Anti-B7-H3 Antibodies and Uses Thereof

Abstract

The present disclosure provides anti-B7-H3 antibodies, including mouse, humanized and human antibodies, as well as those with further optimized CDR sequences. These antibodies are suitable for use in the treatment of diseases such as cancer. One embodiment of the present disclosure provides an antibody or antigen-binding fragment thereof having specificity for human B7-H3 (CD276) protein, comprising a heavy chain variable region (VH) comprising VH CDR1, VH CDR2 and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3.

Description

[Technical field]

[0001] background B7 is a family of integral membrane proteins found on activated antigen-presenting cells (APCs). When paired with either CD28 or CD152 (CTLA-4) surface proteins on T cells, B7 proteins can generate costimulatory or co-inhibitory signals to enhance or reduce the activity of MHC-TCR signals between APCs and T cells, respectively. Binding of B7 on APCs to CTLA-4 on T cells causes inhibition of T cell activity. [Background technology]

[0002] There are two major types of B7 proteins: B7-1 or CD80, and B7-2 or CD86. The proteins CD28 and CTLA-4 (CD152) interact with both B7-1 and B7-2, respectively. Other proteins in this family include B7-DC (PD-L2), B7-H1 (PD-L1), B7-H2 (ICOSLG), B7-H3 (CD276), B7-H4 (VTCN1), B7-H5 (VISTA), B7-H6, and B7-H7.

[0003] B7-H3 is a type I transmembrane protein that contains an extracellular immunoglobulin-like variable region (IgV) and constant region (IgC), a transmembrane region, and a short cytoplasmic region. B7-H3 has two splice variants, B7-H3a and B7-H3b. The extracellular domain of B7-H3a contains two immunoglobulin domains of IgV-IgC (also known as 2IgB7-H3), while the extracellular domain of B7-H3b consists of four immunoglobulin domains of IgV-IgC-IgV-IgC (also known as 4IgB7-H3). 4IgB7-H3 is the predominant isoform in humans, while 2IgB7-H3 is the only isoform in mice.

[0004] B7-H3 protein is not or poorly expressed in normal tissues and cells, but is highly expressed in various tumor tissues and is closely correlated with tumor progression, patient survival and prognosis. B7-H3 has been clinically reported to be overexpressed in many types of cancer, especially in non-small cell lung cancer, renal cancer, urothelial carcinoma, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer and pancreatic cancer. In addition, it has been reported in the literature that in prostate cancer, the expression level of B7-H3 is positively correlated with clinicopathological malignant lesions and cancer progression. Similarly, in glioblastoma multiforme, B7-H3 expression is inversely correlated with event-free survival, and in pancreatic cancer, B7-H3 expression is associated with lymph node metastasis and disease progression. Therefore, B7-H3 is a suitable potential therapeutic target. It has been demonstrated that antibodies targeting B7-H3 can enhance infiltrating CD8 positive T cells in tumors and inhibit tumor growth. Summary of the Invention [Means for solving the problem]

[0005] Abstract The present disclosure provides, in various embodiments, antibodies and antigen-binding fragments specific for human B7-H3 protein. Both murine antibodies and their humanized counterparts, as well as fully human antibodies, have been identified. These antibodies generally have superior properties to the benchmark antibody MGA017 (MacroGenics). These antibodies, together with molecules derived from them, such as chimeric antigen receptors (CARs), multispecific antibodies, can be suitably used to treat diseases such as cancer.

[0006] Thus, one embodiment of the present disclosure provides an antibody or antigen-binding fragment thereof having specificity for human B7-H3 (CD276) protein, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3.

[0007] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 25; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 26, 27, or 28; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 29; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 30; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 32.

[0008] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 33; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 34; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 35; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 36; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 37; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 38.

[0009] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 39; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 40; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 41, 42, 43, 44, 45 or 46; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 47, 48 or 49; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 50; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 51.

[0010] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:52; VH CDR2 comprises the amino acid sequence of SEQ ID NO:53; VH CDR3 comprises the amino acid sequence of SEQ ID NO:54; VL CDR1 comprises the amino acid sequence of SEQ ID NO:55; VL CDR2 comprises the amino acid sequence of SEQ ID NO:56; and VL CDR3 comprises the amino acid sequence of SEQ ID NO:57.

[0011] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:58; VH CDR2 comprises the amino acid sequence of SEQ ID NO:59; VH CDR3 comprises the amino acid sequence of SEQ ID NO:60; VL CDR1 comprises the amino acid sequence of SEQ ID NO:61; VL CDR2 comprises the amino acid sequence of SEQ ID NO:62; and VL CDR3 comprises the amino acid sequence of SEQ ID NO:63.

[0012] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 64; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 65 or 66; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 67; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 68; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 69; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 70.

[0013] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 71; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 72, 73 or 74; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 75; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 76; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 77; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 78.

[0014] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:79; VH CDR2 comprises the amino acid sequence of SEQ ID NO:80; VH CDR3 comprises the amino acid sequence of SEQ ID NO:81; VL CDR1 comprises the amino acid sequence of SEQ ID NO:82; VL CDR2 comprises the amino acid sequence of SEQ ID NO:83; and VL CDR3 comprises the amino acid sequence of SEQ ID NO:84.

[0015] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 85; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 86 or 87; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 88; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 89; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 90; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 91.

[0016] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 92; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 93, 94, 95 or 96; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 97; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 98; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 99 or 100; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 101.

[0017] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 102; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 103; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 104; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 105; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 106; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 107.

[0018] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 108; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 109; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 110, 111 or 112; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 113, 114 or 115; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 116; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 117.

[0019] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 182; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 183, 184, 185, or 186; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 187; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 188, 189, 190, or 191; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 192; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 193.

[0020] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:39; VH CDR2 comprises the amino acid sequence of SEQ ID NO:40; VH CDR3 comprises the amino acid sequence of SEQ ID NO:41; VL CDR1 comprises the amino acid sequence of SEQ ID NO:47; VL CDR2 comprises the amino acid sequence of SEQ ID NO:50; and VL CDR3 comprises the amino acid sequence of SEQ ID NO:51.

[0021] In some embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and 159-162, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6 and 163-166. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 159, and the VL comprises the amino acid sequence of SEQ ID NO: 165.

[0022] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 39; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 40; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 41; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 48 or 49; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 50; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 51.

[0023] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 159 and the VL comprises the amino acid sequence of SEQ ID NO: 178. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 159 and the VL comprises the amino acid sequence of SEQ ID NO: 179.

[0024] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:52; VH CDR2 comprises the amino acid sequence of SEQ ID NO:53; VH CDR3 comprises the amino acid sequence of SEQ ID NO:54; VL CDR1 comprises the amino acid sequence of SEQ ID NO:55; VL CDR2 comprises the amino acid sequence of SEQ ID NO:56; and VL CDR3 comprises the amino acid sequence of SEQ ID NO:57.

[0025] In some embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and 167-169, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and 170-173.

[0026] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 33; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 34; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 35; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 36; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 37; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 38.

[0027] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:3 and the VL comprises the amino acid sequence of SEQ ID NO:4.

[0028] In one embodiment, an antibody or antigen-binding fragment thereof having specificity for human B7-H3 (CD276) protein is also provided, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3.

[0029] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 126; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 127 or 128; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 129, 130 or 131; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 132; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 133; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 134.

[0030] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 135; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 136 or 137; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 138; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 139; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 140; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 141.

[0031] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 142; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 143; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 144; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 145; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 146; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 147 or 148.

[0032] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 149; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 150, 151 or 152; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 153 or 154; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 155; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 156; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 157 or 158.

[0033] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 149; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 150; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 153; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 155; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 156; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 157.

[0034] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:118 and the VL comprises the amino acid sequence of SEQ ID NO:119.

[0035] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:120 and the VL comprises the amino acid sequence of SEQ ID NO:121.

[0036] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:122 and the VL comprises the amino acid sequence of SEQ ID NO:123.

[0037] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:124 and the VL comprises the amino acid sequence of SEQ ID NO:125.

[0038] In some embodiments, the antibody or fragment thereof is a bivalent Fab antibody or a fragment selected from the group consisting of F(ab')2, F(ab)2, Fab', Fab, Fv and scFv.

[0039] Multispecific antibodies are also provided that include an antigen-binding fragment of the disclosure and one or more antibodies or antigen-binding fragments that have binding specificity for a target antigen that is not B7-H3.

[0040] Still further provided is a chimeric antigen receptor (CAR), comprising the antigen-binding fragment of the present disclosure, a transmembrane domain, a costimulatory domain, and a CD3ζ intracellular domain.Still further provided is one or more polynucleotides encoding the antibody or its antigen-binding fragment or CAR of the present disclosure.In some embodiments, the polynucleotide is one or more mRNAs.

[0041] Methods and uses for treating diseases such as cancer are also provided. [Brief description of the drawings]

[0042] [Figure 1] Figure 1 shows that the tested antibodies bound to the B7-H3 protein on A375 tumor cells. With the exception of AHP05564, AHP06331, AHP06345, and AHP06358, all of the tested antibodies showed better binding than the clinical benchmark, MGA017.

[0043] [Diagram 2]FIG. 2 shows the internalization potency of the tested antibodies against A375 tumor cells.

[0044] [Diagram 3] FIG. 3 shows the in vitro killing efficacy of B7-H3 ADC against RKO tumor cells.

[0045] [Figure 4] FIG. 4 shows the binding of humanized antibodies to B7-H3 expressed on A375 tumor cells.

[0046] [Diagram 5] FIG. 5 shows the binding activity of the chimeric antibody after removal of potential PTM sites for B7-H3 expressed on A375 tumor cells.

[0047] [Figure 6] FIG. 6 shows the binding activity of PTM-deleted humanized antibodies to A375 tumor cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] Detailed Description definition It should be noted that the term "a" or "an" entity refers to one or more of that entity, e.g., "an antibody" is understood to represent one or more antibodies. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0049] As used herein, "antibody" or "antigen-binding fragment" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen-binding fragment thereof or a single chain. Thus, the term "antibody" includes any protein or peptide containing molecule that includes at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Examples of such include, but are not limited to, the complementarity determining regions (CDRs) of a heavy or light chain or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, or at least a portion of a binding protein.

[0050] The term "antibody fragment" or "antigen-binding fragment" as used herein refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds with the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegeleisen, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0051] The term antibody encompasses a wide variety of classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses within these (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody, such as IgG, IgM, IgA, IgG, or IgE, respectively.

[0052] Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and known to provide functional specialization. Modified versions of each of these classes and isotypes are readily discernible to one of skill in the art in light of this disclosure and are therefore within the scope of this disclosure. All immunoglobulin classes are expressly within the scope of this disclosure, and the following discussion will generally refer to the IgG class of immunoglobulin molecules. With respect to IgG, a standard immunoglobulin molecule contains two identical light chain polypeptides of approximately 23,000 daltons molecular weight and two identical heavy chain polypeptides of 53,000-70,000 daltons molecular weight. The four chains are typically linked by disulfide bonds in a "Y" configuration, with the light chains flanking the heavy chains on either side, beginning at the mouth of the "Y" and continuing through the variable region.

[0053] Antibodies, antigen-binding polypeptides thereof, variants or derivatives of the disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized or chimeric antibodies, single chain antibodies, epitope-binding fragments such as Fab, Fab' and F(ab')2, Fd, Fv, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (sdFv), fragments comprising the VK or VH domains, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to the LIGHT antibodies disclosed herein). Immunoglobulin or antibody molecules of the disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

[0054] As used herein, the term "chimeric antibody" should be taken to mean any antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial, or modified, according to the present disclosure) is obtained from a second species. In certain embodiments, the target binding region or site will be from a non-human source (e.g., mouse or primate) and the constant region is human.

