized by the presence of cancer cells selected from the cells of the following cancers: adrenal tumors, AIDS-related cancers, alveolar soft tissue sarcomas, astrocytomas, bladder cancer, bone cancer, brain and spinal cord cancers, metastatic brain tumors, breast cancer, carotid body tumors, cervical cancer, chondrosarcoma, chordoma, renal chromophobe carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, benign fibrous histiocytoma of the skin, small round cell tumors that promote connective tissue proliferation, ependymoma, Ewing sarcoma, extraosseous myxoid chondrosarcoma, and osteofibromatosis. Developmental disorders, fibrous dysplasia of bone, gallbladder or bile duct cancer, gastric cancer, gestational trophoblastic disease, germ cell tumors, head and neck cancer, hepatocellular carcinoma, islet cell tumors, Kaposi's sarcoma, kidney cancer, leukemia, lipoma / benign lipoma, liposarcoma / malignant lipoma, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine tumors, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid adenoma, pediatric cancers, peripheral nerve sheath tumors, pheochromocytoma, pituitary adenoma, prostate cancer, posterior uveal melanoma. Unveal melanoma), rare blood disorders, kidney metastases, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroid metastases, and uterine cancer.

[0202] In a particularly preferred embodiment, the disease, disorder, or symptom is CD276-positive carcinoma or CD276 variant-positive carcinoma and / or cancer characterized by the presence of CD276-positive vascular cells in a tumor environment. More preferably, the cancer is a solid tumor, characterized by dual expression-dual targeting of CD276 or its variants in cancer cells of the tumor tissue and in vascular cells of the tumor vascular system.

[0203] Alternatively, modulating (e.g., inhibiting) compounds (e.g., ABP, as in one of these inventions) can be used in combination with various antiproliferative therapies, particularly various anticancer therapies, especially when the various antiproliferative therapies are immunotherapies, particularly immunotherapies using ligands of immune checkpoint molecules. Therefore, the composition can be used to treat proliferative diseases in subjects who require co-treatment with immunotherapy, particularly with ligands of immune checkpoint molecules (e.g., combination therapy).

[0204] In such an implementation, the ligand is a ligand that binds to an immune (inhibitory) checkpoint molecule. For example, such checkpoint molecules may be selected from: A2AR, B7-H3, B7-H4, CTLA-4, IDO, KIR, LAG3, PD-1 (or one of its ligands PD-L1 and PD-L2), TIM-3 (or its ligand galactagogue-9), TIGIT, or antigen-binding molecules targeting FLT3, PSMA, or other tumor-associated targets. In a specific implementation, the ligand binds to a checkpoint molecule selected from: CTLA-4, PD-1, and PD-L1. In other, more specific embodiments, the ligand is an antibody selected from the following: ipilimumab, nivolumab, pembrolizumab, BGB-A317, atezolizumab, avelumab, and durvaluma; particularly, an antibody selected from the following: ipilimumab, nivolumab, pembrolizumab, and atezolizumab.

[0205] When the treatment method or use of the present invention (e.g., involving the ABP of the present invention) is used in combination therapy with any other such procedure (e.g., another reagent or cancer immunotherapy, such as a ligand binding to an immune (inhibitory) checkpoint molecule), this method or use as a combination therapy may include embodiments in which such exposure / administration is coexisting. In alternative embodiments, such administration may be continuous; particularly those embodiments in which the ABP of the present invention is administered prior to such other procedure. For example, such a compound of the present invention may be administered continuously over about 14 days of other procedures, such as about 10 days, 7 days, 5 days, 2 days, or 1 day prior to other procedures (e.g.); also including embodiments in which the compound of the present invention may be administered continuously over about 48 hours, 24 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, or 5 minutes prior to other procedures (e.g.).

[0206] In an eighth aspect, the present invention relates to a method for enhancing a cell-mediated immune response against human cells expressing human CD276, the method comprising, in the presence of immune cells such as T cells or natural killer (NK) cells, contacting the cells with (i) an ABP or bispecific ABP according to the first or second aspect, or (ii) a nucleic acid or NAC according to the third or fourth aspect, or (iii) a recombinant host cell according to the fifth aspect and (iv) a pharmaceutical composition according to the sixth aspect, thereby enhancing the cell-mediated immune response against the human cells, preferably cytotoxic.

[0207] In a ninth aspect, the present invention relates to a method for preventing and / or treating proliferative diseases in a subject, the method comprising administering to the subject a therapeutically effective amount of the component described in the seventh aspect; and wherein the proliferative disease is characterized by the expression of CD276 in cells associated with the proliferative disease.

