Anti-B7-H3 antibodies and uses thereof

Abstract

The present disclosure provides anti-B7-H3 antibodies, including murine antibodies, humanized antibodies, and human antibodies, as well as those having further optimized CDR sequences. These antibodies are useful in the treatment of diseases, such as cancer.

Description

Background Art

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

[0002] There are two main types of B7 proteins: B7-1 or CD80, and B7-2 or CD86. Proteins CD28 and CTLA-4 (CD152) each interact with both B7-1 and B7-2. 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 extracellular immunoglobulin-like variable (IgV) and constant (IgC) regions, 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 includes 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 major isotype in humans, while 2IgB7-H3 is the only isotype in mice.

[0004] B7-H3 protein is not expressed or expressed at a low level in normal tissues and cells, but is highly expressed in various tumor tissues and is closely related to tumor progression, patient survival and prognosis. Clinical reports show that B7-H3 is 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 clinical pathological malignancy and cancer progression. Similarly, in glioblastoma multiforme, B7-H3 expression is negatively correlated with event-free survival, while in pancreatic cancer, B7-H3 expression is associated with lymph node metastasis and pathological progression. Therefore, B7-H3 is a suitable potential therapeutic target. Antibodies targeting B7-H3 have been shown to enhance infiltrating CD8-positive T cells in tumors and inhibit tumor growth. Summary of the invention

[0005] In various embodiments, the present disclosure provides antibodies and antigen-binding fragments specific for human B7-H3 protein. Murine antibodies and their humanized counterparts and fully human antibodies have been identified. These antibodies generally have properties superior to the benchmark antibody MGA017 (MacroGenics). These antibodies and molecules derived therefrom, such as chimeric antigen receptors (CARs), multispecific antibodies, can be appropriately used to treat diseases, such as cancer.

[0006] Accordingly, one embodiment of the present disclosure provides an antibody or an antigen-binding fragment thereof, which is specific to human B7-H3 (CD276) protein and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises VH CDR1, VH CDR2 and VH CDR3, and the light chain variable region comprises VL CDR1, VL CDR2 and VLCDR3.

[0007] In some embodiments, 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 VH CDR3 comprises the amino acid sequence of SEQ ID NO: 29; the 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; and the VLCDR3 comprises the amino acid sequence of SEQ ID NO: 32.

[0008] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO:33; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:34; the VH CDR3 comprises the amino acid sequence of SEQ ID NO:35; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:36; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:37; and the 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, the VH CDR1 comprises the amino acid sequence of SEQ ID NO:52; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:53; the VH CDR3 comprises the amino acid sequence of SEQ ID NO:54; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:55; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:56; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:57.

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

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

[0013] In some embodiments, 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 VH CDR3 comprises the amino acid sequence of SEQ ID NO:75; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:76; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:77; and the VLCDR3 comprises the amino acid sequence of SEQ ID NO:78.

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

[0015] In some embodiments, 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 VH CDR3 comprises the amino acid sequence of SEQ ID NO:88; the 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; and the 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 VLCDR1 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, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 102; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 103; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 104; the 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; and the VLCDR3 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, 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 SEQ ID NO: 187; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 188, 189, 190 or 191; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 192; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 193.

[0020] 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; 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.

[0021] In some embodiments, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 159-162, and VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 163-166. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 159, and 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 acids 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 acids of SEQ ID NO: 159, and the VL comprises the amino acid sequence of SEQ ID NO: 179.

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

[0025] In some embodiments, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 167-169, and VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 170-173.

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

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

[0028] In one embodiment, an antibody or an antigen-binding fragment thereof is also provided, which is specific to human B7-H3 (CD276) protein and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises VH CDR1, VH CDR2 and VH CDR3, and the light chain variable region comprises 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, 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 VH CDR3 comprises the amino acid sequence of SEQ ID NO: 138; the VLCDR1 comprises the amino acid sequence of SEQ ID NO: 139; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 140; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 141.

[0031] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 142; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 143; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 144; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 145; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 146; and the VLCDR3 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, 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; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 153; 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 VLCDR3 comprises the amino acid sequence of SEQ ID NO: 157.

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

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

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

[0037] In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:124, and 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] Also provided are multispecific antibodies comprising an antigen-binding fragment of the disclosure and one or more antibodies or antigen-binding fragments having binding specificity for a non-B7-H3 target antigen.

[0040] Still further provides a chimeric antigen receptor (CAR), which comprises an antigen binding fragment, a transmembrane domain, a co-stimulatory domain and a CD3 ξ intracellular domain of the present disclosure. One or more polynucleotides encoding antibodies or their antigen binding fragments or CARs of the present disclosure are further provided. In some embodiments, the polynucleotide is one or more mRNAs.

[0041] Also provided are methods and uses for treating diseases such as cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Binding of the tested antibodies to the B7-H3 protein on A375 tumor cells is shown. All antibodies tested except AHP05564, AHP06331, AHP06345 and AHP06358 showed better binding than the clinical benchmark MGA017.

[0043] Figure 2 The internalization efficacy of the tested antibodies on A375 tumor cells is shown.

[0044] Figure 3 The in vitro killing efficacy of B7-H3 ADC against RKO tumor cells was shown.

[0045] Figure 4 Binding of the humanized antibody to B7-H3 expressed on A375 tumor cells is shown.

[0046] Figure 5 The binding activity of the chimeric antibody to B7-H3 expressed on A375 tumor cells after removal of potential PTM sites is shown.

[0047] Figure 6 The binding activity of the PTM-depleted humanized antibody to A375 tumor cells is shown. DETAILED DESCRIPTION definition

[0048] It should be noted that the term "a or an" entity refers to one or more entities; for example, "antibody" is understood to represent one or more antibodies. Therefore, the terms "a or an", "one or more", and "at least one" can be used interchangeably herein.

[0049] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody and any antigen-binding fragment or a single chain thereof. Therefore, the term "antibody" includes any protein or peptide containing a molecule that contains at least a portion of an immunoglobulin molecule that has biological activity that binds to an antigen. Such examples include, but are not limited to, a heavy or light chain complementary determining region (CDR) 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 is a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of the structure, an antibody fragment will bind to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegeleisen, and bispecific antibodies. The term "antibody fragment" also includes any synthetic or genetically engineered proteins that act as antibodies by binding to a specific antigen to form a complex.

[0051] The term antibody includes various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will recognize that the heavy chain is classified as γ, μ, α, δ, or ε, with some subclasses (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody, IgG, IgM, IgA, IgG, or IgE, respectively.

[0052] Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and are known to confer functional specialization. In view of the present disclosure, it is easy for those skilled in the art to distinguish modified versions of each of these classes and isotypes, and accordingly, they are within the scope of the present disclosure. All immunoglobulin classes are obviously within the scope of the present disclosure, and the following discussion will generally relate to the IgG class of immunoglobulin molecules. Regarding IgG, a standard immunoglobulin molecule includes two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000 daltons. These four chains are usually connected in a "Y" configuration by disulfide bonds, wherein the light chain begins at the opening of the "Y" and extends all the way to the variable region to surround the heavy chain.

[0053] The antibodies, antigen-binding polypeptides, variants or derivatives disclosed herein 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 VK or VH domains, fragments produced by Fab expression libraries, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies for LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecule disclosed herein can be any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules.