[0055] The antibody disclosed herein can be of any animal origin, including birds and mammals.Preferably, the antibody is human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibody.In some embodiments, the variable region can be of chondrichthyan origin (e.g., from shark).

[0056] As used herein, the term "recombinant" when referring to a polypeptide or polynucleotide, refers to a form of a polypeptide or polynucleotide that does not occur in nature, a non-limiting example of which may be made by combining polynucleotides that do not normally occur together.

[0057] Hybridoma techniques can be performed under conditions of different "stringency". Generally, low stringency hybridization reactions are carried out at about 40°C in about 10xSSC, or a solution of equivalent ionic strength / temperature. Medium stringency hybridization is typically carried out at about 50°C in about 6xSSC, and high stringency hybridization reactions are generally carried out at about 60°C in about 1xSSC. Hybridization reactions can also be performed under "physiological conditions", which are well known to those of skill in the art. Non-limiting examples of physiological conditions include the temperature, ionic strength, pH and Mg normally found in cells. 2+ Concentration. Anti-B7-H3 antibody

[0058] As demonstrated in the accompanying experimental examples, the inventors were able to generate anti-B7-H3 antibodies 35A12B11, 62H9H5, 72D1D11, 81C8A1, 97E6B2, 106A5B3, 126C10B10, 216E7A9, 227E12D1, 294A3C4, 312E1E2 and 429H9F10 (Table 1), all of which have higher binding affinity to human B7-H3 protein than the benchmark antibody MGA017 (MacroGenics).

[0059] Meanwhile, fully human antibodies have been identified from customized phage antibody libraries, including AHP05564, AHP06331, AHP06345 and AHP06358 (Table 2). These human antibodies also have the unique properties of MGA017, highlighting their promise.

[0060] Likely due to improved binding affinity, all of the chimeric versions of the mouse antibodies led to a higher degree of cellular internalization than MGA017 when assessed with A375 cells (Figure 2A).Surprisingly, two of the fully human antibodies, AHP06331 and AHP06358, also performed much better than MGA017 at high concentrations, despite their relatively modest performance at low concentrations (Figure 2B).

[0061] Antibody-drug conjugates (ADCs) of these antibodies were also prepared and tested, and again, at least the ADCs of 72D1D11, 81C8A1, 62H9H5, 106A5B3, 126C10B10 and 227E12D outperformed the vc-MMAE conjugate MGA017 (Figure 3), further supporting the superior properties of these new antibodies.

[0062] Based on the binding competition assay, the antibodies were categorized into five different epitope binning groups, Aα, Aβ, Aγ, B and C, as shown in Table 3B. The Aα group includes MGA017 along with 62H9H5, 81C8A1, 106A5B3, 216E7A9, and 227E12D1, although the majority of the antibodies are not in this group. The Aβ group includes 72D1D11, 97E6B2, 126C10B10, and 429H9F10, the Aγ group includes 35A12B11, 294A3C4, and AHP06358, group B includes 312E1E12 and AHP06345, and group C includes AHP06331 and AHP05564.

[0063] Thus, according to one embodiment of the present disclosure, an antibody or an antigen-binding fragment thereof is provided. In some embodiments, the antibody or an antigen-binding fragment thereof has binding properties to human B7-H3 protein. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3.

[0064] In one embodiment, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are of an antibody identified herein, e.g., any of 35A12B11, 62H9H5, 72D1D11, 81C8A1, 97E6B2, 106A5B3, 126C10B10, 216E7A9, 227E12D1, 294A3C4, 312E1E2, and 429H9F10, or any of AHP05564, AHP06331, AHP06345, and AHP06358.

[0065] In some embodiments, with reference to antibody 72D1D11, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 39, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 40, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 41, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 47, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 50, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 51.

[0066] The antibody or fragment may be murine, chimeric or humanized. Exemplary antibodies include those shown in Table 5. In some embodiments, the antibody or fragment has a VH with the amino acid sequence of SEQ ID NO: 5 (murine), 159, 160, 161 or 162 (humanized). In some embodiments, the antibody or fragment has a VL with the amino acid sequence of SEQ ID NO: 6 (murine), 163, 164, 165 or 166 (humanized). Exemplary pairings of VH / VL sequences are shown in Table 5B. For example, in one embodiment, the antibody or fragment comprises a VH of SEQ ID NO: 159 and a VL of 165. In another embodiment, the antibody or fragment comprises a VH of SEQ ID NO: 159 and a VL of 163.

[0067] Sequence analysis revealed that the VH CDR3 sequence (SEQ ID NO: 41) and the VL CDR1 sequence (SEQ ID NO: 47) contain residues that can potentially be post-translationally modified. To avoid the risk of post-translational modifications (PTMs) and thus simplify production, the present disclosure has designed and tested certain de-risked versions of VH CDR3, including SEQ ID NOs: 42, 43, 44, 45 and 46, and VL CDR1, including SEQ ID NOs: 48 and 49.

[0068] In some embodiments, therefore, antibodies and fragments are provided having CDR sequences in which VH CDR1 comprises the amino acid sequence of SEQ ID NO: 39, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 40, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 41, 42, 43, 44, 45 or 46, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 47, 48 or 49, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 50 and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 51.

[0069] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 39, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 40, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 41, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 47, 48 or 49, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 50, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 51.

[0070] Exemplary antibody sequences with or without PTM-derisked CDRs are also provided. As shown in Table 9. In some embodiments, the antibody or fragment has a VH with the amino acid sequence of SEQ ID NO: 159, 174, 175, 176, or 177. In some embodiments, the antibody or fragment has a VL with the amino acid sequence of SEQ ID NO: 165, 178, or 179. Exemplary pairings of VH / VL sequences are shown in Table 8. For example, in one embodiment, the antibody or fragment comprises a VH of SEQ ID NO: 159 and a VL of 178. In another embodiment, the antibody or fragment comprises a VH of SEQ ID NO: 159 and a VL of 179.

[0071] In some embodiments, antibodies and antigen-binding fragments are provided that bind to the same epitope on B7-H3 as 72D1D11. In some embodiments, antibodies and antigen-binding fragments are provided that compete with 72D1D11 for binding to B7-H3.

[0072] In some embodiments, with reference to antibody 81C8A1, VH CDR1 comprises the amino acid sequence of SEQ ID NO:52, VH CDR2 comprises the amino acid sequence of SEQ ID NO:53, VH CDR3 comprises the amino acid sequence of SEQ ID NO:54, VL CDR1 comprises the amino acid sequence of SEQ ID NO:55, VL CDR2 comprises the amino acid sequence of SEQ ID NO:56, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:57.

[0073] The antibody or fragment may be murine, chimeric or humanized. Exemplary antibodies include those shown in Table 6. In some embodiments, the antibody or fragment has a VH with the amino acid sequence of SEQ ID NO: 7 (murine), 167, 168 or 169 (humanized). In some embodiments, the antibody or fragment has a VL with the amino acid sequence of SEQ ID NO: 8 (murine), 170, 171, 172 or 173 (humanized). Exemplary pairings of VH / VL sequences are shown in Table 6B. For example, in one embodiment, the antibody or fragment comprises a VH of SEQ ID NO: 167 and a VL of 172. In another embodiment, the antibody or fragment comprises a VH of SEQ ID NO: 167 and a VL of 173. In another embodiment, the antibody or fragment comprises a VH of SEQ ID NO: 168 and a VL of 171. In another embodiment, the antibody or fragment comprises a VH of SEQ ID NO: 168 and a VL of 172. In another embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO:168 and a VL of 173.

[0074] In some embodiments, antibodies and antigen-binding fragments that bind to the same epitope on B7-H3 as 81C8A1 are also provided. In some embodiments, antibodies and antigen-binding fragments that compete with 81C8A1 for binding to B7-H3 are also provided.

[0075] In some embodiments, with reference to antibody 35A12B11, VH CDR1 comprises the amino acid sequence of SEQ ID NO:25, VH CDR2 comprises the amino acid sequence of SEQ ID NO:26, VH CDR3 comprises the amino acid sequence of SEQ ID NO:29, VL CDR1 comprises the amino acid sequence of SEQ ID NO:30, VL CDR2 comprises the amino acid sequence of SEQ ID NO:31, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:32.

[0076] Sequence analysis revealed that the VH CDR2 sequence (SEQ ID NO: 26) contains residues that can potentially be post-translationally modified. To avoid the risk of post-translational modifications (PTMs) and thus simplify manufacturing, the present disclosure has designed and tested certain de-risked versions of VH CDR2, including SEQ ID NOs: 27 and 28.

[0077] In some embodiments, therefore, antibodies and fragments are provided having CDR sequences in which VH CDR1 comprises the amino acid sequence of SEQ ID NO: 25, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 26, 27 or 28, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 29, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 30, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31 and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 32.

[0078] In one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO: 1 and a VL of SEQ ID NO: 2. In some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on B7-H3 as 35A12B11. In some embodiments, antibodies and antigen-binding fragments are also provided that compete with 35A12B11 for binding to B7-H3.

[0079] In some embodiments, with reference to antibody 62H9H5, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 33, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 34, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 35, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 36, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 37, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 38.

[0080] In one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO: 3 and a VL of SEQ ID NO: 4. In some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on B7-H3 as 62H9H5. In some embodiments, antibodies and antigen-binding fragments are also provided that compete with 62H9H5 for binding to B7-H3.

[0081] In some embodiments, with reference to antibody 97E6B2, VH CDR1 comprises the amino acid sequence of SEQ ID NO:58, VH CDR2 comprises the amino acid sequence of SEQ ID NO:59, VH CDR3 comprises the amino acid sequence of SEQ ID NO:60, VL CDR1 comprises the amino acid sequence of SEQ ID NO:61, VL CDR2 comprises the amino acid sequence of SEQ ID NO:62, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:63.

[0082] In one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO: 9 and a VL of 10. In some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on B7-H3 as 97E6B2. In some embodiments, antibodies and antigen-binding fragments are also provided that compete with 97E6B2 for binding to B7-H3.

[0083] In some embodiments, with reference to antibody 106A5B3, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 64, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 65, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 67, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 68, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 69, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 70.

[0084] Sequence analysis revealed that the VH CDR2 sequence (SEQ ID NO:65) contains residues that can potentially be post-translationally modified. To avoid the risk of post-translational modifications (PTMs) and thus simplify production, the present disclosure has designed and tested a particular de-risked version of VH CDR2, comprising SEQ ID NO:66.

[0085] In some embodiments, therefore, antibodies and fragments are provided having CDR sequences in which VH CDR1 comprises the amino acid sequence of SEQ ID NO: 64, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 65 or 66, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 67, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 68, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 69, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 70.

[0086] In one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO: 11 and a VL of 12. In some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on B7-H3 as 106A5B3. In some embodiments, antibodies and antigen-binding fragments are also provided that compete with 106A5B3 for binding to B7-H3.

[0087] In some embodiments, with reference to antibody 126C10B10, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 71, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 72, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 75, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 76, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 77, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 78.

[0088] Sequence analysis revealed that the VH CDR2 sequence (SEQ ID NO: 72) contains residues that can potentially be post-translationally modified. To avoid the risk of post-translational modifications (PTMs) and thus simplify production, the present disclosure has designed and tested certain de-risked versions of VH CDR2, including SEQ ID NOs: 73 and 74.

