Anti-B7-H3 Antibodies and Their Uses

Abstract

This disclosure provides anti-B7-H3 antibodies, including murine antibodies, humanized antibodies, and human antibodies, as well as those with further optimized CDR sequences. These antibodies are suitable for treating 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 the 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 signals between APCs and T cells, respectively. The binding of B7 on APCs to CTLA-4 on T cells results in the inhibition of T cell activity.

[0002] There are mainly two types of B7 proteins: B7-1 or CD80, and B7-2 or CD86. The 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 an extracellular immunoglobulin-like variable region (IgV) and constant region (IgC), a transmembrane region, and a short cytoplasmic region. B7-H3 has two splice variants - B7-H3a and B7-H3b. The extracellular domain of B7-H3a consists of two immunoglobulin domains of IgV-IgC (also known as 2IgB7-H3), while the extracellular domain of B7-H3b is composed 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] The B7-H3 protein is not expressed or is expressed at low levels 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 have shown that B7-H3 is overexpressed in multiple types of cancers, 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 also been reported in the literature that in prostate cancer, the expression level of B7-H3 is positively correlated with clinicopathological 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 related to lymph node metastasis and pathological progression. Therefore, B7-H3 is a suitable potential therapeutic target. It has been demonstrated that antibodies targeting B7-H3 can enhance infiltrating CD8-positive T cells in tumors and inhibit tumor growth. Summary of the Invention

[0005] In various embodiments, the present disclosure provides antibodies and antigen-binding fragments that are specific for human B7-H3 protein. Mouse antibodies, 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), bispecific antibodies, can be suitably used for treating diseases, such as cancer.

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

[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 VL CDR3 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 VL CDR3 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 VL CDR1 comprises the amino acid sequence of SEQ ID NO:98; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:99 or 100; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:101.

[0017] In some embodiments, 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.

[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 NO: 5 and 159 - 162, and VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 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, VH comprises the amino acid of SEQ ID NO:159, and VL comprises the amino acid sequence of SEQ ID NO:178. In some embodiments, VH comprises the amino acid of SEQ ID NO:159, and 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 NO:7 and 167 - 169, and VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: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 for human B7-H3 (CD276) protein and comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising VH CDR1, VH CDR2 and VH CDR3, and the light chain variable region comprising VL CDR1, VL CDR2 and VL CDR3.

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

[0030] In some embodiments, 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.

[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 VL CDR3 comprises the amino acid sequence of SEQ ID NO: 147 or 148.

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

[0033] In some embodiments, 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.

[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 that comprise an antigen-binding fragment disclosed herein and one or more antibodies or antigen-binding fragments having binding specificity for a non-B7-H3 target antigen.

[0040] There is still further provided a chimeric antigen receptor (CAR) comprising an antigen-binding fragment, a transmembrane domain, a co-stimulatory domain, and a CD3ξ intracellular domain disclosed herein. There is still further provided one or more polynucleotides encoding an antibody or an antigen-binding fragment thereof or a CAR disclosed herein. In some embodiments, the polynucleotide is one or more mRNAs.

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

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

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

[0044] Figure 3 Shows the in vitro killing efficacy of B7-H3 ADC on RKO tumor cells.

[0045] Figure 4 Shows the binding of the humanized antibody to B7-H3 expressed on A375 tumor cells.

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

[0047] Figure 6 Shows the binding activity of the humanized antibody with removed PTM to A375 tumor cells. DETAILED DESCRIPTION

[0048] DEFINITIONS

[0049] 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. Thus, the terms "a or an", "one or more", and "at least one" may be used interchangeably herein.

[0050] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. An antibody can be a whole antibody and any antigen-binding fragment or its single chain. Thus, the term "antibody" includes any protein or peptide molecule that contains at least a portion of a molecule that includes an immunoglobulin molecule having biological activity of binding to an antigen. Such examples include, but are not limited to, complementarity determining regions (CDRs) of the heavy or light chain or ligand-binding portions thereof, variable regions of the heavy or light chain, constant regions of the heavy or light chain, framework (FR) regions or any portion thereof, or at least a portion of a binding protein.