[0054] As used herein, the term "chimeric antibody" will be considered to refer to any antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be complete, 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 a human constant region.

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

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

[0057] Hybridoma technology can be carried out under different "stringency" conditions. In general, low stringency hybridization reactions are carried out at about 40°C in a solution of about 10x SSC or equivalent ionic strength / temperature. Moderate stringency hybridizations are usually carried out at about 50°C in about 6x SSC, and high stringency hybridization reactions are usually carried out at about 60°C in about 1x SSC. Hybridization reactions can also be carried out under "physiological conditions" well known to those skilled in the art. Non-limiting examples of physiological conditions are the temperature, ionic strength, pH, and Mg2+ concentrations typically found in cells. Anti-B7-H3 Antibody

[0058] As shown 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 affinities to human B7-H3 protein than the benchmark antibody MGA017 (MacroGenics).

[0059] At the same time, fully human antibodies were identified from the custom phage antibody library, including AHP05564, AHP06331, AHP06345, and AHP06358 (Table 2). These human antibodies also have unique properties for MGA017, highlighting the promise of these human antibodies.

[0060] Likely due to the improved binding affinity, all chimeric versions of the murine antibodies resulted in higher cellular internalization than MGA017 when assessed using A375 cells ( Figure 2 A). Surprisingly, two fully human antibodies, AHP06331 and AHP06358, also greatly outperformed MGA017 at high concentrations, although their performance at lower concentrations was relatively modest ( Figure 2 B).

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

[0062] Based on the binding competition assay, the antibodies were classified into five different epitope bin groups, Aα, Aβ, Aγ, B, and C, as shown in Table 3B. The Aα group included MGA017, as well as 62H9H5, 81C8A1, 106A5B3, 216E7A9, and 227E12D1, while most antibodies did not belong to this group. The Aβ group included 72D1D11, 97E6B2, 126C10B10, and 429H9F10; the Aγ group included 35A12B11, 294A3C4, and AHP06358; the B group included 312E1E12 and AHP06345; and the C group included AHP06331 and AHP05564.

[0063] Therefore, according to one embodiment of the present disclosure, an antibody or an antigen-binding fragment thereof is provided. In some embodiments, the antibody or its antigen-binding fragment has binding specificity to human B7-H3 protein. In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprises VH CDR1, VH CDR2 and VH CDR3, and the light chain variable region comprises 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 those of any antibody identified herein, e.g., 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 respect to antibody 72D1D11, VH CDR1 comprises the amino acid sequence of SEQ ID NO:39, VHCDR2 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 can be murine, chimeric or humanized. Example antibodies include those shown in Table 5. In some embodiments, the antibody or fragment has a VH whose amino acid sequence is SEQ ID NO: 5 (murine), 159, 160, 161 or 162 (humanized). In some embodiments, the antibody or fragment has a VL whose amino acid sequence is SEQ ID NO: 6 (murine), 163, 164, 165 or 166 (humanized). Example VH / VL sequence pairings are shown in Table 5B. For example, in one embodiment, the antibody or fragment thereof includes a VH of SEQ ID NO: 159 and a VL of SEQ ID NO: 165. In another embodiment, the antibody or fragment thereof includes a VH of SEQ ID NO: 159 and a VL of SEQ ID NO: 163.

[0067] Sequence analysis revealed that the VH CDR3 sequence (SEQ ID NO: 41) and the VL CDR1 sequence (SEQ ID NO: 47) include residues that may be modified post-translationally. In order to avoid the risk of post-translational modification (PTM) and thus simplify manufacturing, the present disclosure designed and tested certain de-risked versions of VH CDR3, including SEQ ID NO: 42, 43, 44, 45 and 46, and de-risked versions of VL CDR1, including SEQ ID NO: 48 and 49.

[0068] Thus, in some embodiments, antibodies and fragments having CDR sequences are provided, wherein 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, 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, 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.

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

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

[0072] In some embodiments, with respect to antibody 81C8A1, VH CDR1 comprises the amino acid sequence of SEQ ID NO:52, VHCDR2 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 can be murine, chimeric or humanized. Example antibodies include those shown in Table 6. In some embodiments, the antibody or fragment has a VH whose amino acid sequence is SEQ ID NO: 7 (murine), 167, 168 or 169 (humanized). In some embodiments, the antibody or fragment has a VL whose amino acid sequence is SEQ ID NO: 8 (murine), 170, 171, 172 or 173 (humanized). Example VH / VL sequence pairings are shown in Table 6B. For example, in one embodiment, the antibody or fragment thereof includes a VH of SEQ ID NO: 167 and a VL of SEQ ID NO: 172. In another embodiment, the antibody or fragment thereof includes a VH of SEQ ID NO: 167 and a VL of SEQ ID NO: 173. In another embodiment, the antibody or fragment thereof includes a VH of SEQ ID NO: 168 and a VL of SEQ ID NO: 171. In another embodiment, the antibody or fragment thereof includes a VH of SEQ ID NO: 168 and a VL of SEQ ID NO: 172. In another embodiment, the antibody or fragment thereof comprises the VH of SEQ ID NO:168 and the VL of SEQ ID NO:173.

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

[0075] In some embodiments, with respect 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, VLCDR1 comprises the amino acid sequence of SEQ ID NO:30, VL CDR2 comprises the amino acid sequence of SEQ ID NO:31, and VLCDR3 comprises the amino acid sequence of SEQ ID NO:32.

[0076] Sequence analysis revealed that the VH CDR2 sequence (SEQ ID NO: 26) includes residues that may be modified post-translationally. To avoid post-translational modification (PTM) risks and thus simplify manufacturing, the present disclosure designed and tested certain de-risked versions of VH CDR2, including SEQ ID NO: 27 and 28.

[0077] Thus, in some embodiments, antibodies and fragments having CDR sequences are provided, wherein 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 the VH of SEQ ID NO: 1 and the VL of SEQ ID NO: 2. In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on B7-H3 as 35A12B11 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 35A12B11 for binding to B7-H3 are also provided.

[0079] In some embodiments, with respect to antibody 62H9H5, VH CDR1 comprises the amino acid sequence of SEQ ID NO:33, VHCDR2 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 the VH of SEQ ID NO: 3 and the VL of SEQ ID NO: 4. In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on B7-H3 as 62H9H5 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 62H9H5 for binding to B7-H3 are also provided.

[0081] In some embodiments, with respect to antibody 97E6B2, VH CDR1 comprises the amino acid sequence of SEQ ID NO:58, VHCDR2 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 the VH of SEQ ID NO: 9 and the VL of SEQ ID NO: 10. In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on B7-H3 as 97E6B2 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 97E6B2 for binding to B7-H3 are also provided.

[0083] In some embodiments, for antibody 106A5B3, VH CDR1 comprises the amino acid sequence of SEQ ID NO:64, VHCDR2 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) includes residues that may be modified post-translationally. To avoid post-translational modification (PTM) risks and thus simplify manufacturing, the present disclosure designed and tested certain de-risked versions of VH CDR2, including SEQ ID NO:66.

[0085] Accordingly, in some embodiments, antibodies and fragments having CDR sequences are provided, wherein 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 the VH of SEQ ID NO: 11 and the VL of SEQ ID NO: 12. In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on B7-H3 as 106A5B3 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 106A5B3 for binding to B7-H3 are also provided.