[0089] In some embodiments, therefore, antibodies and fragments are provided having CDR sequences in which VH CDR1 comprises the amino acid sequence of SEQ ID NO: 71, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 72, 73 or 74, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 75, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 76, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 77 and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 78.

[0090] In one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO: 13 and a VL of 14. In some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on B7-H3 as 126C10B10. In some embodiments, antibodies and antigen-binding fragments are also provided that compete with 126C10B10 for binding to B7-H3.

[0091] In some embodiments, with reference to antibody 216E7A9, VH CDR1 comprises the amino acid sequence of SEQ ID NO:79, VH CDR2 comprises the amino acid sequence of SEQ ID NO:80, VH CDR3 comprises the amino acid sequence of SEQ ID NO:81, VL CDR1 comprises the amino acid sequence of SEQ ID NO:82, VL CDR2 comprises the amino acid sequence of SEQ ID NO:83, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:84.

[0092] In one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO: 15 and a VL of 16. In some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on B7-H3 as 216E7A9. In some embodiments, antibodies and antigen-binding fragments are also provided that compete with 216E7A9 for binding to B7-H3.

[0093] In some embodiments, with reference to antibody 227E12D1, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 85, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 86, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 88, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 89, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 90, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 91.

[0094] Sequence analysis revealed that the VH CDR2 sequence (SEQ ID NO:86) contains residues that can potentially be post-translationally modified. To avoid the risk of post-translational modifications (PTMs) and thus simplify manufacturing, the present disclosure has designed and tested a particular de-risked version of VH CDR2, including SEQ ID NO:87.

[0095] In some embodiments, therefore, antibodies and fragments are provided having CDR sequences in which VH CDR1 comprises the amino acid sequence of SEQ ID NO: 85, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 86 or 87, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 88, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 89, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 90, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 91.

[0096] In one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO: 17 and a VL of SEQ ID NO: 18. In some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on B7-H3 as 227E12D1. In some embodiments, antibodies and antigen-binding fragments are also provided that compete with 227E12D1 for binding to B7-H3.

[0097] In some embodiments, with reference to antibody 294A3C4, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 92, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 93, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 97, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 98, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 99, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 101.

[0098] Sequence analysis revealed that the VH CDR2 sequence (SEQ ID NO: 93) and the VL CDR2 (SEQ ID NO: 99) contain residues that can potentially be post-translationally modified. To avoid the risk of post-translational modifications (PTMs) and thus simplify manufacturing, the present disclosure has designed and tested certain de-risked versions of the VH CDR2, including SEQ ID NOs: 94, 95, and 96, and the VL CDR2, including SEQ ID NO: 100.

[0099] In some embodiments, therefore, antibodies and fragments are provided having CDR sequences in which VH CDR1 comprises the amino acid sequence of SEQ ID NO: 92, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 93, 94, 95 or 96, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 97, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 98, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 99 or 100, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 101.

[0100] In one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO: 19 and a VL of 20. In some embodiments, antibodies and antigen-binding fragments that bind to the same epitope on B7-H3 as 294A3C4 are also provided. In some embodiments, antibodies and antigen-binding fragments that compete with 294A3C4 for binding to B7-H3 are also provided.

[0101] In some embodiments, with reference to antibody 312E1E2, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 102, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 103, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 104, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 105, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 106, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 107.

[0102] In one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO: 21 and a VL of 22. In some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on B7-H3 as 312E1E2. In some embodiments, antibodies and antigen-binding fragments are also provided that compete with 312E1E2 for binding to B7-H3.

[0103] In some embodiments, with reference to antibody 429H9F10, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 108, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 109, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 110, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 113, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 116, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 117.

[0104] Sequence analysis revealed that the VH CDR3 sequence (SEQ ID NO: 110) and VL CDR1 (SEQ ID NO: 113) contain residues that can potentially be post-translationally modified. To avoid the risk of post-translational modifications (PTMs) and thus simplify production, the present disclosure has designed and tested certain de-risked versions of VH CDR3 comprising SEQ ID NOs: 111 and 112, and VL CDR1 comprising SEQ ID NOs: 114 and 115.

[0105] In some embodiments, therefore, antibodies and fragments are provided having CDR sequences in which VH CDR1 comprises the amino acid sequence of SEQ ID NO: 108, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 109, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 110, 111 or 112, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 113, 114 or 115, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 116 and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 117.

[0106] In one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO: 23 and a VL of 24. In some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on B7-H3 as 429H9F10. In some embodiments, antibodies and antigen-binding fragments are also provided that compete with 429H9F10 for binding to B7-H3.

[0107] In some embodiments, with reference to antibody 43G8A9, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 182; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 183; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 187; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 188; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 192; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 193.

[0108] Sequence analysis revealed that the VH CDR2 sequence (SEQ ID NO: 183) and VL CDR1 (SEQ ID NO: 188) contain residues that can potentially be post-translationally modified. To avoid the risk of post-translational modifications (PTMs) and thus simplify production, the present disclosure has designed and tested certain de-risked versions of VH CDR2 comprising SEQ ID NOs: 184, 185 and 186, and VL CDR1 comprising SEQ ID NOs: 188, 189, 190 and 191.

[0109] In some embodiments, therefore, antibodies and fragments are provided having CDR sequences in which: VH CDR1 comprises the amino acid sequence of SEQ ID NO: 182; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 183, 184, 185, or 186; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 187; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 188, 189, 190, or 191; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 192; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 193.

[0110] In one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO: 180 and a VL of 181. In some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on B7-H3 as 43G8A9. In some embodiments, antibodies and antigen-binding fragments are also provided that compete with 43G8A9 for binding to B7-H3.

[0111] In some embodiments, with reference to antibody AHP05564, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 126, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 127, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 129, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 132, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 133, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 134.

[0112] Sequence analysis revealed that the VH CDR2 (SEQ ID NO: 127) and VH CDR3 sequences (SEQ ID NO: 129) contain residues that can potentially be post-translationally modified. To avoid the risk of post-translational modifications (PTMs) and thus simplify manufacturing, the present disclosure has designed and tested certain de-risked versions of VH CDR2 comprising SEQ ID NO: 128, and VH CDR3 comprising SEQ ID NOs: 130 and 131.

[0113] In some embodiments, therefore, antibodies and fragments are provided having CDR sequences in which VH CDR1 comprises the amino acid sequence of SEQ ID NO: 126, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 127 or 128, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 129, 130 or 131, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 132, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 133, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 134.

[0114] In one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO: 118 and a VL of 119. In some embodiments, antibodies and antigen-binding fragments that bind to the same epitope on B7-H3 as AHP05564 are also provided. In some embodiments, antibodies and antigen-binding fragments that compete with AHP05564 for binding to B7-H3 are also provided.

[0115] In some embodiments, with reference to antibody AHP06331, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 135, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 136, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 138, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 139, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 140, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 141.

[0116] Sequence analysis revealed that VH CDR2 (SEQ ID NO: 136) contains residues that can potentially be post-translationally modified. To avoid the risk of post-translational modifications (PTMs) and thus simplify production, the present disclosure has designed and tested a particular de-risked version of VH CDR2, including SEQ ID NO: 137.

[0117] In some embodiments, therefore, antibodies and fragments are provided having CDR sequences in which VH CDR1 comprises the amino acid sequence of SEQ ID NO: 135, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 136 or 137, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 138, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 139, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 140, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 141.

[0118] In one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO: 120 and a VL of 121. In some embodiments, antibodies and antigen-binding fragments that bind to the same epitope on B7-H3 as AHP06331 are also provided. In some embodiments, antibodies and antigen-binding fragments that compete with AHP06331 for binding to B7-H3 are also provided.

[0119] In some embodiments, with reference to antibody AHP06345, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 142, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 143, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 144, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 145, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 146, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 147.

[0120] Sequence analysis revealed that VL CDR3 (SEQ ID NO: 147) contains residues that can potentially be post-translationally modified. To avoid the risk of post-translational modifications (PTMs) and thus simplify production, the present disclosure has designed and tested a particular de-risked version of VL CDR3, including SEQ ID NO: 148.

[0121] In some embodiments, therefore, antibodies and fragments are provided having CDR sequences in which VH CDR1 comprises the amino acid sequence of SEQ ID NO: 142, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 143, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 144, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 145, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 146, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 147 or 148.

[0122] In one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO: 122 and a VL of 123. In some embodiments, antibodies and antigen-binding fragments that bind to the same epitope on B7-H3 as AHP06345 are also provided. In some embodiments, antibodies and antigen-binding fragments that compete with AHP06345 for binding to B7-H3 are also provided.

[0123] In some embodiments, with reference to antibody AHP06358, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 149, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 150, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 153, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 155, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 156, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 157.

[0124] Sequence analysis revealed that VH CDR2 (SEQ ID NO: 150), VH CDR3 (SEQ ID NO: 153) and VL CDR3 (SEQ ID NO: 157) contain residues that can potentially be post-translationally modified. To avoid the risk of post-translational modifications (PTMs) and thus simplify production, the present disclosure has designed and tested certain de-risked versions of VH CDR2 comprising SEQ ID NO: 151 and 152, VH CDR3 comprising SEQ ID NO: 154, and VL CDR3 comprising SEQ ID NO: 158.

[0125] In some embodiments, therefore, antibodies and fragments are provided having CDR sequences, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 149, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 150, 151 or 152, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 153 or 154, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 155, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 156 and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 157 or 158.

[0126] In one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO: 124 and a VL of 125. In some embodiments, antibodies and antigen-binding fragments that bind to the same epitope on B7-H3 as AHP06358 are also provided. In some embodiments, antibodies and antigen-binding fragments that compete with AHP06358 for binding to B7-H3 are also provided.

[0127] In some embodiments, antibodies and antigen-binding fragments are also provided that comprise CDR sequences derived from those of the disclosure with one, two or three amino acid substitutions, deletions and / or additions.

[0128] In some embodiments, the anti-B7-H3 antibody is a modified mAb that comprises a modified heavy chain constant region, such as an afucosylated heavy chain, that binds with higher affinity to activating Fcγ receptors and mediates enhanced ADCC compared to the unmodified mAb. In some embodiments, the anti-B7-H3 antibody comprises a heavy chain that is of a human IgG1 variant that includes a single L234Y, L235Q, G236W, S239D / M, F243L, H268D, D270E, R292P, S298A, Y300L, V305I, K326D, A330L / M, I332E, K334A / E, P396L (all EU numbering), or a combination thereof, that enhances ADCC function. Antibody-drug conjugates

[0001] In vitro and in vivo data showed that antibody-drug conjugates (ADCs) derived from the new antibodies had then greater anti-tumor activity than the benchmark antibody MGA017, and therefore they are suitable for use in ADCs. In some embodiments, the antibody or fragment may be conjugated to a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent, or PEG. In one embodiment, the antibody or fragment of the present disclosure is covalently attached to a drug moiety. The drug moiety may be a reactive group that has a conjugation point on the antibody, or may be modified to include the group. For example, the drug moiety may be attached by alkylation (e.g., at the epsilon-amino lysine or N-terminus of the antibody), reductive amination of oxidized carbohydrates, transesterification of hydroxyl and carboxyl groups, amidation of amino or carboxyl groups, and conjugation to thiols. In some embodiments, the number of conjugated drug moieties per antibody molecule, p, is in the range of 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 on average. In some embodiments, p is in the range of 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 on average. In other embodiments, p is in the range of 1, 2, 3, 4, 5, 6, 7, or 8 on average. In some embodiments, p is in the range of about 1 to about 20, about 1 to about 10, about 2 to about 10, about 2 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, or about 1 to about 2 on average. In some embodiments, p is in the range of about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, or about 2 to about 3. For example, when chemical activation of a protein results in the formation of a free thiol group, the protein can be conjugated with a sulfhydryl-reactive agent. In one aspect, the agent is substantially specific for free thiol groups. Such agents include, for example, maleimides, haloacetamides (e.g., iodo, bromo, or chloro), haloesters (e.g., iodo, bromo, or chloro), halomethylketones (e.g., iodo, bromo, or chloro), benzyl halides (e.g., iodide, bromide, or chloride), vinylsulfones, and pyridylthios.