[0051] 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 structure, an antibody fragment binds to the same antigen that the whole antibody recognizes. The term "antibody fragment" includes aptamers, spiegeleisen, and bispecific antibodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.

[0052] The term antibody includes a wide variety of polypeptide classes that can be biochemically distinguished. Those skilled in the art will recognize that heavy chains are 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 to be IgG, IgM, IgA, IgG, or IgE, respectively.

[0053] Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and are known to confer functional specialization. Given this disclosure, those skilled in the art can readily discern modified versions of each of these classes and isotypes and, accordingly, they are within the scope of this disclosure. All immunoglobulin classes are clearly within the scope of this disclosure, and the following discussion will generally refer to the IgG class of immunoglobulin molecules. With respect to IgG, a standard immunoglobulin molecule includes two identical light chain polypeptides having a molecular weight of approximately 23,000 daltons and two identical heavy chain polypeptides having a molecular weight of 53,000 - 70,000 daltons. These four chains are typically linked by disulfide bonds in a "Y" configuration, where the light chains start at the opening of the "Y" and extend through the variable regions to surround the heavy chains.

[0054] 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 generated from Fab expression libraries, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against LIGHT antibodies disclosed herein). The immunoglobulins or antibody molecules disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules.

[0055] As used herein, the term "chimeric antibody" will be taken to mean any antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which can be full-length, partial or modified according to the 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.

[0056] The antibodies disclosed herein can be from any animal source, including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken antibodies. In some embodiments, the variable regions may be of chondricthoid origin (e.g., from sharks).

[0057] As used herein, the term "recombinant," when referring to a polypeptide or polynucleotide, means a form of polypeptide or polynucleotide that does not occur naturally, non-limiting examples of which can be produced by combining polynucleotides that do not normally occur together.

[0058] Hybridoma technology can be carried out under different "stringency" conditions. Generally, low-stringency hybridization reactions are carried out at about 40 °C in a solution of about 10x SSC or equivalent ionic strength / temperature. Medium-stringency hybridizations are typically carried out at about 50 °C in about 6x SSC, and high-stringency hybridization reactions are typically 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+ concentration normally found in cells.

[0059] Anti-B7-H3 antibody

[0060] As shown in the attached 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 for human B7-H3 protein than the benchmark antibody MGA017 (MacroGenics).

[0061] Meanwhile, fully human antibodies were identified from a custom phage antibody library, including AHP05564, AHP06331, AHP06345, and AHP06358 (Table 2). These human antibodies also have unique properties relative to MGA017, highlighting the promise of these human antibodies.

[0062] Presumably due to the increased binding affinity, when evaluated with A375 cells, all chimeric versions of the murine antibodies resulted in higher cellular internalization than MGA017( Figure 2 A). Surprisingly, two fully human antibodies, AHP06331 and AHP06358, were also significantly superior to MGA017 at high concentrations, although they performed relatively modestly at lower concentrations( Figure 2 B).

[0063] Antibody-drug conjugates (ADCs) of these antibodies were also prepared and tested. Similarly, the ADCs of at least 72D1D11, 81C8A1, 62H9H5, 106A5B3, 126C10B10, and 227E12D were superior to vc-MMAE-conjugated MGA017( Figure 3 ), further confirming the superior properties of these new antibodies.

[0064] Based on binding competition assays, the antibodies were classified into five different epitope binning groups, Aα, Aβ, Aγ, B, and C, as shown in Table 3B. Group Aα includes MGA017, as well as 62H9H5, 81C8A1, 106A5B3, 216E7A9, and 227E12D1, while most antibodies do not belong to this group. Group Aβ includes 72D1D11, 97E6B2, 126C10B10, and 429H9F10; Group Aγ includes 35A12B11, 294A3C4, and AHP06358; Group B includes 312E1E12 and AHP06345; and Group C includes AHP06331 and AHP05564.