[0087] In some embodiments, for 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, VLCDR1 comprises the amino acid sequence of SEQ ID NO:76, VL CDR2 comprises the amino acid sequence of SEQ ID NO:77, and VLCDR3 comprises the amino acid sequence of SEQ ID NO:78.

[0088] Sequence analysis revealed that the VH CDR2 sequence (SEQ ID NO: 72) includes residues that may be modified post-translationally. To avoid post-translational modification (PTM) risks and thus simplify manufacturing, the present disclosure designed and tested certain de-risked versions of VH CDR2, including SEQ ID NO: 73 and 74.

[0089] Accordingly, in some embodiments, antibodies and fragments having CDR sequences are provided, wherein 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 the VH of SEQ ID NO: 13 and the VL of SEQ ID NO: 14. In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on B7-H3 as 126C10B10 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 126C10B10 for binding to B7-H3 are also provided.

[0091] In some embodiments, with respect to antibody 216E7A9, VH CDR1 comprises the amino acid sequence of SEQ ID NO:79, VHCDR2 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 the VH of SEQ ID NO: 15 and the VL of SEQ ID NO: 16. In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on B7-H3 as 216E7A9 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 216E7A9 for binding to B7-H3 are also provided.

[0093] In some embodiments, with respect 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, VLCDR1 comprises the amino acid sequence of SEQ ID NO:89, VL CDR2 comprises the amino acid sequence of SEQ ID NO:90, and VLCDR3 comprises the amino acid sequence of SEQ ID NO:91.

[0094] Sequence analysis revealed that the VH CDR2 sequence (SEQ ID NO:86) includes residues that may be modified post-translationally. To avoid post-translational modification (PTM) risks and thus simplify manufacturing, the present disclosure designed and tested certain de-risked versions of VH CDR2, including SEQ ID NO:87.

[0095] Accordingly, in some embodiments, antibodies and fragments having CDR sequences are provided, wherein 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 the VH of SEQ ID NO: 17 and the VL of SEQ ID NO: 18. In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on B7-H3 as 227E12D1 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 227E12D1 for binding to B7-H3 are also provided.

[0097] In some embodiments, with respect to antibody 294A3C4, VH CDR1 comprises the amino acid sequence of SEQ ID NO:92, VHCDR2 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 VL CDR2 (SEQ ID NO: 99) include residues that may be modified post-translationally. To avoid the risk of post-translational modification (PTM) and thus simplify manufacturing, the present disclosure designed and tested certain de-risked versions of VH CDR2, including SEQ ID NO: 94, 95 and 96, and de-risked versions of VL CDR2, including SEQ ID NO: 100.

[0099] Thus, in some embodiments, antibodies and fragments having CDR sequences are provided, wherein 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 the VH of SEQ ID NO: 19 and the VL of SEQ ID NO: 20. In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on B7-H3 as 294A3C4 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 294A3C4 for binding to B7-H3 are also provided.

[0101] In some embodiments, for 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, VLCDR1 comprises the amino acid sequence of SEQ ID NO: 105, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 106, and VLCDR3 comprises the amino acid sequence of SEQ ID NO: 107.

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

[0103] In some embodiments, with respect 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, VLCDR1 comprises the amino acid sequence of SEQ ID NO: 113, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 116, and VLCDR3 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) include residues that may be modified post-translationally. In order to avoid the risk of post-translational modification (PTM) and thus simplify manufacturing, the present disclosure designed and tested certain de-risked versions of VH CDR3, including SEQ ID NO: 111 and 112, and de-risked versions of VL CDR1, including SEQ ID NO: 114 and 115.

[0105] Thus, in some embodiments, antibodies and fragments having CDR sequences are provided, wherein 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, VLCDR2 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 the VH of SEQ ID NO: 23 and the VL of SEQ ID NO: 24. In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on B7-H3 as 429H9F10 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 429H9F10 for binding to B7-H3 are also provided.

[0107] In some embodiments, with respect to antibody 43G8A9, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 182; VHCDR2 comprises the amino acid sequence of SEQ ID NO: 183; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 187; VLCDR1 comprises the amino acid sequence of SEQ ID NO: 188; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 192; and VLCDR3 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) include residues that may be modified post-translationally. In order to avoid the risk of post-translational modification (PTM) and thus simplify manufacturing, the present disclosure designed and tested certain de-risked versions of VH CDR2, including SEQ ID NO: 184, 185 and 186, and de-risked versions of VL CDR1, including SEQ ID NO: 188, 189, 190 and 191.

[0109] Thus, in some embodiments, antibodies and fragments having CDR sequences are provided, wherein 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 the VH of SEQ ID NO: 180 and the VL of SEQ ID NO: 181. In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on B7-H3 as 43G8A9 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 43G8A9 for binding to B7-H3 are also provided.

[0111] In some embodiments, with respect 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, VLCDR1 comprises the amino acid sequence of SEQ ID NO: 132, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 133, and VLCDR3 comprises the amino acid sequence of SEQ ID NO: 134.

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

[0113] Accordingly, in some embodiments, antibodies and fragments having CDR sequences are provided, wherein 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, VLCDR2 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 the VH of SEQ ID NO: 118 and the VL of SEQ ID NO: 119. In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on B7-H3 as AHP05564 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with AHP05564 for binding to B7-H3 are also provided.

[0115] In some embodiments, with respect 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, VLCDR1 comprises the amino acid sequence of SEQ ID NO: 139, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 140, and VLCDR3 comprises the amino acid sequence of SEQ ID NO: 141.

[0116] Sequence analysis revealed that VH CDR2 (SEQ ID NO: 136) includes residues that may be modified post-translationally. To avoid post-translational modification (PTM) risks and thus simplify manufacturing, the present disclosure designed and tested certain de-risked versions of VH CDR2, including SEQ ID NO: 137.

[0117] Thus, in some embodiments, antibodies and fragments having CDR sequences are provided, wherein 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 the VH of SEQ ID NO: 120 and the VL of SEQ ID NO: 121. In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on B7-H3 as AHP06331 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with AHP06331 for binding to B7-H3 are also provided.

[0119] In some embodiments, with respect 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, VLCDR1 comprises the amino acid sequence of SEQ ID NO: 145, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 146, and VLCDR3 comprises the amino acid sequence of SEQ ID NO: 147.

[0120] Sequence analysis revealed that VL CDR3 (SEQ ID NO: 147) includes residues that may be modified post-translationally. To avoid post-translational modification (PTM) risks and thus simplify manufacturing, the present disclosure designed and tested certain de-risked versions of VL CDR3, including SEQ ID NO: 148.

[0121] Accordingly, in some embodiments, antibodies and fragments having CDR sequences are provided, wherein 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 the VH of SEQ ID NO: 122 and the VL of SEQ ID NO: 123. In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on B7-H3 as AHP06345 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with AHP06345 for binding to B7-H3 are also provided.

[0123] In some embodiments, with respect 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, VLCDR1 comprises the amino acid sequence of SEQ ID NO: 155, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 156, and VLCDR3 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) include residues that may be modified post-translationally. In order to avoid the risk of post-translational modification (PTM) and thus simplify manufacturing, the present disclosure designed and tested certain de-risked versions of VH CDR2, including SEQ ID NO: 151 and 152, de-risked versions of VHCDR3, including SEQ ID NO: 154, and de-risked versions of VL CDR3, including SEQ ID NO: 158.