[0005] The drug can be linked to the antibody or fragment by a linker. Suitable linkers include, for example, cleavable and non-cleavable linkers. Cleavable linkers are typically susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include, for example, peptide linkers that can be cleaved by intracellular proteases, such as lysosomal or endosomal proteases. In exemplary embodiments, the linker can be a dipeptide linker, such as a valine-citrulline (val-cit), phenylalanine-lysine (phe-lys) linker, or a maleimidocapronic-valine-citruline-p-aminobenzyloxycarbonyl (mc-Val-Cit-PABA) linker. Another linker is sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (smcc). Sulfo-smcc conjugation occurs through a maleimide group, which reacts with sulfhydryls (thiols, -SH), while the sulfo-NHS ester is reactive to primary amines (such as those found in lysine and protein or peptide N-termini). Yet another linker is maleimidocaproyl (mc). Other suitable linkers include linkers that are hydrolyzable at a particular pH or pH range, such as hydrazone linkers. Additional suitable cleavable linkers include disulfide linkers. Linkers, such as mc linkers, can be covalently attached to antibodies to such an extent that the antibody must be degraded intracellularly to release the drug.

[0006] The linker may contain a group for linking to an antibody. For example, the linker may contain an amino, hydroxyl, carboxyl or sulfhydryl reactive group (e.g., maleimide, haloacetamide (e.g., iodo, bromo or chloro), haloester (e.g., iodo, bromo or chloro), halomethylketone (e.g., iodo, bromo or chloro), benzyl halide (e.g., iodide, bromide or chloride), vinylsulfone and pyridylthio). In some embodiments, the drug moiety is a cytotoxic or cytostatic agent, an immunosuppressant, a radioisotope, a toxin, etc. The conjugate can be used to inhibit tumor or cancer cell multiplication, to cause apoptosis in tumor or cancer cells, or to treat cancer in patients. Thus, the conjugate can be used in a variety of situations for the treatment of cancer in animals. The conjugate can be used to deliver drugs to tumor or cancer cells. Without being bound by theory, in some embodiments, the conjugate can bind to or associate with cancer cells that express GPC3, and the conjugate and / or drug can be taken up into tumor or cancer cells by receptor-mediated endocytosis.

[0008] Once inside the cell, one or more specific peptide sequences in the conjugate (e.g., in the linker) are cleaved by hydrolysis by one or more tumor or cancer cell-associated proteases, resulting in the release of the drug. The released drug is then free to migrate into the cell and induce cytotoxic or cytostatic or other activity. In some embodiments, the drug is cleaved from the antibody outside the tumor or cancer cell, and then the drug penetrates the cell or acts on the cell surface.

[0009] Examples of drug moieties or payloads include DM1 (maytansine, N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)- or N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine), mc-MMAD (6-maleimidocaproyl-monomethylauristatin-D or N-methyl-L-valyl-N-[(1S,2R)-2-methoxy-4-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[[(1 S)-2-phenyl-1-(2-thiazolyl)ethyl]amino]propyl]-1-pyrrolidinyl]-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-(9Cl)-L-valinamide), mc-MMAF (maleimidocaproyl-monomethylauristatin F or N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-N-methyl-L-valyl-L-valyl-(3R,4S,5S)-3-methoxy-5-methyl mc-Val-Cit-PABA-MMAE (6-Maleimidocaproyl-ValcCit-(p-aminobenzyloxycarbonyl)-monomethylauristatin E or N-[[[4-[[N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-L-valyl-N5-(αR,βR,2S)-β-methoxy-α-methyl-2-pyrrolidinepropanoyl-L-phenylalanine) DM1 is a derivative of the tubulin inhibitor maytansine, while MMAD, MMAE and MMAF are derivatives of auristatins.In some embodiments, the drug moiety is selected from the group consisting of mc-MMAF and mc-Val-Cit-PABA-MMAE, hi some embodiments, the drug moiety is a maytansinoid or an auristatin.

[0010] The antibody or fragment may be conjugated or fused to a therapeutic agent, which may include a detectable label, such as a radioactive label, an immunomodulatory agent, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent which may be a drug or a toxin, an ultrasound enhancing agent, a non-radioactive label, combinations of these, and other such agents known in the art.

[0011] The antibody can be detectably labeled by coupling with a chemiluminescent compound. The presence of the chemiluminescent-tagged antigen-binding polypeptide is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

[0012] Fluorescent metals, e.g. 152Antibodies can also be detectably labeled using Eu, or others of the lanthanide series. These metals can be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).Techniques for conjugating various moieties to antibodies are well known and are described, for example, in Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al.(eds.), pp. 243-56 (Alan R. Liss, Inc. (1985);Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al., (eds.), Marcel Dekker, Inc., pp. 623- 53 (1987);Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al.(eds.), pp. 475-506 (1985);"Analysis, Results, And Future Developments in Antibodies See, "Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. (52:119-58 (1982)). multifunctional molecules

[0129] A multifunctional molecule comprising an antibody or antigen-binding fragment specific for B7-H3, such as those disclosed herein, and one or more antibodies or antigen-binding fragments having specificity for a second antigen.

[0130] In some embodiments, the second antigen is a protein expressed on immune cells, such as T cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, phagocytes, natural killer cells, eosinophils, basophils and mast cells.

[0131] In some embodiments, the second antigen is against CD3, CD47, PD1, PD-L1, LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, CEA, HER2, CMET, 4-1BB, OX40, SIRPA CD16, CD28, ICOS, CTLA4, BTLA, TIGIT, HVEM, CD27, VEGFR, or VEGF.

[0132] Different formats of bispecific antibodies are also provided. In some embodiments, each of the anti-B7-H3 fragment and the second fragment is independently selected from a Fab fragment, a single chain variable fragment (scFv), or a single domain antibody. In some embodiments, the bispecific antibody further comprises an Fc fragment.

[0133] Bifunctional molecules are also provided that do not simply comprise antibodies or antigen-binding fragments. As tumor antigen targeting molecules, antibodies or antigen-binding fragments specific to B7-H3, such as those described herein, can be combined with immunocytokines or ligands, optionally via peptide linkers. The linked immunocytokines or ligands include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, GM-CSF, TNF-α, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT and GITRL. Such bifunctional molecules can combine immune checkpoint blocking effects with tumor site local immune regulation. Chimeric Antigen Receptor

[0134] In one embodiment, a chimeric antigen receptor (CAR) is also provided, comprising the antibody or fragment thereof of the present disclosure as a targeting unit. In some embodiments, the CAR comprises the antibody or fragment thereof of the present disclosure, a transmembrane domain, a costimulatory domain, and a CD3ζ intracellular domain.

[0135] The transmembrane domain can be designed to be fused to the extracellular domain, including antibody or fragment, optionally via hinge domain. It can also be fused to an intracellular domain, such as a costimulatory domain. In some embodiments, the transmembrane domain can include the natural transmembrane region of the costimulatory domain (e.g., the TM region of CD28T or 4-1BB used as costimulatory domain) or the natural transmembrane domain of the hinge region (e.g., the TM region of CD8alpha or CD28T used as hinge domain).

[0136] In some embodiments, a transmembrane domain may comprise a sequence that spans a cell membrane, but extends into the cytoplasm of the cell and / or into the extracellular space. For example, a transmembrane may comprise a membrane-spanning sequence that itself may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids that extend into the cytoplasm of the cell and / or into the extracellular space. Thus, a transmembrane domain may comprise a region that spans the membrane, and may further comprise amino acids that extend beyond the inner or outer surface of the membrane itself, and still be considered to be a "transmembrane domain".

[0137] In some embodiments, the transmembrane domain is fused to the cytoplasmic domain via a short linker. Optionally, a short peptide or polypeptide linker, preferably between 2 and 10 amino acids in length, can form the link between the transmembrane domain and the proximal cytoplasmic signaling domain of the chimeric receptor. A glycine-serine doublet (GS), a glycine-serine-glycine triplet (GSG), or an alanine-alanine-alanine triplet (AAA) would be a suitable linker.

[0138] In some embodiments, the CAR further comprises a costimulatory domain. In some embodiments, the costimulatory domain is located between the transmembrane domain and the activation domain. Examples of costimulatory domains include CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8a, CD8, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (T FRSF7), CD28, CD28T, CD29(ITGB1), CD30(TNFRSF8), CD40(TNFRSF5), CD48(SLAMF2), CD49a(ITGA1), CD49d(ITGA4), CD49f(ITGA6), CD66a(CEACAM1), CD66b(CEACAM8), CD66c(CEACAM6), CD66d(CEACAM3), CD66e(CEACAM5), CD69(CLEC2), CD79A(B cell antigen receptor complex-associated alpha chain), CD79B(B cell antigen receptor complex-associated beta chain), CD84(SLAMF5), CD96(Tactile), CD100(SEMA4D), CD103 (ITGAE), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD158A(KIR2DL1), CD158B1(KI R2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1), CD158D(KIRDL4), CD158F1(KIR2DL5A), C D158F2(KIR2DL5B), CD158K(KTR3DL2), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD 229(SLAMF3), CD244(SLAMF4), CD247(CD3-zeta), CD258(LIGHT), CD268(BAFFR), CD270(T FSF14), CD272(BTLA), CD276(B7-H3), CD279(PD-1), CD314(KG2D), CD319(SLAMF7), CD335(K-p46 ), CD336(K-p44), CD337(K-p30), CD352(SLAMF6), CD353(SLAMF8), CD355(CRTAM), CD357(TNFRSF 18), inducible T cell costimulatory factor (ICOS), LFA-1 (CD 11a / CD 18), KG2C, DAP-10, ICAM-1, Kp80 (KLRF1), IL-2R beta, IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, activating NK cell receptor, Toll ligand receptor, and fragments or combinations thereof.

[0139] In some embodiments, the cytoplasmic portion of the CAR also comprises a signaling / activation domain. In one embodiment, the signaling / activation domain is a CD3 zeta domain or an amino acid sequence having at least about 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a CD3 zeta domain. Methods for expressing or preparing polynucleotides, mRNA, and antibodies

[0140] The present disclosure also provides a polynucleotide or nucleic acid molecule encoding the antibody, variant or derivative thereof, or CAR of the present disclosure. The polynucleotide of the present disclosure may encode the entire heavy and light chain variable regions of the antigen-binding polypeptide, variant or derivative thereof on the same polynucleotide molecule or on separate polynucleotide molecules. In addition, the polynucleotide of the present disclosure may encode portions of the heavy and light chain variable regions of the antigen-binding polypeptide, variant or derivative thereof on the same polynucleotide molecule or on separate polynucleotide molecules. It may be encoded on the molecule.

[0141] In some embodiments, the polynucleotide is an mRNA molecule. In some embodiments, the mRNA can be introduced into a target cell for expression of the antibody or fragment thereof.

[0142] mRNA can be synthesized according to any of a variety of known methods. For example, mRNA can be synthesized by in vitro transcription (IVT). Briefly, IVT is typically carried out using linear or circular template DNA, containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor. Stringent conditions vary depending on specific applications.