[0208] As described above, in one aspect, this document provides cells capable of expressing the ABP described above, such as (recombinant) host cells or hybridoma cells. Alternatively, this document provides cells comprising at least one NAC encoding the ABP or a component of the ABP described above. The cells of the present invention can be used in the methods provided herein to generate the ABP and / or NACA of the present invention.

[0209] Therefore, in other respects, the present invention relates to a method for generating a recombinant cell line capable of expressing ABP specific to CD276 or a CD276 variant, the method comprising the following steps:

[0210] • Provide suitable host cells;

[0211] • Provide at least one gene construct containing a coding sequence encoding the ABP of the present invention;

[0212] • Introducing the gene construct into the suitable host cell; and

[0213] Optionally, the gene construct may be expressed in the suitable host cell, provided that ABP expression is permitted.

[0214] In another aspect, this document provides a method for generating the ABP as described above, which includes, for example, culturing one or more types of cells of the present invention while allowing the expression of the ABP.

[0215] Therefore, in another aspect, the present invention relates to a method for generating an ABP specific to CD276 or a CD276 variant, the method comprising the following steps:

[0216] • Provide hybridoma cells or (host) cells capable of expressing the ABP according to the invention, such as recombinant cell lines comprising at least one gene construct containing a coding sequence encoding the compound or ABP; and

[0217] • Culture the hybridoma cells or host cells while allowing ABP expression.

[0218] As used herein, the terms “of the present invention,” “according to the present invention,” “according to the present invention,” etc., are intended to refer to all aspects and embodiments of the invention described and / or listed herein.

[0219] As used herein, the term “comprising” is interpreted to encompass both “including” and “consisting of”, both meanings being specific and therefore specific to the embodiments disclosed separately according to the invention. When used herein, “and / or” is considered a specific disclosure of each of two specified features or components, wherein the other is present or absent. For example, “A and / or B” is considered a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were separately stated herein. In the context of this invention, the terms “about” and “approximately” represent precise ranges that will be understood by those skilled in the art to still ensure the technical effect of the stated feature. This term typically indicates a deviation from the indicated numerical value of 20%, 15%, 10%, for example, 5%. As will be understood by those skilled in the art, the specific deviation of such a numerical value for a given technical effect will depend on the nature of the technical effect. For example, natural or biotechnological effects may generally have a larger such deviation than artificial or engineered effects. As will be understood by those skilled in the art, the specific deviation of such a numerical value for a given technical effect will depend on the nature of the technical effect. For example, natural or biotechnological effects may generally have a larger such deviation than artificial or engineered effects. When referring to a noun, quantifiers are not used, including the plural form of the noun, unless otherwise specified.

[0220] It should be understood that applying the teachings of this invention to specific problems or environments, including variations of the invention or additional features thereof (such as other aspects and embodiments), based on the teachings contained herein, will be within the capabilities of those skilled in the art.

[0221] Unless the context otherwise requires, the description and definition of the above features are not limited to any particular aspect or embodiment of the invention, but are equally applicable to all aspects and embodiments described.

[0222] All references, patents and publications cited in this article are incorporated herein in their entirety through citation.

[0223] In view of the foregoing, it should be understood that the present invention also relates to the following embodiments, listed one by one:

[0224] Clause 1: An isolated antigen-binding protein (ABP) that specifically binds to CD276 protein or a variant thereof, wherein the isolated ABP is capable of inducing antibody-dependent cell-mediated cytotoxicity in cells expressing CD276.

[0225] Clause 2: The isolated ABP as described in Clause 1, wherein the isolated ABP specifically binds to the immunoglobulin-like V (IgV) or C (IgC) domain or FG loop region of the CD276 protein or a variant thereof.

[0226] Clause 3: ABP isolated according to Clause 1 or 2, wherein the CD276 protein is the human CD276 protein.

[0227] Clause 4: The isolated ABP according to any one of Clauses 1 to 3, wherein the isolated ABP specifically binds to the CD276 epitope, said epitope comprising an amino acid position of human CD276 selected from (i) Q179, (ii) G130, (iii) R127 and (iv) G43, A115 and / or F120; preferably wherein the ABP specifically binds to the (antibody) epitope comprising Q179 of human CD276.

[0228] Clause 5: The isolated ABP according to any one of Clauses 1 to 4, comprising at least one complementarity-determining region (CDR) 3, said complementarity-determining region (CDR) 3 having at least 80% sequence identity with a sequence selected from SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31, 35 and 39 or having no more than three or two, preferably one, amino acid substitution, deletion or insertion amino acid sequence.

[0229] Clause 6: A separate ABP as described in Clause 5, wherein the ABP further comprises at least one CDR1 and at least one CDR2.

[0230] Clause 7: ABP isolated according to any one of Clauses 1 to 6, wherein the ABP is an antibody or an antigen-binding fragment thereof.