[0125] Accordingly, in some embodiments, antibodies and fragments having CDR sequences are provided, 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 the VH of SEQ ID NO: 124 and the VL of SEQ ID NO: 125. In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on B7-H3 as AHP06358 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof 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 include CDR sequences derived from the CDR sequences of the disclosure, having one, two or three amino acid substitutions, deletions and / or additions.

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

[0001] In vitro and in vivo data showed that antibody-drug conjugates (ADCs) derived from the novel antibodies had higher anti-tumor activity than the benchmark antibody MGA017, making them suitable for ADCs.

[0001] 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.

[0002] In one embodiment, the antibody or fragment of the present disclosure is covalently attached to a drug moiety. The drug moiety can be or be modified to include a group that reacts with a conjugation site on the antibody. For example, the drug moiety can be attached by alkylation (e.g., at the ε-amino lysine or N-terminus of the antibody), reductive amination of oxidized carbohydrates, transesterification between hydroxyl and carboxyl groups, amidation of amino or carboxyl groups, and conjugation with thiols.

[0003] In some embodiments, the number of drug moieties conjugated per antibody molecule, p, is on average 1 to 8; 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p is on average 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, p is on average 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, p is on average 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. In some embodiments, p is 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.

[0004] For example, when chemical activation of the protein results in the formation of free thiol groups, the protein can be conjugated with a thiol-reactive agent. In one aspect, the reagent has substantial specificity for free thiol groups. Such reagents include, for example, maleimide, haloacetamide (e.g., iodine, bromine or chlorine), halogenated ester (e.g., iodine, bromine or chlorine), halomethyl ketone (e.g., iodine, bromine or chlorine), benzyl halide (e.g., iodide, bromide or chloride), vinyl sulfone and pyridine sulfide.

[0005] The drug can be connected to the antibody or fragment through a linker. Suitable linkers include, for example, cleavable and non-cleavable linkers. Cleavable linkers are generally easy to cleave under intracellular conditions. Suitable cleavable linkers include, for example, peptide linkers that can be cleaved by intracellular proteases, such as lysosomal proteases or endosomal proteases. In an exemplary embodiment, the linker can be a dipeptide linker, such as valine-citrulline (val-cit), phenylalanine-lysine (phe-lys) linker or maleimidocaproic acid-valine-citrulline-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 that reacts with a sulfhydryl group (thiol, -SH), while its sulfo-NHS ester is reactive to primary amines (such as lysine and protein or peptide N-terminus). Yet another linker is maleimidocaproyl (mc). Other suitable linkers include linkers that can be hydrolyzed at a specific pH or pH range, such as hydrazone linkers. Other suitable cleavable linkers include disulfide linkers. The linker can be covalently bound to the antibody to such an extent that the antibody must be degraded in the cell to release the drug, such as mc linkers, etc.

[0006] The linker can include a group for attachment to the antibody. For example, the linker can include an amino, hydroxyl, carboxyl or thiol reactive group (e.g., maleimide, haloacetamide (e.g., iodine, bromine or chloride), halogenated ester (e.g., iodine, bromine or chloride), halomethyl ketone (e.g., iodine, bromine or chloride), benzyl halide (e.g., iodide, bromide or chloride), vinyl sulfone and pyridine sulfide).

[0007] In some embodiments, the drug moiety is a cytotoxic or cytostatic agent, an immunosuppressant, a radioisotope, a toxin, or the like. The conjugate can be used to inhibit the proliferation of tumor cells or cancer cells, cause apoptosis of tumor or cancer cells, or be used to treat cancer in patients. Therefore, the conjugate can be used in various scenarios for treating animal cancer. The conjugate can be used to deliver drugs to tumor cells or cancer cells. Without being bound by theory, in some embodiments, the conjugate binds or associates with cancer cells expressing GPC3, and the conjugate and / or the drug can be taken up in tumor cells or cancer cells through receptor-mediated endocytosis.

[0008] Once inside the cell, one or more specific peptide sequences in the conjugate (e.g., in the linker) are hydrolyzed and cleaved by one or more tumor cell or cancer cell-associated proteases, resulting in drug release. The released drug then migrates freely within the cell and induces cytotoxicity or cell inhibition or other activity. In some embodiments, the drug is cleaved from the antibody outside the tumor cell or cancer cell, and the drug subsequently penetrates the cell, or acts on the cell surface.

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

[0010] The antibodies or fragments 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 thereof, and other such agents known in the art.

[0011] The antibody can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent labeled antigen-binding polypeptide is then determined by detecting the presence of luminescence generated during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters.

[0012] Antibodies can also be stained with fluorescent metals such as 152Eu or other lanthanide metals are detectably labeled. These metals can be attached to antibodies using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).Techniques for conjugating various moieties to antibodies are well known, see, e.g., 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 And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985)); Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future 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] Multifunctional molecules 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 specific 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 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] Also provided are different forms of bispecific antibodies. 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] Also provided are bifunctional molecules including not only antibodies or antigen binding fragments.As tumor antigen targeting molecules, antibodies or antigen binding fragments (such as those described herein) specific to B7-H3 can be optionally combined with immune cytokines or ligands through peptide linkers.The immune cytokines or ligands connected 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.These bifunctional molecules can combine immune checkpoint blocking with local immunomodulation at the tumor site. Chimeric Antigen Receptor

[0134] In one embodiment, a chimeric antigen receptor (CAR) is also provided, which includes an antibody or a fragment thereof disclosed herein as a targeting unit. In certain embodiments, CAR includes an antibody or a fragment thereof disclosed herein, a transmembrane domain, a costimulatory domain, and a CD3 ξ intracellular domain.

[0135] The membrane-spanning domain can be designed to be fused with an extracellular domain including an antibody or a fragment, optionally through a hinge domain. It can also be fused with an intracellular domain, such as a costimulatory domain. In certain embodiments, the membrane-spanning domain may include a natural membrane-spanning region of a costimulatory domain (e.g., a TM region of CD28T or 4-1BB as a costimulatory domain) or a natural membrane-spanning domain of a hinge region (e.g., a TM region of CD8α or CD28T as a hinge domain).

[0136] In some embodiments, a transmembrane domain may include a sequence that spans the cell membrane but extends into the cytoplasm and / or extracellular space of the cell. For example, a transmembrane may include a transmembrane sequence, which itself may also include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids that extend into the cytoplasm and / or extracellular space of the cell. Thus, a transmembrane domain includes a transmembrane region, but may further include one or more amino acids that extend beyond the inner or outer surface of the membrane itself; such a sequence may still be considered 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 a connection between the transmembrane domain and the proximal cytoplasmic signaling domain of the chimeric receptor. Glycine-serine doublets (GS), glycine-serine-glycine triplet (GSG) or alanine-alanine-alanine triplet (AAA) provide suitable linkers.