[0143] In some embodiments, for the preparation of mRNA encoding an antibody, the template DNA is transcribed in vitro. A suitable template DNA typically has a promoter for in vitro transcription, such as a T3, T7 or SP6 promoter, followed by the desired nucleotide sequence for the mRNA encoding the desired antibody (e.g., encoding a heavy or light chain), and a termination signal.

[0144] Using standard methods, the mRNA sequence encoding the desired antibody (e.g., encoding a heavy or light chain) can be determined and incorporated into the template DNA. For example, starting from the desired amino acid sequence (e.g., the desired heavy or light chain sequence), a virtual back-translation is performed based on the degenerate genetic code. An optimization algorithm can then be used to select the appropriate codons. Typically, the G / C content can be optimized on the one hand to achieve as high a G / C content as possible, and on the other hand to take into account as much as possible the frequency of tRNA according to the codon usage frequency. The optimized RNA sequence can be established and displayed, for example with the aid of a suitable display device, and compared with the original (wild type) sequence. The secondary structure can also be analyzed to calculate the stabilizing and destabilizing properties or, respectively, regions of the RNA.

[0145] mRNA can be synthesized as unmodified or modified mRNA.Typically, mRNA is modified to improve stability.Modification of mRNA can include, for example, modification of nucleotide of RNA.Thus, modified mRNA can include, for example, backbone modification, sugar modification or base modification.In some embodiments, the mRNA encoding the antibody (e.g., mRNA encoding the heavy and light chains) can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil)), as well as modified nucleotides such as purines and pyrimidine analogs. Analogs or derivatives, such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, These include, but are not limited to, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, 13-D-mannosyl-queosine, wybutoxosine, as well as phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine.The preparation of such analogs is known to those skilled in the art, for example from U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530 and 5,700,642, the disclosures of which are incorporated herein by reference in their entirety.

[0146] In some embodiments, mRNA (e.g., mRNA encoding heavy and light chains) may contain RNA backbone modification. Typically, backbone modification is a modification in which the backbone phosphate of the nucleotide contained in RNA is chemically modified. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonate, methylphosphoramidate, phosphoramidate, phosphorothioate (e.g., cytidine 5'-O-(1-thiophosphate)), boranophosphate, positively charged guanidinium, etc., which means that phosphodiester bond is replaced by other anionic, cationic or neutral groups.

[0147] In some embodiments, the mRNA (e.g., mRNA encoding the heavy and light chains) may contain sugar modifications. Exemplary sugar modifications are chemical modifications of the sugars of the nucleotides they contain, such as 2'-deoxy-2'-fluoro-oligoribonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamine-oligoribonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyloligoribonucleotides, 2'-deoxy-2'-fluoro-2'-deoxyuridine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate, 2'-amino ... and 2'-C-alkyl oligoribonucleotides, and their isomers (2'-aracytidine 5'-triphosphate, 2'-aruridine 5'-triphosphate), or azido triphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0148] In some embodiments, the mRNA (e.g., mRNA encoding the heavy and light chains) may contain modifications of the base of a nucleotide (base modification). Modified nucleotides containing base modifications are also referred to as base-modified nucleotides. Examples of such base-modified nucleotides include 2-amino-6-chloropurine riboside 5'-triphosphate, 2-aminoadenosine 5'-triphosphate, 2-thiocytidine 5'-triphosphate, 2-thiouridine 5'-triphosphate, 4-thiouridine 5'-triphosphate, 5-aminoallylcytidine 5'-triphosphate, 5-aminoallyluridine 5'-triphosphate, 5-bromocytidine 5'-triphosphate, 5-bromouridine 5'-triphosphate, 5-iodocytidine 5'-triphosphate, 5-iodouridine 5'-triphosphate, 5-methylcytidine 5'-triphosphate, 5-methyluridine 5'-triphosphate, and 6-azacytidine 5'-triphosphate. , 6-azauridine 5'-triphosphate, 6-chloropurine riboside 5'-triphosphate, 7-deazaadenosine 5'-triphosphate, 7-deazaguanosine 5'-triphosphate, 8-azaadenosine 5'-triphosphate, 8-azidoadenosine 5'-triphosphate, benzimidazole riboside 5'-triphosphate, N1-methyladenosine 5'-triphosphate, N1-methylguanosine 5'-triphosphate, N6-methyladenosine 5'-triphosphate, O6-methylguanosine 5'-triphosphate, pseudouridine 5'-triphosphate, puromycin 5'-triphosphate or xanthosine 5'-triphosphate.

[0149] Typically, mRNA synthesis involves the addition of a "cap" to the N-terminal (5') end and a "tail" to the C-terminal (3') end. The presence of the cap is important in making the mRNA resistant to nucleases found in most eukaryotic cells. The presence of the "tail" helps protect the mRNA from exonuclease degradation.

[0150] Thus, in some embodiments, the mRNA (e.g., mRNA encoding the heavy and light chains) includes a 5' cap structure. The 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate by guanylyltransferase, resulting in a 5'5'5 triphosphate linkage; then, the 7-nitrogen of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp(5'(A,G(5')ppp(5)A and G(5)ppp(5')G.

[0151] In some embodiments, the mRNA (e.g., mRNA encoding the heavy and light chains) comprises a 3' poly(A) tail structure. The polyA tail at the 3' end of the mRNA typically comprises about 10-300 adenosine nucleotides (e.g., about 10-200 adenosine nucleotides, about 10-175 adenosine nucleotides, about 10-150 adenosine nucleotides, about 10-125 adenosine nucleotides, 10-100 adenosine nucleotides, about 10-75 adenosine nucleotides, about 20-70 adenosine nucleotides, or about 20-60 adenosine nucleotides). In some embodiments, the mRNA encoding the antibody (e.g., mRNA encoding the heavy and light chains) comprises a 3' poly(C) tail structure. A suitable poly-C tail at the 3' end of an mRNA typically comprises about 10-200 cytosine nucleotides (e.g., about 10-150 cytosine nucleotides, about 10-100 cytosine nucleotides, about 20-70 cytosine nucleotides, about 20-60 cytosine nucleotides, or about 10-40 cytosine nucleotides). The poly-C tail can be added to or replace the poly-A tail.

[0152] In some embodiments, the mRNA (e.g., mRNAs encoding the heavy and light chains) comprises a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region comprises one or more elements that affect mRNA stability or translation, e.g., an iron response element. In some embodiments, the 5' untranslated region can be between about 50 and 500 nucleotides in length (e.g., between about 50 and 400 nucleotides in length, between about 50 and 300 nucleotides in length, between about 50 and 200 nucleotides in length, or between about 50 and 100 nucleotides in length).

[0153] In some embodiments, the 5' region of the mRNA (e.g., mRNA encoding the heavy and light chains) comprises a sequence encoding a signal peptide, such as those described herein. In certain embodiments, a signal peptide derived from human growth hormone (hGH) is incorporated into the 5' region. Typically, the sequence encoding the signal peptide is linked directly or indirectly to the sequence encoding the heavy or light chain at the N-terminus.

[0154] This technology can be used to deliver any antibody known in the art and that can be raised against a desired antigen using standard methods. The invention can be used to deliver monoclonal antibodies, polyclonal antibodies, antibody mixtures or cocktails, human or humanized antibodies, chimeric antibodies, or bispecific antibodies.

[0155] Methods for making antibodies are well known in the art and are described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptide of the present disclosure are fully human. Fully human antibodies can be made using techniques described in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen administration but has an inactive endogenous locus. Exemplary techniques that can be used to make such antibodies are described in U.S. Patent Nos. 6,150,584, 6,458,592, and 6,420,140, ​​which are incorporated herein by reference in their entirety. Treatment and Use

[0156] As described herein, the antibodies, variants, antibody-drug conjugates, chimeric antigen receptors (CARs) and CAR cells, encoding polynucleotides or derivatives of the disclosure can be used in certain treatment and diagnostic methods.

[0157] The present disclosure further relates to antibody-based therapy, including administering the antibodies or fragments, antibody-drug conjugates, chimeric antigen receptors (CARs) and CAR cells, encoding polynucleotides or derivatives of the present disclosure to patients, such as animals, mammals and humans, to treat one or more of the disorders or conditions described herein. Therapeutic molecules or cells of the present disclosure include, but are not limited to, the antibodies of the present disclosure (including variants and derivatives thereof as described herein), antibody-drug conjugates, chimeric antigen receptors (CARs) and CAR cells, and nucleic acids or polynucleotides encoding the antibodies of the present disclosure (including variants and derivatives thereof as described herein).

[0158] The molecule or cell of the present disclosure can also be used to treat or inhibit cancer.As mentioned above, B7-H3 can be overexpressed in tumor cells, particularly in liver, stomach, pancreas, esophagus, ovary and lung tumors.Inhibition of B7-H3 has been shown to be useful in tumor treatment.

[0159] Thus, in some embodiments, a method for treating cancer in a patient in need thereof is provided. In one embodiment, the method entails administering to the patient an effective amount of the molecule or cell of the present disclosure. In some embodiments, at least one of the cancer cells (e.g., stromal cells) in the patient overexpresses B7-H3.

[0160] Cell therapy, for example, chimeric antigen receptor (CAR) T cell therapy, is also provided in the present disclosure. Suitable cells can be used that are transduced with a vector that encodes or contacts a CAR that includes (or alternatively is engineered to express) the anti-B7-H3 antibody of the present disclosure. Thus, by such contact or manipulation, the cells can be introduced into a cancer patient that requires treatment. The cancer patient can have any type of cancer as disclosed herein. The cells (e.g., T cells) can be, for example, but are not limited to, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or combinations thereof.

[0161] In some embodiments, the cells are isolated from the cancer patient himself or herself. In some embodiments, the cells are provided by a donor or from a cell bank. If the cells are isolated from the cancer patient, unwanted immune responses can be minimized.

[0162] Non-limiting examples of cancer include bladder cancer, breast cancer, colon cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer. In some embodiments, the cancer is one or more of gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, and lung cancer.

[0163] Additional diseases or conditions associated with increased cell survival that may be treated, prevented, diagnosed and / or prognosed by the disclosed antibodies or variants, or derivatives thereof, include, but are not limited to, progression and / or metastasis of malignancies and related disorders, such as leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, and solid tumors, including, but not limited to, sarcomas and Carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody, its variant or derivative used, the age, weight, general health, sex and diet of the patient, as well as the number of administrations, excretion rate, drug combination, and the severity of the particular disease being treated. The judgment of such factors by the medical caregiver is within the ordinary skill of the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by principles of pharmacology and pharmacokinetics well known in the art.

[0164] Methods of administration of the antibody or fragment include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The antigen-binding polypeptide or composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Thus, pharmaceutical compositions containing the antigen-binding polypeptides of the present disclosure can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as a powder, ointment, drops or transdermal patch), bucally, or as an oral spray or nasal drops.

[0165] The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0166] Administration can be systemic or local.In addition, it may be desirable to introduce the antibody of the present disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection, and intraventricular injection can be facilitated by an intraventricular catheter, for example, attached to a reservoir such as an Ommaya reservoir.Pulmonary administration can also be utilized, for example, by using an inhaler or nebulizer, and a formulation containing an aerosolizing agent.

[0167] It may be desirable to administer an antigen-binding polypeptide or composition of the disclosure locally to the area in need of treatment, which can be achieved by, for example and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with wound dressing after surgery, by injection, by catheter, by suppository, or by a deposit, said deposit being of a porous, nonporous or gelatinous material, including membranes, e.g., sialastic membranes, or fibers. Preferably, when administering proteins, including antibodies, of the disclosure, care should be taken to use materials to which the proteins do not absorb.