[0231] Clause 8: The isolated ABP according to any one of Clauses 1 to 7 comprises an antibody heavy chain or an antigen-binding fragment thereof, and / or an antibody light chain or an antigen-binding fragment thereof.

[0232] Clause 9: The isolated ABP according to any one of Clauses 1 to 8 comprises an antibody heavy chain variable region or an antigen-binding fragment thereof, and / or an antibody light chain variable region or an antigen-binding fragment thereof.

[0233] Clause 10: The isolated ABP according to any one of Clauses 1 to 9, comprising antibody heavy chain variable regions CDR1, CDR2 and CDR3 and / or antibody light chain variable regions CDR1, CDR2 and CDR3.

[0234] Clause 11: An isolated ABP according to any one of Clauses 1 to 10, comprising an antibody heavy chain sequence and / or an antibody light chain sequence, or an antigen-binding fragment thereof; wherein, relative to a sequence selected from SEQ ID No: 3, 11, 19, 27 and 35, the antibody heavy chain sequence or fragment thereof comprises having at least 80% sequence identity, or having no more than three or two, preferably one, amino acid substitution, deletion or insertion CDR3, and / or wherein, relative to a sequence selected from SEQ ID No: 7, 15, 23, 31 and 39, the antibody light chain sequence or fragment thereof comprises having at least 80% sequence identity, or having no more than three or two, preferably one, amino acid substitution, deletion or insertion CDR3.

[0235] Clause 12: The isolated ABP according to any one of Clauses 8 to 11, wherein the antibody heavy chain sequence or fragment thereof further comprises, relative to the sequence selected from SEQ ID No: 1, 9, 17, 25 and 33, having at least 80% sequence identity, or having no more than three or two, preferably one, amino acid substitution, deletion or insertion CDR1; and / or having at least 80% sequence identity, or having no more than three or two, preferably one, amino acid substitution, deletion or insertion CDR2, relative to the sequence selected from SEQ ID No: 2, 10, 18, 26 and 34.

[0236] Clause 13: The isolated ABP according to any one of Clauses 8 to 12, wherein the antibody light chain sequence or fragment thereof further comprises, relative to the sequence selected from SEQ ID No: 5, 13, 21, 29 and 37, having at least 80% sequence identity, or having no more than three or two, preferably one, amino acid substitution, deletion or insertion CDR1; and / or having at least 80% sequence identity, or having no more than three or two, preferably one, amino acid substitution, deletion or insertion CDR2, relative to the sequence selected from SEQ ID No: 6, 14, 22, 30 and 38.

[0237] Clause 14: An isolated ABP according to any one of Clauses 1 to 13, comprising at least 80% sequence identity relative to a sequence selected from SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36 and 40, or having no more than three or two, preferably one, amino acid substitution, deletion or insertion of an antibody variable chain sequence.

[0238] Clause 15: An isolated ABP according to any one of Clauses 1 to 14, comprising an antigen-binding fragment of an antibody, wherein the antigen-binding fragment comprises CDR1, CDR2, and CDR3, optionally selected from CDR1, CDR2, and CDR3 sequences having amino acid sequences of SEQ ID No: 1, 2, 3 or 5, 6, 7 or 9, 10, 11 or 13, 14, 15 or 17, 18, 19 or 21, 22, 23 or 25, 26, 27 or 29, 30, 31 or 33, 34, 35 or 37, 38, 39 respectively; and in each case independently, optionally having no more than three or two, preferably one, amino acid substitution, insertion, or deletion compared to these sequences.

[0239] Clause 16: An isolated ABP according to any one of Clauses 10 to 15, wherein CDR1 has an amino acid sequence of SEQ ID No: 1, 5, 9, 13, 17, 21, 25, 29, 33 or 37, CDR2 has an amino acid sequence of SEQ ID No: 2, 6, 10, 14, 18, 22, 26, 30, 34 or 38, and CDR3 has an amino acid sequence of SEQ ID No: 3, 7, 11, 15, 19, 23, 27, 31, 35 or 39; and in each case, independently, optionally having no more than three or two, preferably one, amino acid substitution, insertion or deletion compared to these sequences.

[0240] Clause 17: An isolated ABP according to any one of Clauses 1 to 16, wherein the ABP is an antibody or an antigen-binding fragment thereof, comprising at least one, preferably two, antibody heavy chain sequences and at least one, preferably two, antibody light chain sequences, wherein at least one, preferably two, of the antibody heavy chain sequences and antibody light chain sequences comprises CDR1 to CDR3 sequences comprising the following combinations:

[0241]

[0242] Independently in each case, compared to these sequences, there may optionally be no more than three or two, preferably one, amino acid substitution, insertion or deletion.