[0138] In some embodiments, CAR further comprises a co-stimulatory domain. In some embodiments, the co-stimulatory domain is located between the transmembrane domain and the activation domain. Example co-stimulatory domains include, but are not limited to, CD2, CD3δ, CD3ε, CD3γ, 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 (HECT), CD100 (SEMA4D), CD 103(ITGAE), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD158A(KIR2DL1), CD158B1( KIR2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1), CD158D(KIRDL4), CD158F1(KIR2DL5A) , CD158F2(KIR2DL5B), CD158K(KTR3DL2), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(SLAMF3), CD244(SLAMF4), CD247(CD3-ζ), 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 co-stimulator (ICOS), LFA-1 (CD 1la / CD18), KG2C, DAP-10, ICAM-1, Kp80 (KLRF1), IL-2Rβ, IL-2Rγ, IL-7Rα, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fcγ receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, activated NK cell receptor, Toll ligand receptor and fragments or combinations thereof.

[0139] In some embodiments, the cytoplasmic portion of CAR also includes a signaling / activation domain. In one embodiment, the signaling / activation domain is a CD3 ξ domain, or its amino acid sequence has at least about 80%, 85%, 90%, 95%, 98% or 99% sequence identity with a CD3 ξ domain. Polynucleotides, mRNA and methods of expressing or preparing antibodies

[0140] The disclosure also provides polynucleotides or nucleic acid molecules encoding antibodies, variants or derivatives thereof or CARs disclosed herein. The polynucleotides disclosed herein can encode the entire heavy chain and light chain variable region of antigen-binding polypeptides, variants thereof or derivatives thereof on the same polynucleotide molecule or on a separate polynucleotide molecule. In addition, the polynucleotides disclosed herein can encode the heavy chain and light chain variable region of antigen-binding polypeptides, variants thereof or derivatives thereof on the same polynucleotide molecule or on a separate polynucleotide molecule.

[0141] In some embodiments, the polynucleotide is an mRNA molecule. In some embodiments, the mRNA can be introduced into a target cell to express 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 performed using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitors. The exact conditions will vary depending on the specific application.

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

[0144] Standard methods can be used to determine the encoding of the desired antibody (e.g., encoding heavy chain or light chain) mRNA sequence and incorporate it into the DNA template. For example, starting from the desired amino acid sequence (e.g., desired heavy chain or light chain sequence), virtual reverse translation is performed based on the degenerate genetic code. Then, an optimization algorithm can be used to select suitable codons. Generally, on the one hand, the G / C content can be optimized to achieve the highest possible G / C content, and on the other hand, the tRNA frequency can be taken into account as much as possible according to the codon usage. For example, the RNA sequence optimized can be established and displayed by means of a suitable display device, and compared with the original (wild type) sequence. The secondary structure can also be analyzed to calculate the stability and destabilization characteristics or corresponding regions of RNA.

[0145] mRNA can be synthesized as unmodified or modified mRNA. Generally, mRNA is modified to enhance stability. Modification of mRNA can include, for example, modification of RNA nucleotides. Therefore, modified mRNA can include, for example, backbone modification, sugar modification or base modification. In some embodiments, mRNA encoding an antibody (e.g., mRNA encoding a heavy chain and a light chain) can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including but not limited to purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and modified nucleotide analogs or derivatives of purines and pyrimidines, 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, uracil, dihydrouracil, 2-thiouracil, 4-thiouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thiouracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5- Methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyluracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, 13-D-mannosyl-queosine, whibutoxin and phosphoamide, phosphorothioate, peptide nucleotide, methylphosphonate, 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 hereby incorporated by reference in their entirety.

[0146] In certain embodiments, mRNA (for example, mRNA encoding heavy and light chains) may contain RNA backbone modifications. Generally, backbone modifications are modifications in which the phosphate of the nucleotide backbone included in the RNA is chemically modified. Exemplary backbone modifications generally include, but are not limited to, modifications from groups composed of methylphosphonates, methylphosphoramidates, phosphoramidates, thiophosphates (for example, cytidine 5'-O-(1-thiophosphates)), borophosphates, positively charged guanidine groups, etc., which means replacing phosphodiester bonds with other anions, cations or neutral groups.

[0147] In some embodiments, mRNA (e.g., mRNA encoding heavy and light chains) may contain sugar modifications. Typical sugar modifications are chemical modifications of the sugars of the nucleotides contained therein, including but not limited to sugar modifications selected from the group consisting of: 2'-deoxy-2'-fluoro-oligonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamine-oligonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2' -O-alkyl oligoribonucleotides, 2'-deoxy-2'-C-alkyl oligoribonucleotides (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyl oligoribonucleotides and their isomers (2'-arabinocytidine 5'-triphosphate, 2'-arabinouridine 5'-triphosphate) or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5' triphosphate).

[0148] In some embodiments, mRNA (e.g., mRNA encoding heavy and light chains) may contain modifications of nucleotide bases (base modifications). Modified nucleotides containing base modifications are also referred to as base-modified nucleotides. Examples of such base-modified nucleotides include, but are not limited to, 2-amino-6-chloropurine nucleoside 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, 6-azacytidine 5'-triphosphate, 6-azauridine 5'-triphosphate, 6-chloropurine nucleoside 5'-triphosphate, 7-deazaadenosine 5'-triphosphate, 7-deazaguanosine 5'-triphosphate, 8-azaadenosine 5'-triphosphate, 8-azidoadenosine 5'-triphosphate, benzimidazole nucleoside 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" at the N-terminus (5') and a "tail" at the C-terminus (3'). The presence of the cap is important for providing resistance to nucleases found in most eukaryotic cells. The presence of the "tail" helps protect the mRNA from exonuclease degradation.

[0150] Thus, in some embodiments, an mRNA (e.g., an mRNA encoding a heavy chain and a light chain) includes a 5' cap structure. The 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one terminal phosphate group from the 5' nucleotide, leaving two terminal phosphates; then guanosine triphosphate (GTP) is added to the terminal phosphate by a 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, mRNA (e.g., mRNA encoding heavy and light chains) includes a 3' poly (A) tail structure. The poly A tail at the 3' end of the mRNA generally includes about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 175 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 125 adenosine nucleotides, 10 to 100 adenosine nucleotides, about 10 to 75 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides). In some embodiments, mRNA encoding antibodies (e.g., mRNA encoding heavy and light chains) includes a 3' poly (C) tail structure. Suitable poly-C tails at the 3' end of the mRNA generally include about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). The poly-C tail may be added to the poly-A tail or may replace the poly-A tail.

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

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

[0154] The present technology can be used to deliver any antibody known in the art and antibodies that can be generated against a desired antigen using standard methods. The present 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 preparing antibodies are well known in the art and are described herein. In certain embodiments, the variable and constant regions of the antigen-binding polypeptides disclosed herein are fully human. Fully human antibodies can be prepared using techniques described in the art and 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 an antigenic attack, but whose endogenous loci have been disabled. Exemplary techniques for preparing such antibodies are described in U.S. Patents 6,150,584, 6,458,592, 6,420,140, ​​the entire contents of which are incorporated by reference. Treatment and uses

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

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

[0158] The molecules or cells disclosed herein can also be used to treat or inhibit cancer. As described above, B7-H3 can be overexpressed in tumor cells, particularly liver, stomach, pancreas, esophagus, ovary and lung tumors. Inhibition of B7-H3 has been shown to be useful in treating tumors.