[0168] The amount of the antibody or fragment of the present disclosure that will be effective in treating, inhibiting and preventing inflammatory, immune or malignant diseases, disorders or conditions can be determined by standard clinical techniques.In addition, in vitro assays can be used as necessary to help identify optimal dosage ranges.The exact dose to be used in the formulation will also depend on the route of administration and the severity of disease, disorder or condition, and should be determined according to the judgment of the practitioner and each patient's circumstances.Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0169] As a general proposition, the dosage of the antibody or fragment of the present disclosure administered to a patient is typically between 0.001 mg and 100 mg per kg of the patient's body weight, between 0.01 mg and 20 mg per kg of the patient's body weight, or between 0.5 mg and 10 mg per kg of the patient's body weight. Generally, human antibodies have a longer half-life in the human body than antibodies from other animal species due to the immune response to the foreign polypeptides. Thus, lower dosages and less frequent administration of human antibodies are often possible. Additionally, the dosage and frequency of administration of the antibodies of the present disclosure can be reduced by enhancing antibody uptake and tissue penetration (e.g., into the brain) by modifications such as lipidation.

[0170] In additional embodiments, the compositions of the present disclosure are administered in combination with cytokines. Cytokines that may be administered with the compositions of the present disclosure include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

[0171] In additional embodiments, the compositions of the present disclosure are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy. composition

[0172] The present disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of antibody or fragment, antibody-drug conjugate, chimeric antigen receptor (CAR) and CAR cell, encoding polynucleotide or derivative, and acceptable carrier. In some embodiments, the composition further comprises a second anti-cancer agent (e.g., immune checkpoint inhibitor).

[0173] In specific embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias, for use in animals, and more particularly in humans. Moreover, a "pharmaceutically acceptable carrier" will generally be a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid of any type.

[0174] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be utilized as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, and the like. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates, or phosphates, if desired. Antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and agents for adjusting osmotic tonicity, such as sodium chloride or dextrose, are also contemplated. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The compositions can be formulated as suppositories using traditional binders and carriers, such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with a suitable amount of carrier to provide the form for proper administration to the patient. The formulation must be suitable for the method of administration. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0175] In an embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Optionally, the composition may also include a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the injection site. Generally, these ingredients are supplied either separately or mixed in unit dosage form, for example as a dry frozen powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet indicating the amount of active agent. If the composition is to be administered by injection, it can be dispensed using an injection bottle containing sterile water or saline of pharmaceutical grade. If the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0176] The compounds of the present disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. EXAMPLES

[0177] Example 1 Generation and testing of mouse anti-human B7-H3 antibodies This example describes the generation of a mouse anti-human B7-H3 monoclonal antibody using hybridoma technology.

[0178] Antigen: human B7-H3(4Ig) His-tagged protein. Protein immunization was performed by subcutaneous (sc) and intraperitoneal (ip) injection.

[0179] Immunization: To generate monoclonal antibodies against human B7-H3, Balb / c, C57BL / 6, SJL, and A / J mice were immunized with human B7-H3 (4Ig) protein. To monitor the immune response, titrated sera from the mice were screened by ELISA and flow cytometry as described below. Sera were screened by antibody binding to human B7-H3 protein and B7-H3 overexpressing CHO-K1 cell lines, as well as a negative control CHO-K1 cell line that does not express B7-H3. Animals with sufficient ELISA titers and FACS binding ratios of positive cell lines (B7-H3 overexpressing CHO-K1 cells) to negative cell lines (CHO-K1 expression vector only) were selected for a final boost with B7-H3 protein prior to hybridoma fusion.

[0180] Cell fusion and hybridoma screening: Spleens were isolated from mice with a final boost as described above. Hybridomas were generated by cell fusion with immortalized mouse myeloma cells by electric field-based electrofusion. Fused cells were plated in 96-flat bottom microtiter plates for hybridoma selection. Supernatants were screened by epitope binning using ELISA and FACS using CHO-K1 overexpressing B7-H3 and negative control cell lines.

[0181] Subcloning and screening: Positive primary clones with different epitopes from each fusion were subcloned by limiting dilution to ensure that the subclones were derived from a single parent cell. The subclones were screened using the same approach as the primary clones, and in addition, the culture supernatants of the positive clones were tested by FACS on A375 cells to confirm the binding ability of the antibodies.

[0182] Hybridoma clones were selected for further analysis. The amino acid sequences of the variable regions are provided below in Table 1A. [Table 1A-1] [Table 1A-2]

[0183] The CDR sequences (according to the Kabat system) of these antibodies are listed below in Table 1B. Also shown are variants thereof in which potential sites for post-translational modification (PTM) have been mutated to biological equivalents (e.g., NG→NA, NG→QG, NS→NA, NS→QS, NS→YS, DG→DA, DG→EG, and DS→DA). These substitutions are intended to retain the biological activity of the antibodies while preventing PTM to facilitate manufacturing. [Table 1B-1] Bold / underlined residues indicate mutations to avoid PTM [Table 1B-2] [Table 1B-3]

[0184] Example 2 Panning and screening of a fully human naive phage library This example screened for fully human anti-B7-H3 antibodies from a human naive phage library.

[0185] Antigens: human B7-H3(2Ig)-Avi-biotin, human B7-H3(4Ig)His tag, human B7-H3(4Ig)-Avi-biotin-His.

[0186] Preparation of a fully human naive phage library: A phage library was constructed by using a phagemid vector consisting of antibody gene fragments amplified from PBMCs of healthy human subjects. The library format was a Fab phage library. The library size was 1.04 × 10 11 It was.

[0187] Solid-phase and solution panning of the phage library against human B7-H3 protein: For solution panning, the phage library was first negatively screened by incubation with BSA-coated streptavidin Dynabeads. The resulting phages were incubated with biotinylated B7-H3-His tagged protein and washed with Kingfisher magnetic bead system. Binders were eluted with trypsin. For solid-phase panning, the phage library was first blocked with PBS supplemented with 5% milk. The resulting phages were incubated with B7-H3 His tagged protein and washed with PBST. Binders were eluted with trypsin.

[0188] The eluted phages were then tested for their titer of binding to antigen and co-cultured with E. coli. There were 4 rounds of panning and screening. The titers of output 3 and output 4 increased significantly.

[0189] Single clones were carefully selected from output 3 and 4, and then cultured in 96-well deep plates. The culture supernatants were subjected to IgG enrichment and antigen-binding titer evaluation. A total of 232 positive clones were selected and subjected to sequencing. By post-sequence analysis, 65 unique sequences were identified. All these clones were subjected to ELISA binding analysis, and the sequences of the strong binders are shown in Table 2A below. [Table 2A]

[0190] The CDR sequences (according to the Kabat system) of these antibodies are listed below in Table 2B. Also shown are variants thereof in which potential sites of post-translational modification (PTM) have been mutated to biological equivalents (e.g., NG→NA, NG→QG, NS→NA, NS→QS, NS→YS, DG→DA, DG→EG, and DS→DA). These substitutions are intended to retain the biological activity of the antibodies while preventing PTM to facilitate manufacturing. [Table 2B-1] Bold / underlined residues indicate mutations to avoid PTM [Table 2B-2]

[0191] Example 3 Binding activity to human B7-H3 antigen This example examined the binding activity of antibodies to the human B7-H3 antigen. 3.1 Cell-based binding to human B7-H3

[0192] To assess the binding activity of the clones, the tested antibodies were subjected to FACS assays on A375 or A375.S2 cells.

[0193] In short, the total number is 4 x 10 4 A375 cells were first incubated with 3-fold or 5-fold serially diluted antibodies in FACS buffer for 30 min at 4° C. After washing with FACS buffer, fluorescent dye-conjugated anti-human IgG antibodies were added to each well and incubated for 30 min at 4° C. Samples were washed twice with FACS buffer. Geometric mean fluorescence intensity (MFI) was evaluated by a MACSQuant Analyzer 16.

[0194] Among all these hybridoma clones, 43G8A9 showed comparable binding to MGA017, and the others showed better binding activity than the positive reference MGA017, as shown in Figure 1. The four antibodies derived from the human naive phage library showed weaker binding activity than MGA017. 3.2 Kinetic activity against human B7-H3-4Ig

[0195] The binding of the tested antibodies to human B7-H3 (4Ig) protein (his tag) was tested on Biacore using the capture method. The mAb was captured by a protein A chip. A serial dilution of human B7-H3 protein was injected over the captured antibody for 3 minutes at a flow rate of 30 μL / min. The antigen was left to dissociate for 600 seconds. All experiments were performed on a Biacore T200. The binding traces were fitted in the Biacore T200 evaluation software according to a 1:1 model to calculate the association (ka) and dissociation (kd) rate constants and the equilibrium constant (KD). The results are shown in Table 3 below. [Table 3] 3.3 Binding epitopes of tested antibodies

[0196] The binding epitopes of the tested antibodies were characterized by epitope binning assays using Octet.

[0197] Briefly, human B7-H3 protein at a concentration of 100 nM was captured by the sensor, and a signal of 0.3 nm was obtained. The sensor was then transferred to 100 nM of competing antibodies (72D1D11, AHP05562, AHP06345, CA84D), which were injected into the sensor for 120 seconds for antigen saturation and association. The tested antibodies were then injected for another 120 seconds for antigen competition testing. The sensor was transferred to 100 nM of the tested antibodies for 120 seconds after the baseline step. Data analysis was performed using Octet Analysis Studio 12.2 software. The binding inhibition ratios of the tested antibodies and the competing antibodies are shown in Table 4A below. Based on the results, the binding epitopes of the tested antibodies can be classified as bins Aα, Aβ, Aγ, B and C, as shown in Table 4B. [Table 4A] [Table 4B]

[0198] Example 4 Internalization of tested antibodies This example characterized the internalization of the tested antibodies in A375 cells.

[0199] pHAb thiol dye is a pH-sensitive dye with very low fluorescence at pH>7, and a dramatic increase in fluorescence occurs when it is internalized into endosomes or lysosomes, where the pH is approximately 6.3 or 4.7, respectively. Briefly, pHAb thiol dye-labeled α-hIgG secondary antibody (10 nM) was incubated for 30 min with serial dilutions of antibody starting from 5–50 nM. The mixture was then diluted with 2 × 10 4 A375 cells were added to a 96-well assay plate with pre-seeded cells in each well. After 48 hours of culture, the fluorescent signal was captured on a multimode plate reader (Envision® 2105) or Operetta.

[0200] As shown in Figure 2, almost all hybridoma clones showed better internalization than MGA017. Among the antibodies derived from the human naive phage library, AHP06331 and AHP06358 showed a lower degree of internalization than MGA017 at lower concentrations, but at higher concentrations, the internalization of AHP06331 and AHP06358 was much stronger than MGA017, as suggested by the higher EC50 and higher maximum value of the assay. AHP05564 and AHP05346 showed weaker internalization than MGA017.

[0201] Example 5 In vitro cytotoxicity of B7-H3 ADC This example characterized the killing efficacy of vc-MMAE-labeled B7-H3 ADC against RKO tumor cells.

[0202] The B7-H3 leading hit was labeled with vc-MMAE(L1-D1) at a drug-to-antibody ratio (DAR) of approximately 4.0. The cytotoxicity of the B7-H3 ADC was tested against RKO cells. Briefly, 2×10 4 RKO cells were seeded overnight in each well of a 96-well plate. Serially diluted B7-H3 ADCs were added to the cell cultures, and the mixtures were incubated for 72 or 96 hours at 37°C in a CO2 incubator. At the end of the incubation, 100 μL of CellTiter-Glo reaction reagent was added, and the mixtures were incubated at room temperature for 10 minutes. Luminescence signals were obtained by a multimode plate reader (Envision® 2105).