[0243] Clause 18: An isolated ABP according to any one of Clauses 1 to 17, wherein the ABP is an antibody or an antigen-binding fragment thereof, comprising at least one, preferably two, antibody heavy chain sequences and at least one, preferably two, antibody light chain sequences, wherein each of the antibody heavy chain sequence and antibody light chain sequence comprises a variable region sequence comprising the following combinations:

[0244]

[0245] Independently in each case, compared to these sequences, there may optionally be no more than three or two, preferably one, amino acid substitution, insertion or deletion.

[0246] Clause 19: The isolated ABP according to any one of Clauses 1 to 18, which contains an effector group and / or its labeling.

[0247] Clause 20: A separate ABP pursuant to any one of Clauses 1 to 19, which is separate and / or substantially pure.

[0248] Clause 21: The isolated ABP pursuant to any one of Clauses 1 to 20 is an antibody, such as a monoclonal antibody; or is a fragment of an antibody, such as a fragment of a monoclonal antibody.

[0249] Clause 22: Separated ABP as described in Clause 21, wherein the antibody is a chimeric antibody, such as a human-chimeric antibody.

[0250] Clause 23: ABP isolated according to Clause 21 or 22, wherein the antibody is IgG, IgE, IgD, IgA or IgM immunoglobulin; preferably IgG immunoglobulin.

[0251] Clause 24: The isolated ABP pursuant to any one of Clauses 21 to 23 is an antibody fragment selected from Fab, Fab'-SH, Fv, scFv and F(ab')2.

[0252] Clause 25: An isolated ABP according to any one of Clauses 1 to 24, wherein the ABP is modified or engineered to increase antibody-dependent cell cytotoxicity (ADCC), preferably wherein the ABP is defucosylated.

[0253] Clause 26: The isolated ABP according to any one of Clauses 1 to 25 comprises one or more additional antigen-binding domains that bind to an antigen other than said CD276 or a variant thereof; said antigen is, for example, an antigen present on mammalian T cells, preferably human CD3.

[0254] Clause 27: The isolated ABP as described in Clause 26 is bispecific and preferably contains two binding sites for binding to CD276 and two binding sites for binding to an antigen other than CD276, such as an antigen present on mammalian T cells, most preferably human CD3.

[0255] Clause 28: The isolated ABP as described in Clause 27, wherein the two binding sites that bind to antigens other than CD276 bind to human CD3, and preferably comprises a UCHT1 anti-CD3 scFv construct.

[0256] Clause 29: The isolated ABP according to any one of Clauses 1 to 28 further comprises an enhanced antibody-dependent cell cytotoxicity (ADCC) portion, preferably said portion being an immune cytokine (MIC) such as interleukin-15 (IL-15) or modified IL-15.

[0257] Clause 30: A bispecific antigen-binding protein (ABP) comprising a first antigen-binding domain capable of binding the CD276 antigen or a variant thereof and a second antigen-binding domain capable of binding the human differentiation cluster 3 (CD3) antigen.

[0258] Clause 31: A bispecific ABP as described in Clause 30, comprising no more than or no less than two first antigen-binding domains and two second antigen-binding domains, optionally wherein the bispecific ABP is in the form of IgSc.

[0259] Clause 32: A bispecific ABP according to any one of Clauses 30 or 31, wherein at least one amino acid residue of the CH2 domain capable of mediating binding to the Fc receptor in the antibody is missing or mutated.

[0260] Clause 33: A bispecific ABP according to any one of Clauses 30 to 32, wherein the first antigen-binding domain comprises an ABP or its antigen-binding domain according to any one of Clauses 1 or 29.

[0261] Clause 34: A bispecific ABP according to any one of Clauses 30 to 34, having the activity of binding to T cells and tumor cells expressing CD276, or tumor cells adjacent to cells expressing CD276, such as tumor vascular cells, preferably wherein the antibody increases the recruitment of cytotoxic cells to tumor cells expressing CD276 by binding to CD276 and CD3.

[0262] Clause 35: A bispecific ABP according to any one of Clauses 30 to 34, wherein the second antigen-binding domain comprises the heavy chain variable region and the light chain variable region of UCHT1.

[0263] Clause 36: An isolated nucleic acid comprising an ABP encoding any one of Clauses 1 to 29, or an antigen-binding fragment or monomer of an ABP, such as a heavy or light chain, or a sequence encoding a bispecific ABP according to any one of Clauses 30 to 35.

[0264] Clause 37: A nucleic acid construct (NAC) comprising the nucleic acid as described in Clause 36 and one or more additional sequence features that allow the encoded ABP or bispecific ABP, or a component of said ABP or bispecific ABP (e.g., antibody heavy or light chain), to be expressed in cells.