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

[0160] The disclosure also provides cell therapy, such as chimeric antigen receptor (CAR) T cell therapy. Suitable cells can be used, which are transduced with a vector encoding CAR or contacted with CAR, and the CAR comprises the anti-B7-H3 antibody disclosed herein (or alternatively engineered to express the anti-B7-H3 antibody disclosed herein). After such contact or engineering, the cells can then be introduced into cancer patients in need of treatment. Cancer patients may suffer from any type of cancer disclosed herein. Cells (such as T cells) can be, for example, tumor infiltrating T lymphocytes, CD4+T cells, CD8+T cells, or a combination thereof, but are not limited thereto.

[0161] In some embodiments, cells are isolated from cancer patients themselves. In some embodiments, cells are provided by donors or cell banks. When cells are isolated from cancer patients, undesirable immune responses can be minimized.

[0162] Non-limiting examples of cancer include bladder cancer, breast cancer, colorectal 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] Other diseases or conditions associated with increased cell survival that can be treated, prevented, diagnosed and / or prognosed with the antibodies of the present disclosure, 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 myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia)) and chronic leukemias (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors, including, but not limited to, sarcomas and carcinomas, such as fibrosarcomas, myxosarcoma, liposarcoma, chondrosarcoma, Osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, 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, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, melanoma, neuroblastoma, and retinoblastoma. The specific dosage and treatment regimen for any particular patient will depend on various factors, including the specific antibody used, its variant or derivative, the patient's age, weight, general health, sex and diet, as well as the time of administration, excretion rate, drug combination and the severity of the specific disease being treated. The judgment of the medical caregiver to such factors is within the scope of those skilled in 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 pharmacology and pharmacokinetic principles well known in the art.

[0164] The method of applying the antibody or fragment includes but is not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. Antigen-binding polypeptides or compositions can be applied by any convenient approach, such as by infusion or push injection, absorbed by epithelial or mucocutaneous linings (e.g., oral mucosa, rectum and intestinal mucosa, etc.), and can be applied together with other bioactive agents. Therefore, the pharmaceutical composition containing the antigen-binding polypeptide of the present disclosure can be applied orally, rectally, parenterally, intracisternal, intravaginal, intraperitoneal, locally (such as by powders, ointments, drops or transdermal patches), cheeks or as oral or nasal sprays.

[0165] As used herein, the term "parenteral" refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intraarticular injection and infusion.

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

[0167] It is desirable to administer the antigen-binding polypeptide or composition of the present disclosure locally to the area in need of treatment; this can be achieved, for example but not limited to, by local infusion during surgery, topical application (e.g., in conjunction with a wound dressing after surgery), by injection, by catheter, by suppository, or by implant, the implant being a porous, non-porous or gelatinous material, including a membrane, such as a sialastic membrane or fiber. Preferably, when administering the proteins (including antibodies) of the present disclosure, care must be taken to use materials that the proteins do not absorb.

[0168] The amount of the antibody or fragment of the present disclosure that effectively treats, inhibits and prevents inflammatory, immune or malignant diseases, disorders or conditions can be determined by standard clinical techniques. In addition, in vitro assays can be optionally used to help determine the optimal dosage range. The exact dosage to be used in the formulation will also depend on the route of administration and the severity of the disease, disorder or condition, and should be determined according to the physician's judgment and the circumstances of each patient. The effective dose can be inferred from a dose-response curve derived from an in vitro or animal model test system.

[0169] As a general suggestion, the dosage of the antibody or fragment of the present disclosure administered to the patient is usually 0.001mg / kg to 100mg / kg patient weight, between 0.01mg / kg and 20mg / kg patient weight, or between 0.5mg / kg and 10mg / kg patient weight. In general, due to the immune response to exogenous polypeptides, the half-life of human antibodies in the human body is longer than the half-life of antibodies from other species. Therefore, the dosage of human antibodies can usually be reduced and the frequency of administration can be reduced. In addition, the dosage and frequency of administration of the antibody of the present disclosure can be reduced by modifying (e.g., lipidation) to enhance the uptake and tissue penetration (e.g., into the brain) of the antibody.

[0170] In another embodiment, the compositions of the present disclosure are administered in combination with cytokines. Cytokines that can 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 further embodiments, the compositions of the disclosure are administered in combination with other therapeutic or preventive regimens, such as radiation therapy. Composition

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

[0173] In certain embodiments, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia, for use in animals, and more particularly in humans. In addition, a "pharmaceutically acceptable carrier" is generally a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or any type of formulation auxiliary.

[0174] The term "carrier" refers to a diluent, adjuvant, excipient or vehicle used together with the therapeutic agent. This pharmaceutical carrier can be a sterile liquid, such as water and oil, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and dextrose and glycerol aqueous solutions can also be used as liquid carriers, particularly for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition can also contain a small amount of wetting agent or emulsifier, or pH buffer, such as acetate, citrate or phosphate. Antibacterial agents such as benzyl alcohol or methyl parabens are also contemplated; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for adjusting tonicity such as sodium chloride or dextrose. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The compositions can be formulated as suppositories, containing conventional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described by EW Martin in Remington's Pharmaceutical Sciences, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of an antigen-binding polypeptide, preferably in purified form, and an appropriate amount of a carrier to provide the patient with a form for appropriate administration. The formulation should be suitable for the mode of administration. Parenteral preparations can be packaged in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0175] In an embodiment, the composition is formulated as a pharmaceutical composition suitable for intravenous administration to a human being according to conventional procedures. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lidocaine, to relieve pain at the injection site. Typically, the components are provided separately or mixed together in a unit dosage form, for example, as a dry lyophilized powder or anhydrous concentrate, in a sealed container (e.g., ampoule or pouch) indicating the amount of the active agent. When the composition is administered by infusion, it can be distributed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided to mix the components before administration.

[0176] The compounds of the present disclosure can be formulated in neutral or salt form. Pharmaceutically acceptable salts include salts formed with anions, such as salts derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations, such as salts derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. Examples Example 1. Generation and testing of mouse anti-human B7-H3 antibodies

[0177] This example describes the generation of mouse anti-human B7-H3 monoclonal antibodies using hybridoma technology.

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

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

[0180] Cell fusion and hybridoma screening: Spleens were isolated from terminally boosted mice 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 selection of hybridomas. Supernatants were screened by epitope binning by ELISA and FACS using CHO-K1 overexpressing B7-H3 and negative control cell lines.

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

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

[0183] The CDR sequences of these antibodies (according to the Kabat system) are listed in Table 1B below. Also shown are their variants, in which potential sites of post-translational modification (PTM) are mutated to bioequivalents (e.g., NG=>NA, NG=>QG, NS=>NA, NS=>QS, NS=>YS, DG=>DA, DG=>EG and DS=>DA). These substitutions are considered to retain the biological activity of the antibody while preventing PTM to facilitate manufacturing. Table 1B. CDR sequences (with optional mutations to avoid PTM, Kabat numbering) Bold / underlined residues indicate mutations that avoid PTMs Example 2: Whole Person Natural Phage library panning and screening

[0184] In this example, fully human anti-B7-H3 antibodies were screened from a natural human 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 natural phage library: The 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 .

[0187] Solid phase and solution panning of phage libraries against human B7-H3 protein: For solution panning, the phage library was first negatively screened by incubation with BSA-coated streptavidin Dynabeads. The resulting phage was incubated with biotinylated B7-H3-His tag protein and washed with Kingfisher magnetic bead system. The binders were eluted with trypsin. For solid phase panning, the phage library was first blocked with PBS supplemented with 5% milk. The resulting phage was incubated with B7-H3His tag protein and washed with PBST. The binders were eluted with trypsin.