[0203] As shown in Figure 3, 72D1D11, 81C8A1, 62H9H5, 106A5B3, 126C10B10, and 227E12D1 mediated stronger cytotoxicity for RKO cell killing compared to MGA017. AHP06358 showed comparable cell killing to MGA017.

[0204] Example 6 Humanization of B7-H3 antibody The variable region genes of antibodies 72D1D11 and 81C8A1 were utilized to generate humanized mAbs. The amino acid sequences of the VH and VK of 72D1D11 and 81C8A1 were compared to available databases of human Ig gene sequences to find the best overall match to the human germline Ig sequence.

[0205] For the light chain of 72D1D11, IGKV2-40 * 01 and IGKV2-28 * 01 was the best matched germline, and for the heavy chain of 72D1D11, IGHV7-4-1 * 02 was selected as the humanized backbone. Then, a humanized 72D1D11 CDR-grafted antibody was designed, and CDR-L1, L2 and L3 were ligated to IGKV2-40 * 01 and IGKV2-28 *01 framework sequence, and CDR-H1, H2 and H3 were grafted onto IGHV7-4-1 * The grafted antibody was grafted onto the framework sequence of humanized heavy chain 02. A 3D model was then generated to determine the amino acids in the original murine framework regions that are essential for antibody binding and conformation. Based on the analysis, back mutations were performed on the grafted antibody, resulting in the generation of four additional humanized heavy chains and four additional light chains.

[0206] For the light chain of 81C8A1, IGKV6-21 * 01 was the best matched germline, and for the heavy chain of 81C8A1, IGHV2-70 * 04 was selected as the humanized backbone. Then, a humanized 81C8A1 CDR-grafted antibody was designed, and CDR-L1, L2 and L3 were ligated to IGKV6-21 * 01 framework sequence, and CDR-H1, H2 and H3 were grafted onto IGHV2-70 * The grafted antibody was grafted onto the framework sequence of 04. A 3D model was then generated to determine the amino acids in the original murine framework regions that are essential for antibody binding and conformation. Based on the analysis, back mutations were performed on the grafted antibody, resulting in the generation of three additional humanized heavy chains and four additional light chains.

[0207] The resulting humanized sequences are listed in Tables 5-6. [Table 5A] [Table 5B] [Table 6A] [Table 6B]

[0208] Example 7 Binding activity of humanized antibodies Flow cytometry was performed to confirm the binding activity of the humanized antibodies to A375 cells.

[0209] In short, the total number is 4 x 10 4 A375 cells were first incubated with 3-fold serial dilutions of antibodies starting at 30 nM in FACS buffer for 30 min at 4° C. After washing with FACS buffer, PE-conjugated anti-human IgG antibody (eBioscience™, Invitrogen) was added to each well and incubated for 30 min at 4° C. Samples were washed twice with FACS buffer. The geometric mean fluorescence intensity (MFI) of PE was evaluated by a MACSQuant Analyzer 16.

[0210] As shown in Figure 4, all humanized 72D1D11 antibodies were comparable to the chimeric antibodies in terms of human B7-H3 binding. For the humanized 81C8A1 antibodies, 81C8A1-z3, 81C8A1-z4, 81C8A1-z7, and 81C8A1-z8 showed comparable binding to the chimeric antibody.

[0211] Example 8 PTM removal of chimeric 72D1D11 It has been observed that NG residues exist on 72D1D11 VL CDR1 and DG residues exist on 72D1D11 VH CDR3 (Kabat numbering), which are at risk of post-translational modification (PTM) and pose a challenge for future manufacturing. Therefore, this example mutated NG on VL CDR1 to QG or NA and DG on VH CDR3 to EG to prevent PTM. The sequences of potential PTM removal sites are listed in Table 7. [Table 7]

[0212] As shown in Figure 5, 72D1D11-p1 / p2 showed comparable human B7-H3 binding to the corresponding parent antibody in A375 cells, whereas 72D1D11-p3 showed some loss of human B7-H3 binding in A375 cells.

[0213] Example 9 Confirmation of PTM removal of humanized 72D1D11 Therefore, this example confirmed the sequences of humanized 72D1D11 (72D1D11-z3) with potential PTM sites removed (VL CDR1 NG mutated to NA or QG; VH CDR3 DG mutated to RG or LG or AG). The sequences are listed in Table 8. [Table 8] [Table 9]

[0214] As shown in Figure 6A, 72D1D11-z3p1 showed comparable human B7-H3 binding to parent antibody 72D1D11-z3 in A375 cells. 72D1D11-z3p2 showed some loss of human B7-H3 binding in A375 cells. As shown in Figure 6B, 72D1D11-z3p6, 72D1D11-z3p7, and 72D1D11-z3p8 lost human B7-H3 binding in A375 cells. * * *

[0215] The present disclosure is not limited in scope by the specific embodiments described, which are intended as single illustrations of individual aspects of the present disclosure, and any compositions or methods that are functionally equivalent are within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of the present disclosure without departing from the spirit and scope of the present disclosure. Thus, the present disclosure encompasses modifications and variations of the present disclosure, provided that they fall within the scope of the appended claims and their equivalents.

[0216] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. An antibody or its antigen-binding fragment having specificity for the human B7-H3 (CD276) protein, comprising a heavy chain variable region (VH) including VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) including VL CDR1, VL CDR2, and VL CDR3, (a) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 39; The aforementioned VH CDR2 comprises the amino acid sequence of SEQ ID NO: 40; The VH CDR3 comprises the amino acid sequence of sequence number 41, 42, 43, 44, 45, or 46; The VL CDR1 comprises the amino acid sequence of SEQ ID NO: 47, 48, or 49; The aforementioned VL CDR2 comprises the amino acid sequence of SEQ ID NO: 50; The VL CDR3 contains the amino acid sequence of SEQ ID NO:

51. (b) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 52; The aforementioned VH CDR2 comprises the amino acid sequence of SEQ ID NO: 53; The aforementioned VH CDR3 contains the amino acid sequence of SEQ ID NO: 54; The aforementioned VL CDR1 comprises the amino acid sequence of SEQ ID NO: 55; The aforementioned VL CDR2 comprises the amino acid sequence of SEQ ID NO: 56; The VL CDR3 contains the amino acid sequence of SEQ ID NO: 57, (c) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 33; The aforementioned VH CDR2 contains the amino acid sequence of SEQ ID NO: 34; The aforementioned VH CDR3 contains the amino acid sequence of SEQ ID NO: 35; The aforementioned VL CDR1 comprises the amino acid sequence of SEQ ID NO: 36; The aforementioned VL CDR2 contains the amino acid sequence of SEQ ID NO: 37; The VL CDR3 contains the amino acid sequence of SEQ ID NO:

38. (d) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 25; The VH CDR2 comprises the amino acid sequence of SEQ ID NO: 26, 27, or 28; The aforementioned VH CDR3 comprises the amino acid sequence of SEQ ID NO: 29; The aforementioned VL CDR1 comprises the amino acid sequence of SEQ ID NO: 30; The VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31; The VL CDR3 contains the amino acid sequence of SEQ ID NO:

32. (e) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 58; The aforementioned VH CDR2 comprises the amino acid sequence of SEQ ID NO: 59; The aforementioned VH CDR3 comprises the amino acid sequence of SEQ ID NO: 60; The aforementioned VL CDR1 comprises the amino acid sequence of SEQ ID NO: 61; The aforementioned VL CDR2 contains the amino acid sequence of SEQ ID NO: 62; The VL CDR3 contains the amino acid sequence of SEQ ID NO:

63. (f) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 64; The VH CDR2 comprises the amino acid sequence of SEQ ID NO: 65 or 66; The aforementioned VH CDR3 contains the amino acid sequence of SEQ ID NO: 67; The aforementioned VL CDR1 comprises the amino acid sequence of SEQ ID NO: 68; The aforementioned VL CDR2 contains the amino acid sequence of SEQ ID NO: 69; The VL CDR3 contains the amino acid sequence of SEQ ID NO:

70. (g) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 71; The VH CDR2 comprises the amino acid sequence of SEQ ID NO: 72, 73, or 74; The aforementioned VH CDR3 contains the amino acid sequence of SEQ ID NO: 75; The aforementioned VL CDR1 comprises the amino acid sequence of SEQ ID NO: 76; The aforementioned VL CDR2 contains the amino acid sequence of SEQ ID NO: 77; The aforementioned VL CDR3 contains the amino acid sequence of SEQ ID NO:

78. (h) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 79; The aforementioned VH CDR2 comprises the amino acid sequence of SEQ ID NO: 80; The aforementioned VH CDR3 comprises the amino acid sequence of SEQ ID NO: 81; The aforementioned VL CDR1 comprises the amino acid sequence of SEQ ID NO: 82; The aforementioned VL CDR2 contains the amino acid sequence of SEQ ID NO: 83; The VL CDR3 contains the amino acid sequence of SEQ ID NO: 84, (i) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 85; The VH CDR2 comprises the amino acid sequence of SEQ ID NO: 86 or 87; The aforementioned VH CDR3 comprises the amino acid sequence of SEQ ID NO: 88; The aforementioned VL CDR1 comprises the amino acid sequence of SEQ ID NO: 89; The VL CDR2 comprises the amino acid sequence of SEQ ID NO: 90; The VL CDR3 contains the amino acid sequence of SEQ ID NO: 91, (j) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 92; The VH CDR2 comprises the amino acid sequence of SEQ ID NO: 93, 94, 95, or 96; The aforementioned VH CDR3 contains the amino acid sequence of SEQ ID NO: 97; The aforementioned VL CDR1 comprises the amino acid sequence of SEQ ID NO: 98; The VL CDR2 comprises the amino acid sequence of SEQ ID NO: 99 or 100; The VL CDR3 contains the amino acid sequence of SEQ ID NO:

101. (k) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 102; The aforementioned VH CDR2 comprises the amino acid sequence of SEQ ID NO: 103; The aforementioned VH CDR3 comprises the amino acid sequence of SEQ ID NO: 104; The aforementioned VL CDR1 comprises the amino acid sequence of SEQ ID NO: 105; The VL CDR2 comprises the amino acid sequence of SEQ ID NO: 106; The VL CDR3 contains the amino acid sequence of SEQ ID NO:

107. (l) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 108; The aforementioned VH CDR2 comprises the amino acid sequence of SEQ ID NO: 109; The VH CDR3 comprises the amino acid sequence of SEQ ID NO: 110, 111, or 112; The VL CDR1 comprises the amino acid sequence of sequence number 113, 114, or 115; The VL CDR2 comprises the amino acid sequence of SEQ ID NO: 116; The VL CDR3 contains the amino acid sequence of SEQ ID NO: 117, or (m) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 182; The VH CDR2 comprises the amino acid sequence of SEQ ID NO: 183, 184, 185, or 186; The VH CDR3 comprises the amino acid sequence of Sequence ID No. 187; The VL CDR1 comprises the amino acid sequence of SEQ ID NO: 188, 189, 190, or 191; The aforementioned VL CDR2 comprises the amino acid sequence of SEQ ID NO: 192; The VL CDR3 contains the amino acid sequence of SEQ ID NO:

193. An antibody or its antigen-binding fragment.

2. (c) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 39; The aforementioned VH CDR2 comprises the amino acid sequence of SEQ ID NO: 40; The VH CDR3 comprises the amino acid sequence of sequence number 41, 42, 43, 44, 45, or 46; The VL CDR1 comprises the amino acid sequence of SEQ ID NO: 47, 48, or 49; The aforementioned VL CDR2 comprises the amino acid sequence of SEQ ID NO: 50; The VL CDR3 contains the amino acid sequence of SEQ ID NO:

51. The antibody or antigen-binding fragment according to claim 1.

3. The aforementioned VH CDR1 comprises the amino acid sequence of SEQ ID NO: 39; The aforementioned VH CDR2 comprises the amino acid sequence of SEQ ID NO: 40; The aforementioned VH CDR3 comprises the amino acid sequence of SEQ ID NO: 41; The aforementioned VL CDR1 contains the amino acid sequence of SEQ ID NO: 47; The aforementioned VL CDR2 comprises the amino acid sequence of SEQ ID NO: 50; The VL CDR3 contains the amino acid sequence of SEQ ID NO:

51. The antibody or antigen-binding fragment according to claim 2.

4. The antibody or antigen-binding fragment according to claim 3, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and 159-162, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6 and 163-166.