[0265] Clause 38: A recombinant host cell comprising the nucleic acid described in Clause 36 or the NAC described in Clause 37.

[0266] Clause 39: A pharmaceutical composition comprising: (i) an ABP or a bispecific ABP according to any one of Clauses 1 to 35, or (ii) a nucleic acid according to Clause 36 or a NAC according to Clause 37, or (iii) a recombinant host cell according to Clause 38 and a pharmaceutically acceptable carrier, stabilizer and / or excipient.

[0267] Clause 40: A component used as a medicament, wherein the component is selected from: ABP or bispecific ABP according to any one of Clauses 1 to 35, isolated nucleic acid according to Clause 36 or NAC according to Clause 37, recombinant host cell according to Clause 38 and medicament composition according to Clause 39.

[0268] Clause 41: Components for use as described in Clause 40, wherein the component is for enhancing T cell-mediated killing and / or inhibiting the proliferation of CD276-positive tumors or tumor-associated cells, or CD276 variant-positive tumor cells or tumor-associated cells.

[0269] Clause 42: A component for use according to any one of Clauses 40 to 41, wherein the component is used for the diagnosis, prevention and / or treatment of proliferative diseases, wherein the proliferative diseases are related to the expression of CD276 and are preferably cancers, such as cancers selected from (preferably CD276-positive cancers).

[0270] Clause 43: A method for enhancing a cell-mediated immune response against human cells expressing human CD276, comprising, in the presence of immune cells such as T cells or natural killer (NK) cells, contacting said cells with an ABP according to any one of Clauses 1 to 29, or a bispecific ABP according to any one of Clauses 30 to 35, or a nucleic acid encoding said ABP or a bispecific ABP according to Clause 36, thereby enhancing the cell-mediated immune response against said human cells, preferably cytotoxic.

[0271] Clause 44: A method for preventing and / or treating a proliferative disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the component according to Clause 40; and wherein the proliferative disease is characterized by the expression of CD276 in cells associated with the proliferative disease. Attached Figure Description

[0272] Figure 1: Showing the antibodies and antibody constructs of the present invention; (A) Summary of epitope mapping results for the newly generated B7-H3 antibodies. The scheme shows the crystal structure of the B7-H3 antigen (Vigdorovich V et al., Structure 2013), with arrows indicating the binding sites of various proprietary B7-H3 mAbs. Note that the 7C4 antibody contained in CC-3 binds to epitopes close to the cell membrane. Specified antibodies no longer bind to CD276 mutants carrying specified amino acid exchanges. These changes are selected based on the differences between non-human primate and human CD276 sequences. (B) Graph of monospecific antibodies (left), immune cytokines (MIC proteins, middle), and bispecific IgGsc antibodies (right). Note the use of monospecific antibodies with wild-type human IgG1 Fc moiety or its SDIE-modified form (as shown).

[0273] Figure 2 The image shows a Biacore® sensor plot confirming the binding of the B7-H3 antigen to the specified B7-H3xCD3 bsAbs. Note that constructs containing 7C4 or 8H8 exhibit particularly long dissociation rates.

[0274] Figure 3: Shows the binding of different B7-H3xCD3 bsAbs to B7-H3 and CD3 expressed on NALM-16 (A) and Jurkat cells (B), respectively, as determined by flow cytometry. Note that in all constructs, the affinity of the anti-CD3 antibody was reduced to decrease off-target T cell activation, while the binding to the target antigen B7-H3 was significantly higher for all constructs.

[0275] Figure 4 This study demonstrates NK cell activation and tumor cell loss induced by various monospecific CD276 antibodies (A, B) or MIC proteins containing the SDIE-modified Fc-moment (C, D). Methods: Tumor cells (LNCap) were incubated with human PBMCs and the antibodies shown. After 3 days, NK cell activation and tumor cell loss were measured by flow cytometry.

[0276] Figure 5 This study demonstrates NK cell activation, proliferation, and tumor cell loss induced by various CD276 antibodies (A to C) or MIC proteins containing the Fc-fraction of oil-containing wild-type human IgG1 (D to F). Methods: Tumor cells (LNCap) were incubated with human PBMCs and the antibodies shown. After 3 days, NK cell activation and tumor cell loss were measured by flow cytometry.