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

[0189] Individual clones were picked from outputs 3 and 4 and then cultured in 96-well deep plates. The culture supernatants were evaluated for IgG concentration and antigen binding titer. A total of 232 positive clones were selected and sequenced. Post-sequence analysis identified 65 unique sequences. All of these clones were subjected to ELISA binding analysis, and the sequences of the strong binders are shown in Table 2A below. Table 2A. Antibody variable region sequences

[0190] The CDR sequences of these antibodies (according to the Kabat system) are listed in Table 2B below. Also shown are their variants, in which potential sites of post-translational modification (PTM) are mutated to bioequivalents (e.g., NG=>NA, NG=>QG, NS=>NA, NS=>QS, NS=>YS, DG=>DA, DG=>EG and DS=>DA). These substitutions are considered to retain the biological activity of the antibody while preventing PTM to facilitate manufacturing. Table 2B. CDR sequences (with optional mutations to avoid PTMs, Kabat numbering) Bold / underlined residues indicate mutations that avoid PTMs Example 3. Binding activity to human B7-H3 antigen

[0191] This example tests the binding activity of the antibody to human B7-H3 antigen. 3.1 Cell-based binding to human B7-H3

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

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

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

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

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

[0197] In short, the human B7-H3 protein with a concentration of 100nM was captured on the sensor, generating a signal of 0.3nm. Then, the sensor was transferred to 100nM competing antibodies (72D1D11, AHP05562, AHP06345, CA84D), which were injected into the sensor for antigen saturation and association for 120s. Then, the test antibody was injected for antigen competition test for another 120s. After the baseline step, the sensor was transferred to 100nM test antibody for 120s. Data analysis was performed using Octet Analysis Studio12.2 software. The binding inhibition ratio between the test antibody and the competing antibody is shown in Table 4A below. According to the results, the binding epitopes of the test antibodies can be classified into bins Aα, Aβ, Aγ, B and C, as shown in Table 4B. Table 4A. Inhibition ratios by Octet assay Table 4B. Epitope binning results warehouse Antibody Aα MGA017, 62H9H5, 81C8A1, 106A5B3, 216E7A9, 227E12D1 Aβ 72D1D11, 97E6B2, 126C10B10, 429H9F10 Aγ 35A12B11, 294A3C4, AHP06358, 43G8A9 B 312E1E12、AHP06345 C AHP06331、AHP05564 Example 4. Internalization of test antibodies

[0198] This example characterizes the internalization of a test antibody in A375 cells.

[0199] pHAb thiol dyes are pH-sensitive dyes that have very low fluorescence at pH>7 and a dramatic increase in fluorescence when internalized into endosomes or lysosomes at pH approximately 6.3 or 4.7, respectively. Briefly, pHAb thiol dye-labeled α-hIgG secondary antibody (10 nM) was incubated with serially diluted antibodies starting from 5-50 nM for 30 min. The mixture was added to a 96-well assay plate, with 2 × 10 4 A375 cells. After 48 hours of culture, the cells were plated on a multimode plate reader ( 2105) or Operetta to capture the fluorescence signal.

[0200] like Figure 2 As shown in Figure 2, almost all hybridoma clones showed better internalization than MGA017. Among the antibodies derived from human natural phage libraries, AHP06331 and AHP06358 were internalized less than MGA017 at lower concentrations, but at higher concentrations, AHP06331 and AHP06358 were internalized much more strongly than MGA017, as indicated by higher EC50 and higher top value in the assay. AHP05564 and AHP05346 were internalized less than MGA017. Example 5. In vitro cytotoxicity of B7-H3 ADC

[0201] This example characterizes the killing efficacy of vc-MMAE-labeled B7-H3 ADC on RKO tumor cells.

[0202] The B7-H3 lead hit was labeled with vc-MMAE (L1-D1) with a drug to antibody ratio (DAR) of approximately 4.0. The cytotoxicity of the B7-H3 ADC was tested on RKO cells. Briefly, 2×10 4 RKO cells were seeded in each well of a 96-well plate overnight. Serial dilutions of B7-H3 ADC were added to the cell culture, and the mixture was incubated at 37 °C in a CO2 incubator for 72 h or 96 h. At the end of the incubation, 100 μL CellTiter-Glo reagent was added, and the mixture was incubated at room temperature for 10 min. The cells were then plated using a multimode plate reader ( 2105) to obtain a luminescent signal.

[0203] like Figure 3As shown, 72D1D11, 81C8A1, 62H9H5, 106A5B3, 126C10B10, and 227E12D1 mediated stronger cytotoxicity against RKO cells compared to MGA017. AHP06358 showed comparable cell killing to MGA017. Example 6. Humanization of B7-H3 Antibody

[0204] The variable region genes of antibodies 72D1D11 and 81C8A1 were used to generate humanized mAbs. The amino acid sequences of VH and VK of 72D1D11 and 81C8A1 were compared with existing human Ig gene sequence databases to find the best overall matching human germline Ig sequences.

[0205] For the light chain of 72D1D11, IGKV2-40*01 and IGKV2-28*01 were the best-fitting germlines, and for the heavy chain of 72D1D11, IGHV7-4-1*02 was selected as the humanized framework. The humanized 72D1D11 CDR-grafted antibody was then designed, in which CDR-L1, L2, and L3 were grafted onto the framework sequences of IGKV2-40*01 and IGKV2-28*01, and CDR-H1, H2, and H3 were grafted onto the framework sequences of IGHV7-4-1*02. A 3D model was then generated to determine the amino acids in the original mouse framework region that were critical for antibody binding and conformation. Based on the analysis, the grafted antibodies were back-mutated to produce 4 additional humanized heavy chains and 4 additional light chains.

[0206] For the light chain of 81C8A1, IGKV6-21*01 was the best fitting germline, and for the heavy chain of 81C8A1, IGHV2-70*04 was selected as the humanized framework. The humanized 81C8A1 CDR-grafted antibody was then designed, in which CDR-L1, L2, and L3 were grafted onto the framework sequence of IGKV6-21*01, and CDR-H1, H2, and H3 were grafted onto the framework sequence of IGHV2-70*04. A 3D model was then generated to determine the amino acids in the original mouse framework region that were critical for antibody binding and conformation. Based on the analysis, the grafted antibodies were backmutated, resulting in 3 additional humanized heavy chains and 4 additional light chains.

[0207] The sequences of the resulting humanized sequences are listed in Tables 5-6. Table 5A. Humanization of 72D1D11 Table 5B. Humanized antibodies from 72D1D11 Table 6A. Humanization of 81C8A1 Table 6B. Humanized antibodies from 81C8A1 Example 7. Binding activity of humanized antibodies

[0208] Flow cytometry was performed to confirm the binding activity of the humanized antibody to A375 cells.