[0065] Accordingly, in one embodiment of the present disclosure, an antibody or an antigen-binding fragment thereof is provided. In some embodiments, the antibody or an antigen-binding fragment thereof has binding specificity for human B7-H3 protein. In some embodiments, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising VH CDR1, VH CDR2, and VH CDR3, and the light chain variable region comprising VL CDR1, VL CDR2, and VL CDR3.

[0066] In one embodiment, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are those of any of the antibodies identified herein, such as 35A12B11, 62H9H5, 72D1D11, 81C8A1, 97E6B2, 106A5B3, 126C10B10, 216E7A9, 227E12D1, 294A3C4, 312E1E2, and 429H9F10, or any one of AHP05564, AHP06331, AHP06345, and AHP06358.

[0067] In some embodiments, with respect to antibody 72D1D11, VH CDR1 comprises the amino acid sequence of SEQ ID NO:39, VH CDR2 comprises the amino acid sequence of SEQ ID NO:40, VH CDR3 comprises the amino acid sequence of SEQ ID NO:41, VL CDR1 comprises the amino acid sequence of SEQ ID NO:47, VL CDR2 comprises the amino acid sequence of SEQ ID NO:50, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:51.

[0068] The antibody or fragment can be murine, chimeric, or humanized. Exemplary antibodies include those shown in Table 5. In some embodiments, the antibody or fragment has a VH with an amino acid sequence of SEQ ID NO:5 (murine), 159, 160, 161, or 162 (humanized). In some embodiments, the antibody or fragment has a VL with an amino acid sequence of SEQ ID NO:6 (murine), 163, 164, 165, or 166 (humanized). Exemplary VH / VL sequence pairings are shown in Table 5B. For example, in one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO:159 and a VL of SEQ ID NO:165. In another embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO:159 and a VL of SEQ ID NO:163.

[0069] 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 after translation. To avoid the risk of post-translational modification (PTM) and thus simplify manufacturing, certain de-risked versions of VH CDR3 were designed and tested in this disclosure, including SEQ ID NO:42, 43, 44, 45, and 46, as well as de-risked versions of VL CDR1, including SEQ ID NO:48 and 49.

[0070] 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.

[0071] 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.

[0072] Example antibody sequences with or without PTM de-risked CDRs are also provided. As shown in Table 9. In some embodiments, the antibody or fragment has a VH with an amino acid sequence of SEQ ID NO:159, 174, 175, 176, or 177. In some embodiments, the antibody or fragment has a VL with an amino acid sequence of 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 its fragment comprises a VH of SEQ ID NO:159 and a VL of SEQ ID NO:178. In another embodiment, the antibody or its fragment comprises a VH of SEQ ID NO:159 and a VL of SEQ ID NO:179.

[0073] 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.

[0074] In some embodiments, with respect to antibody 81C8A1, VH CDR1 comprises the amino acid sequence of SEQ ID NO:52, VH CDR2 comprises the amino acid sequence of SEQ ID NO:53, VH CDR3 comprises the amino acid sequence of SEQ ID NO:54, VL CDR1 comprises the amino acid sequence of SEQ ID NO:55, VL CDR2 comprises the amino acid sequence of SEQ ID NO:56, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:57.

[0075] The antibody or fragment can be murine, chimeric, or humanized. Exemplary antibodies include those shown in Table 6. In some embodiments, the antibody or fragment has a VH with an amino acid sequence of SEQ ID NO:7 (murine), 167, 168, or 169 (humanized). In some embodiments, the antibody or fragment has a VL with an amino acid sequence of SEQ ID NO:8 (murine), 170, 171, 172, or 173 (humanized). Exemplary VH / VL sequence pairings are shown in Table 6B. For example, in one embodiment, the antibody or its fragment comprises a VH of SEQ ID NO:167 and a VL of SEQ ID NO:172. In another embodiment, the antibody or its fragment comprises a VH of SEQ ID NO:167 and a VL of SEQ ID NO:173. In another embodiment, the antibody or its fragment comprises a VH of SEQ ID NO:168 and a VL of SEQ ID NO:171. In another embodiment, the antibody or its fragment comprises a VH of SEQ ID NO:168 and a VL of SEQ ID NO:172. In another embodiment, the antibody or its fragment comprises a VH of SEQ ID NO:168 and a VL of SEQ ID NO:173.