[0277] Figure 6 : Demonstrates T cell activation and tumor cell killing induced by various CD276 antibodies incorporated into the form of bispecific antibodies in IgGsc (see Figure 1BMethods: B7-H3 cells expressing NALM-16 tumor cells were incubated with PBMCs from healthy donors in the presence of increased concentrations of the designated B7-H3xCD3 construct or a control bsAb with no relevant specificity. CD4 and CD8 T cell activation and tumor cell killing were measured by flow cytometry after 3 days (B). T cell proliferation induction was assessed using a 3H-thymidine incorporation assay (C). It was noted that CC-3 (7C4xCD3)-mediated T cell stimulation was significantly better in all assays compared to other B7-H3xCD3 bsAbs. A long-duration xCELLigence tumor lysis assay was performed on B7-H3 cells expressing LNCaP tumor cells and PBMCs from healthy donors in the presence of the designated B7-H3xCD3 bsAb (D). It was noted that CC-3 (7C4xCD3)-mediated T cell activation and tumor cell killing were better compared to other B7-H3xCD3 bsAbs.

[0278] Figure 7 The in vivo antitumor activity of various CD276 antibodies incorporating bispecific IgGsc antibodies was demonstrated. Figure 1B Methods: LNCaP cells were subcutaneously injected into the right flank of female NSG mice to establish tumors with a diameter of 5 mm. Then (day 1) on days 1, 8, and 15 (indicated by arrows), PBMCs (2 µg per dose) with or without B7-H3xCD3 antibody, or unrelated control bsAb, were administered intravenously. Tumor growth was measured twice weekly, and mice were euthanized when the tumor diameter reached 15 mm. It was noted that CC-3 (7C4xCD3) was again significantly more effective than 8D9xCD3 in this in vivo setting, confirming the provided in vitro data.

[0279] Sequence display:

[0280] Table 1: Antibody sequences of the present invention:

[0281]

[0282]

[0283]

[0284] Abbreviations: VH: "Variable Heavy Chain", VL: "Variable Light Chain"; VL: "Complementarity Determinant Region"

[0285] SEQ ID NO: 41 shows the amino acid sequence of human CD276 isotype 1:

[0286]

[0287] SEQ ID NO: 42 shows the amino acid sequence of human CD276 isotype 2:

[0288]

[0289]

[0290] SEQ ID NO: 43 shows the amino acid sequence of human CD276 isotype 3:

[0291]

[0292] SEQ ID NO: 44 shows the amino acid sequence of human CD276 isotype 4:

[0293]

[0294]

[0295] Example

[0296] Certain aspects and embodiments of the invention will now be described by way of example and with reference to the description, drawings, and tables set forth herein. These embodiments of the methods, uses, and other aspects of the invention are merely representative and should not be construed as limiting the scope of the invention to only such representative embodiments.

[0297] In the course of this invention, a series of seven antibodies were generated after mice were immunized with recombinant CD276 protein using a hybridization and screening procedure. The binding epitopes of the antibodies were then characterized (see...). Figure 1A ), and selected representative antibodies for each epitope to construct the following derivatives using recombinant antibody technology (see Figure 1B ):

[0298] • Monospecific antibodies containing the wild-type or SDIE-modified human IgG1 Fc-fraction

[0299] • Immune cytokine (MIC) proteins containing a modified IL-15 moiety and a wild-type or SDIE-modified human IgG1 Fc moiety.

[0300] • Previously developed bispecific antibodies in the form of IgGsc with CD276xCD3 specificity

[0301] The antitumor activity of different constructs was then tested in vitro and in vivo, and the CD276 conjugates with optimal activity were identified according to the following examples.

[0302] The example shows:

[0303] Example 1: Antibody generation

[0304] Balb / c mice were immunized with recombinant CD276 protein, and spleen cells were fused according to a standard protocol. Six-month-old female BALB / c mice were immunized with a soluble CD276-Fc fusion protein. The protein used consisted of the extracellular domain of human CD276 isotype 2 (NM_001024736.1) with Met1-Thr461, fused to the N-terminus of the Fc region of human IgG1. 50 µg of the Fc fusion protein was repeatedly administered intraperitoneally in 100 µl PBS per immunization. Mice were immunized every 10 days for a total of 3 to 4 immunizations, with the final intravenous administration 4 days prior to fusion. Binding to CD276-transfected mouse cells was assessed by flow cytometry, and the supernatant of the fusion cells was screened for specific antibody production. A total of seven hybridoma cell lines were then recloned and grown in advanced DMEM medium supplemented with 1.25% FCS. The antibody was purified from the culture supernatant by protein affinity chromatography and size exclusion chromatography (SEC) on a Superdex S200. Purity was assessed by SDS-PAGE and analytical SEC. The sequences of the generated antibodies are provided in Table 1.