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

[0210] like Figure 4 As shown, all humanized 72D1D11 antibodies were comparable to the chimeric antibody in terms of human B7-H3 binding. For the humanized 81C8A1 antibodies, 81C8A1-z3, 81C8A1-z4, 81C8A1-z7, 81C8A1-z8 showed comparable binding to the chimeric antibody. Example 8. PTM removal of chimeric 72D1D11

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

[0212] like Figure 5 As shown, 72D1D11-p1 / p2 showed comparable human B7-H3 binding to the corresponding parental antibodies on A375 cells. 72D1D11-p3 showed a slight loss of human B7-H3 binding on A375 cells. Example 9. Confirmation of PTM removal of humanized 72D1D11

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

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

[0215] The scope of the present disclosure is not limited by the particular embodiments described, which are intended to be a single illustration of the various 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 may be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure is intended to cover modifications and variations of the present disclosure as long as they are 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 an antigen-binding fragment thereof, which is specific to human B7-H3 (CD276) protein and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises VH CDR1, VH CDR2 and VH CDR3, and the light chain variable region comprises VL CDR1, VL CDR2 and VL CDR3, wherein: (a) 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, (b) the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 52; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:53; the VH CDR3 comprises the amino acid sequence of SEQ ID NO:54; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:55; The VL CDR2 comprises the amino acid sequence of SEQ ID NO:56; and The VL CDR3 comprises the amino acid sequence of SEQ ID NO: 57, (c) the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 33; The VH CDR2 comprises the amino acid sequence of SEQ ID NO: 34; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 35; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:36; The VL CDR2 comprises the amino acid sequence of SEQ ID NO: 37; and The VL CDR3 comprises 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 VH CDR3 comprises the amino acid sequence of SEQ ID NO:29; The 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; and The VL CDR3 comprises the amino acid sequence of SEQ ID NO: 32, (e) the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 58; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:59; The VH CDR3 comprises the amino acid sequence of SEQ ID NO: 60; The VL CDR1 comprises the amino acid sequence of SEQ ID NO:61; The VL CDR2 comprises the amino acid sequence of SEQ ID NO: 62; and The VL CDR3 comprises 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 VH CDR3 comprises the amino acid sequence of SEQ ID NO: 67; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 68; The VL CDR2 comprises the amino acid sequence of SEQ ID NO: 69; and The VL CDR3 comprises 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 VH CDR3 comprises the amino acid sequence of SEQ ID NO: 75; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:76; The VL CDR2 comprises the amino acid sequence of SEQ ID NO: 77; and The VL CDR3 comprises the amino acid sequence of SEQ ID NO: 78, (h) the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 79; The VH CDR2 comprises the amino acid sequence of SEQ ID NO: 80; the VH CDR3 comprises the amino acid sequence of SEQ ID NO:81; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:82; The VL CDR2 comprises the amino acid sequence of SEQ ID NO: 83; and The VL CDR3 comprises 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 VH CDR3 comprises the amino acid sequence of SEQ ID NO:88; the 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; and The VL CDR3 comprises 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 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, (k) the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 102; The VH CDR2 comprises the amino acid sequence of SEQ ID NO: 103; The VH CDR3 comprises the amino acid sequence of SEQ ID NO: 104; the 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; and The VL CDR3 comprises the amino acid sequence of SEQ ID NO: 107, (1) 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, 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 SEQ ID NO: 187; The VL CDR1 comprises the amino acid sequence of SEQ ID NO: 188, 189, 190 or 191; The VL CDR2 comprises the amino acid sequence of SEQ ID NO: 192; and The VL CDR3 comprises the amino acid sequence of SEQ ID NO:

193.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein: (c) 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.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein: 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; 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.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 159-162, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 163-166.

5. The antibody or antigen-binding fragment thereof according to claim 3, wherein 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.

6. The antibody or antigen-binding fragment thereof according to claim 2, wherein: 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.

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

178.

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

179.

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

57.

10. The antibody or antigen-binding fragment thereof 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 antibody or antigen-binding fragment thereof according to claim 1, wherein: the VH CDR1 comprises the amino acid sequence of SEQ ID NO:33; The VH CDR2 comprises the amino acid sequence of SEQ ID NO: 34; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 35; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:36; The VL CDR2 comprises the amino acid sequence of SEQ ID NO: 37; and The VL CDR3 comprises the amino acid sequence of SEQ ID NO:

38.

12. The antibody or antigen-binding fragment thereof according to claim 9, wherein 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.

13. An antibody or antigen-binding fragment thereof, which is specific to human B7-H3 (CD276) protein and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises VH CDR1, VH CDR2 and VH CDR3, and the light chain variable region comprises VL CDR1, VL CDR2 and VL CDR3, wherein: (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 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, (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 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, (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 VH CDR3 comprises the amino acid sequence of SEQ ID NO: 138; The VL CDR1 comprises the amino acid sequence of SEQ ID NO: 139; The VL CDR2 comprises the amino acid sequence of SEQ ID NO: 140; and The VL CDR3 comprises 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 VH CDR2 comprises the amino acid sequence of SEQ ID NO: 143; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 144; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 145; The VL CDR2 comprises the amino acid sequence of SEQ ID NO: 146; and The VL CDR3 comprises the amino acid sequence of SEQ ID NO: 147 or 148.

14. The antibody or antigen-binding fragment thereof according to claim 13, wherein: 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.

15. The antibody or antigen-binding fragment thereof according to claim 14, wherein: 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; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 153; 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.

16. The antibody or antigen-binding fragment thereof according to claim 15, wherein 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.

17. The antibody or antigen-binding fragment thereof according to claim 13, wherein: 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.

18. The antibody or antigen-binding fragment thereof according to claim 17, wherein 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.

19. The antibody or antigen-binding fragment thereof according to claim 13, wherein: 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 VH CDR3 comprises the amino acid sequence of SEQ ID NO: 138; The VL CDR1 comprises the amino acid sequence of SEQ ID NO: 139; The VL CDR2 comprises the amino acid sequence of SEQ ID NO: 140; and The VL CDR3 comprises the amino acid sequence of SEQ ID NO:

141.

20. The antibody or antigen-binding fragment thereof according to claim 19, wherein 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.

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

22. The antibody or antigen-binding fragment thereof according to claim 21, wherein 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.

23. The antibody or fragment thereof according to any one of claims 1-22, wherein 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.

24. A multispecific antibody comprising an antigen-binding fragment according to any one of claims 1-23 and one or more antibodies or antigen-binding fragments having binding specificity for a non-B7-H3 target antigen.

25. A chimeric antigen receptor (CAR) comprising an antigen binding fragment according to any one of claims 1-23, a transmembrane domain, a co-stimulatory domain and a CD3ξ intracellular domain.

26. One or more polynucleotides encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-24 or the CAR according to claim 20.

27. The one or more polynucleotides of claim 26 which are one or more mRNAs.

28. The one or more polynucleotides of claim 27, wherein the mRNA is chemically modified.

29. A cell comprising one or more polynucleotides according to any one of claims 26-28.

30. An antibody-drug conjugate comprising: (a) an antibody or fragment thereof according to any one of claims 1 to 24; and (b) a conjugate moiety conjugated to the antibody or fragment thereof, wherein the conjugate moiety is selected from: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme or a combination thereof.

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

32. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1-24, a CAR according to claim 25, one or more polynucleotides according to any one of claims 26-28, a cell according to claim 29, or an antibody-drug conjugate according to claim 30.

33. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-24, the CAR according to claim 25, the one or more polynucleotides according to any one of claims 26-28, the cell according to claim 29, or the antibody-drug conjugate according to claim 30 for the preparation of a medicament for treating cancer.

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