5. The antibody or antigen-binding fragment according to claim 3, wherein VH comprises the amino acid sequence of SEQ ID NO: 159 and VL comprises the amino acid sequence of SEQ ID NO:

165.

6. The aforementioned VH CDR1 comprises the amino acid sequence of SEQ ID NO: 39; The aforementioned VH CDR2 comprises the amino acid sequence of SEQ ID NO: 40; The aforementioned VH CDR3 comprises the amino acid sequence of SEQ ID NO: 41; The VL CDR1 comprises the amino acid sequence of SEQ ID NO: 48 or 49; The aforementioned VL CDR2 comprises the amino acid sequence of SEQ ID NO: 50; The VL CDR3 contains the amino acid sequence of SEQ ID NO:

51. The antibody or antigen-binding fragment according to claim 2.

7. The antibody or antigen-binding fragment according to claim 6, wherein VH comprises the amino acid sequence of SEQ ID NO: 159 and VL comprises the amino acid sequence of SEQ ID NO:

178.

8. The antibody or antigen-binding fragment according to claim 6, wherein VH comprises the amino acid sequence of SEQ ID NO: 159 and VL comprises the amino acid sequence of SEQ ID NO:

179.

9. The aforementioned VH CDR1 comprises the amino acid sequence of SEQ ID NO: 52; The aforementioned VH CDR2 comprises the amino acid sequence of SEQ ID NO: 53; The aforementioned VH CDR3 contains the amino acid sequence of SEQ ID NO: 54; The aforementioned VL CDR1 comprises the amino acid sequence of SEQ ID NO: 55; The aforementioned VL CDR2 comprises the amino acid sequence of SEQ ID NO: 56; The VL CDR3 contains the amino acid sequence of SEQ ID NO: 57, The antibody or antigen-binding fragment according to claim 1.

10. The antibody or antigen-binding fragment according to claim 9, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 167-169, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 170-173.

11. The aforementioned VH CDR1 comprises the amino acid sequence of SEQ ID NO: 33; The aforementioned VH CDR2 contains the amino acid sequence of SEQ ID NO: 34; The aforementioned VH CDR3 contains the amino acid sequence of SEQ ID NO: 35; The aforementioned VL CDR1 comprises the amino acid sequence of SEQ ID NO: 36; The aforementioned VL CDR2 contains the amino acid sequence of SEQ ID NO: 37; The VL CDR3 contains the amino acid sequence of SEQ ID NO:

38. The antibody or antigen-binding fragment according to claim 1.

12. The antibody or antigen-binding fragment according to claim 9, wherein VH comprises the amino acid sequence of SEQ ID NO: 3 and VL comprises the amino acid sequence of SEQ ID NO:

4.

13. An antibody or its antigen-binding fragment having specificity for the human B7-H3 (CD276) protein, comprising a heavy chain variable region (VH) including VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) including VL CDR1, VL CDR2, and VL CDR3, (a) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 149; The VH CDR2 comprises the amino acid sequence of SEQ ID NO: 150, 151, or 152; The VH CDR3 comprises the amino acid sequence of SEQ ID NO: 153 or 154; The aforementioned VL CDR1 comprises the amino acid sequence of SEQ ID NO: 155; The aforementioned VL CDR2 comprises the amino acid sequence of SEQ ID NO: 156; The VL CDR3 comprises the amino acid sequence of SEQ ID NO: 157 or 158, (b) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 126; The VH CDR2 comprises the amino acid sequence of SEQ ID NO: 127 or 128; The VH CDR3 comprises the amino acid sequence of SEQ ID NO: 129, 130, or 131; The VL CDR1 comprises the amino acid sequence of SEQ ID NO: 132; The aforementioned VL CDR2 contains the amino acid sequence of SEQ ID NO: 133; The VL CDR3 contains the amino acid sequence of SEQ ID NO:

134. (c) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 135; The VH CDR2 comprises the amino acid sequence of SEQ ID NO: 136 or 137; The aforementioned VH CDR3 contains the amino acid sequence of SEQ ID NO: 138; The VL CDR1 comprises the amino acid sequence of SEQ ID NO: 139; The aforementioned VL CDR2 comprises the amino acid sequence of SEQ ID NO: 140; The VL CDR3 contains the amino acid sequence of SEQ ID NO: 141, or (d) The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 142; The aforementioned VH CDR2 comprises the amino acid sequence of SEQ ID NO: 143; The aforementioned VH CDR3 comprises the amino acid sequence of SEQ ID NO: 144; The aforementioned VL CDR1 comprises the amino acid sequence of SEQ ID NO: 145; The aforementioned VL CDR2 comprises the amino acid sequence of SEQ ID NO: 146; The VL CDR3 contains the amino acid sequence of SEQ ID NO: 147 or 148. An antibody or its antigen-binding fragment.

14. The aforementioned VH CDR1 comprises the amino acid sequence of SEQ ID NO: 149; The VH CDR2 comprises the amino acid sequence of SEQ ID NO: 150, 151, or 152; The VH CDR3 comprises the amino acid sequence of SEQ ID NO: 153 or 154; The aforementioned VL CDR1 comprises the amino acid sequence of SEQ ID NO: 155; The aforementioned VL CDR2 comprises the amino acid sequence of SEQ ID NO: 156; The VL CDR3 contains the amino acid sequence of SEQ ID NO: 157 or 158. The antibody or antigen-binding fragment according to claim 13.

15. The aforementioned VH CDR1 comprises the amino acid sequence of SEQ ID NO: 149; The aforementioned VH CDR2 comprises the amino acid sequence of SEQ ID NO: 150; The aforementioned VH CDR3 comprises the amino acid sequence of SEQ ID NO: 153; The aforementioned VL CDR1 comprises the amino acid sequence of SEQ ID NO: 155; The aforementioned VL CDR2 comprises the amino acid sequence of SEQ ID NO: 156; The VL CDR3 contains the amino acid sequence of SEQ ID NO: 157, The antibody or antigen-binding fragment according to claim 14.

16. The antibody or antigen-binding fragment according to claim 15, wherein VH comprises the amino acid sequence of SEQ ID NO: 124 and VL comprises the amino acid sequence of SEQ ID NO:

125.

17. The aforementioned VH CDR1 comprises the amino acid sequence of SEQ ID NO: 126; The VH CDR2 comprises the amino acid sequence of SEQ ID NO: 127 or 128; The VH CDR3 comprises the amino acid sequence of SEQ ID NO: 129, 130, or 131; The VL CDR1 comprises the amino acid sequence of SEQ ID NO: 132; The aforementioned VL CDR2 contains the amino acid sequence of SEQ ID NO: 133; The VL CDR3 contains the amino acid sequence of SEQ ID NO:

134. The antibody or antigen-binding fragment according to claim 13.

18. The antibody or antigen-binding fragment according to claim 17, wherein VH comprises the amino acid sequence of SEQ ID NO: 118 and VL comprises the amino acid sequence of SEQ ID NO:

119.

19. The aforementioned VH CDR1 comprises the amino acid sequence of SEQ ID NO: 135; The VH CDR2 comprises the amino acid sequence of SEQ ID NO: 136 or 137; The aforementioned VH CDR3 contains the amino acid sequence of SEQ ID NO: 138; The VL CDR1 comprises the amino acid sequence of SEQ ID NO: 139; The aforementioned VL CDR2 comprises the amino acid sequence of SEQ ID NO: 140; The VL CDR3 contains the amino acid sequence of SEQ ID NO:

141. The antibody or antigen-binding fragment according to claim 13.

20. The antibody or antigen-binding fragment according to claim 19, wherein VH comprises the amino acid sequence of SEQ ID NO: 120 and VL comprises the amino acid sequence of SEQ ID NO:

121.

21. The aforementioned VH CDR1 comprises the amino acid sequence of SEQ ID NO: 142; The aforementioned VH CDR2 comprises the amino acid sequence of SEQ ID NO: 143; The aforementioned VH CDR3 comprises the amino acid sequence of SEQ ID NO: 144; The aforementioned VL CDR1 comprises the amino acid sequence of SEQ ID NO: 145; The aforementioned VL CDR2 comprises the amino acid sequence of SEQ ID NO: 146; The VL CDR3 contains the amino acid sequence of SEQ ID NO: 147 or 148. The antibody or antigen-binding fragment according to claim 13.

22. The antibody or antigen-binding fragment according to claim 21, wherein VH comprises the amino acid sequence of SEQ ID NO: 122 and VL comprises the amino acid sequence of SEQ ID NO:

123.

23. The antibody or fragment thereof according to claim 1, wherein it is a bivalent Fab antibody, or a fragment selected from the group consisting of F(ab')2, F(ab)2, Fab', Fab, Fv, and scFv.

24. A multispecific antibody comprising the antigen-binding fragment described in claim 1 and one or more antibodies or antigen-binding fragments having binding specificity to a target antigen other than B7-H3.

25. A chimeric antigen receptor (CAR) comprising the antigen-binding fragment described in claim 1, a transmembrane domain, a costimulatory domain, and a CD3ζ intracellular domain.

26. An antibody or antigen-binding fragment thereof as described in claim 1, or one or more polynucleotides encoding a chimeric antigen receptor (CAR) comprising the antigen-binding fragment as described in claim 1, a transmembrane domain, a costimulatory domain, and a CD3ζ intracellular domain.

27. The polynucleotide according to claim 26, which is one or more mRNAs.

28. The polynucleotide according to claim 27, wherein the mRNA is chemically modified.

29. A cell comprising the polynucleotide described in claim 26.

30. (a) the antibody or fragment thereof according to claim 1; and (b) Conjugation portion conjugated to the antibody or fragment thereof The conjugation portion includes a detectable marker, drug, toxin, cytokine, radionuclide, enzyme, or a combination thereof. Antibody-drug conjugate.

31. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 24, a CAR according to claim 25, a polynucleotide according to any one of claims 26 to 28, a cell according to claim 29, or an antibody-drug conjugate according to claim 30, and a pharmaceutically acceptable carrier.

32. A composition for treating cancer in a patient who requires treatment of cancer, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 24, a CAR according to claim 25, a polynucleotide according to any one of claims 26 to 28, a cell according to claim 29, or an antibody-drug conjugate according to claim 30.

33. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 24, a CAR according to claim 25, a polynucleotide according to any one of claims 26 to 28, a cell according to claim 29, or an antibody-drug conjugate according to claim 30 for preparing a pharmaceutical for treating cancer.

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