[0305] Example 2: Epitope mapping of the novel CD276-antibody

[0306] Human CD276 exists in two isoforms, 2IgB7-H3 and 4IgB7-H3, the latter being an exon repeat and constituting the predominant form in humans. The extracellular structure of this protein is characterized by either an IgV-IgC-like (2IgB7-H3) or an IgV1-IgC1-IgV2-IgC2-like (4IgB7-H3) domain. Mice express only the 2IgB7-H3 isoform. For epitope mapping, truncated forms of the 4IgB7-H3 molecule (IgV1-IgC1 and IgV2-IgC2) were first generated as Fc fusion proteins. It was noted that all antibodies recognized both truncated forms. Furthermore, no antibodies cross-reacted with the mouse protein. Therefore, sequence alignment between the highly conserved human and mouse proteins was performed. Finally, variable amino acids in the human IgV1-IgC1-Fc protein were replaced with the corresponding amino acids from the mouse molecule. This produced... Figure 1A The antibody epitope map and groupings are shown. Additionally, a FACS-based competition assay was performed, confirming that antibodies within the same group cross-blocked each other, while antibodies from different groups did not.

[0307] Example 3: Generation and Characterization of Recombinant Antibody Derivatives

[0308] The variable regions of each representative antibody group were sequenced, incorporated into different antibody forms, and their antitumor activity was tested in appropriate functional assays.

[0309] (1) SDIE-optimized monospecific antibodies: ADCC diagrams of three SDIE antibodies. Figure 4 Results for the Fc-enhanced forms of antibodies 7C4 and 8D9 are provided. Antibody 7C4 outperformed other antibodies, showing significantly higher NK cell activation and tumor cell depletion.

[0310] (2) Immune cytokines containing modified IL-15 moiety: Figure 4 and Figure 5 The study demonstrated the NK cell activation and tumor cell killing effects of antibodies MIC 7C4, 8H8, and 8D9. It can be seen that all antibodies performed well, with 7C4 being the most active.

[0311] (3) Binding of bispecific antibodies with CD265xCD3 specificity to Biacore, FACS, and tumor cell killing: Results are shown in Figure 2 Figure 3 and Figures 6 to 7 middle.

[0312] References

[0313] The references are:

[0314] 1) Weiner GJ. Building better monoclonal antibody-based therapeutics. Nat Rev Cancer 2015;15:361-70.

[0315] 2) Osenga KL, Hank JA, Albertini MR, Gan J, Sternberg AG, Eickhoff J, Seeger RC, Matthay KK, Reynolds CP, Twist C, Krailo M, Adamson PC, ReisfeldRA, Gillies SD, Sondel PM; Children's Oncology Group. A phase I clinical trial of the hu14.18-IL2 (EMD 273063) as a treatment for children withrefractory or recurrent neuroblastoma and melanoma: a study of the Children'sOncology Group. Clin Cancer Res. 2006 Mar 15;12(6):1750-9.

[0316] 3) Jung G, Salih HR, Lindner C, Lochmann B. Fusion proteinscomprising a binding protein and an interleukin-15 polypeptide having areduced affinity for IL15Ra and therapeutic uses thereof. EP15157911 (2015)

[0317] 4) Nelson MH, Paulos CM. Novel immunotherapies for hematologicmalignancies. Immunol Rev 2015;263:90-105.

[0318] 5) Ribas A, Wolchok JD. Cancer immunotherapy using checkpointblockade. Science 359, 1350-1355 (2018).

[0319] 6) Gross G, Eshhar Z. Therapeutic Potential of T Cell ChimericAntigen Receptors (CARs) in Cancer Treatment: Counteracting Off-TumorToxicities for Safe CAR T Cell Therapy. Annu Rev Pharmacol Toxicol 56, 59-83(2016).

[0320] 7) Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigenreceptor-modified T cells in chronic lymphoid leukemia. N Engl J Med 365,725-733 (2011).

[0321] 8) Topp, M.S., et al. Targeted therapy with the T-cell-engagingantibody blinatumomab of chemotherapy-refractory minimal residual disease inB-lineage acute lymphoblastic leukemia patients results in high response rateand prolonged leukemia-free survival. J Clin Oncol 29, 2493-2498 (2011).

[0322] 9) Portell, C.A., Wenzell, C.M. & Advani, A.S. Clinical andpharmacologic aspects of blinatumomab in the treatment of B-cell acutelymphoblastic leukemia. Clin Pharmacol 5, 5-11 (2013).

[0323] 10) Riethmuller, G. Symmetry breaking: bispecific antibodies, thebeginnings, and 50 years on. Cancer Immun 12, 12 (2012).

[0324] 11) Pishvaian M, Morse MA, McDevitt J, Norton JD, Ren S, Robbie GJ,Ryan PC, Soukharev S, Bao H, Denlinger CS. Phase 1 Dose Escalation Study ofMEDI-565, a Bispecific T-Cell Engager that Targets Human CarcinoembryonicAntigen, in Patients With Advanced Gastrointestinal Adenocarcinomas. ClinColorectal Cancer. 15:345-351, 2016.

[0325] 12) Kebenko M, Goebeler ME, Wolf M, Hasenburg A, Seggewiss-BernhardtR, Ritter B, Rautenberg B, Atanackovic D, Kratzer A, Rottman JB, Friedrich M,Vieser E, Elm S, Patzak I, Wessiepe D, Stienen S, Fiedler W. A multicenterphase 1 study of solitomab (MT110, AMG 110), a bispecific EpCAM / CD3 T-cellengager (BiTE®) antibody construct, in patients with refractory solidtumors. Oncoimmunology. 7: e1450710, 2018.

[0326] 13) Ganss, R., Ryschich, E., Klar, E., Arnold, B. & Hammerling, G.J.Combination of T-cell therapy and trigger of inflammation induces remodelingof the vasculature and tumor eradication. Cancer Res 62, 1462-1470 (2002).14)Sondel, P.M., Sosman, J.A., Hank, J.A., Kohler, P.C. & Storer, B. Tumor-infiltrating lymphocytes and interleukin-2 in melanomas. N Engl J Med 320,1418-1419 (1989).

Claims

1. An isolated antigen-binding protein (ABP) that specifically binds to CD276 protein or a variant thereof, wherein the isolated ABP is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) against cells expressing CD276 protein, wherein the ABP is an antibody or an antigen-binding fragment thereof, comprising at least one antibody heavy chain sequence and at least one antibody light chain sequence; and the ABP has at least one antigen-binding domain, wherein: It includes the antibody heavy chain CDR1 sequence shown in SEQ ID NO: 33, the antibody heavy chain CDR2 sequence shown in SEQ ID NO: 34, and the antibody heavy chain CDR3 sequence shown in SEQ ID NO: 35; and the antibody light chain CDR1 sequence shown in SEQ ID NO: 36, the antibody light chain CDR2 sequence shown in SEQ ID NO: 37, and the antibody light chain CDR3 sequence shown in SEQ ID NO:

38.

2. The isolated ABP according to claim 1, wherein the ABP is an antibody or an antigen-binding fragment thereof, which consists of two antibody heavy chain sequences and two antibody light chain sequences.

3. The isolated ABP according to claim 1 or 2, wherein the ABP contains the SDIE mutation.

4. The isolated ABP according to claim 1 or 2, comprising one or more additional antigen-binding domains that bind to an antigen other than CD276 or its variants, wherein one or more additional antigen-binding domains that bind to an antigen other than CD276 or its variants bind to human CD3, and comprising a UCHT1 anti-CD3 scFv construct.

5. A bispecific antigen-binding protein (ABP) comprising a first antigen-binding domain capable of binding to the CD276 antigen or a variant thereof, and a second antigen-binding domain capable of binding to an antigen expressed on an immune cell; wherein the first antigen-binding domain is the antigen-binding domain of the ABP according to claim 1, and wherein the second antigen-binding domain comprises a heavy chain variable region and a light chain variable region of UCHT1.

6. The bispecific ABP according to claim 5, having the activity of binding to CD3-expressing T cells and CD276-expressing tumor cells, or tumor cells adjacent to CD276-expressing cells.

7. The bispecific ABP according to claim 6, wherein the tumor cells adjacent to CD276-expressing cells are tumor vascular cells.

8. The bispecific ABP of claim 6, wherein the bispecific ABP increases the recruitment of cytotoxic cells to CD276-expressing cells by binding to CD276 and CD3.

9. An isolated nucleic acid comprising encoding an ABP according to any one of claims 1 to 4, or an antigen-binding fragment or monomer of an ABP, or a sequence encoding a bispecific ABP according to claim 5 or 6.

10. A nucleic acid construct (NAC) comprising the nucleic acid of claim 9 and one or more additional sequence features, said sequence features allowing the expression of the encoded ABP or a bispecific ABP, or a component of said ABP or a bispecific ABP, in cells.

11. A recombinant host cell comprising the nucleic acid according to claim 9 or the NAC according to claim 10.

12. A pharmaceutical composition comprising: (i) an ABP or a bispecific ABP according to any one of claims 1 or 5, or (ii) a nucleic acid according to claim 9 or a NAC according to claim 10, or (iii) a recombinant host cell according to claim 11, and a pharmaceutically acceptable excipient.

13. The pharmaceutical composition according to claim 12, wherein the excipient is a carrier or a stabilizer.

14. Use of a medicament for the treatment or prevention of proliferative diseases characterized by expression of CD276, selected from the components of an ABP or bispecific ABP according to any one of claims 1 or 5, an isolated nucleic acid according to claim 9, or an NAC according to claim 10, a recombinant host cell according to claim 11, and a pharmaceutical composition according to claim 12.

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