[0076] 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.

[0077] 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, 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] Sequence analysis revealed that the VH CDR2 sequence (SEQ ID NO:26) includes residues that may be modified post-translationally. To avoid the risk of post-translational modification (PTM) and thus simplify manufacturing, certain risk-reduced versions of VH CDR2 were designed and tested in this disclosure, including SEQ ID NO:27 and 28.

[0079] Accordingly, in some embodiments, antibodies and fragments with 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.

[0080] In one embodiment, the antibody or its fragment comprises VH of SEQ ID NO:1 and VL of SEQ ID NO:2. In some embodiments, antibodies and their antigen-binding fragments that bind to the same epitope on B7-H3 as 35A12B11 are also provided. In some embodiments, antibodies and their antigen-binding fragments that compete with 35A12B11 for binding to B7-H3 are also provided.

[0081] In some embodiments, with respect to antibody 62H9H5, VH CDR1 comprises the amino acid sequence of SEQ ID NO:33, VH CDR2 comprises the amino acid sequence of SEQ ID NO:34, VH CDR3 comprises the amino acid sequence of SEQ ID NO:35, VL CDR1 comprises the amino acid sequence of SEQ ID NO:36, VL CDR2 comprises the amino acid sequence of SEQ ID NO:37, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:38.

[0082] In one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO:3 and a VL of SEQ ID NO:4. In some embodiments, antibodies and antigen-binding fragments thereof that bind 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.

[0083] In some embodiments, with respect to antibody 97E6B2, VH CDR1 comprises the amino acid sequence of SEQ ID NO:58, VH CDR2 comprises the amino acid sequence of SEQ ID NO:59, VH CDR3 comprises the amino acid sequence of SEQ ID NO:60, VL CDR1 comprises the amino acid sequence of SEQ ID NO:61, VL CDR2 comprises the amino acid sequence of SEQ ID NO:62, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:63.

[0084] In one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO:9 and a VL of SEQ ID NO:10. In some embodiments, antibodies and antigen-binding fragments thereof that bind 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.

[0085] In some embodiments, with respect to antibody 106A5B3, VH CDR1 comprises the amino acid sequence of SEQ ID NO:64, VH CDR2 comprises the amino acid sequence of SEQ ID NO:65, VH CDR3 comprises the amino acid sequence of SEQ ID NO:67, VL CDR1 comprises the amino acid sequence of SEQ ID NO:68, VL CDR2 comprises the amino acid sequence of SEQ ID NO:69, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:70.

[0086] Sequence analysis revealed that the VH CDR2 sequence (SEQ ID NO:65) contains residues that may be modified post-translationally. To avoid the risk of post-translational modification (PTM) and thus simplify manufacturing, certain de-risked versions of VH CDR2, including SEQ ID NO:66, were designed and tested in this disclosure.

[0087] Thus, 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.

[0088] In one embodiment, the antibody or fragment thereof comprises VH of SEQ ID NO:11 and 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.

[0089] In some embodiments, with respect to antibody 126C10B10, VH CDR1 comprises the amino acid sequence of SEQ ID NO:71, VH CDR2 comprises the amino acid sequence of SEQ ID NO:72, VH CDR3 comprises the amino acid sequence of SEQ ID NO:75, VL CDR1 comprises the amino acid sequence of SEQ ID NO:76, VL CDR2 comprises the amino acid sequence of SEQ ID NO:77, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:78.

[0090] Sequence analysis revealed that the VH CDR2 sequence (SEQ ID NO:72) contains residues that may be modified post-translationally. To avoid the risk of post-translational modification (PTM) and thus simplify manufacturing, certain risk-reduced versions of VH CDR2 were designed and tested in this disclosure, including SEQ ID NO:73 and 74.

[0091] Thus, 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.

[0092] In one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO:13 and a VL of SEQ ID NO:14. In some embodiments, antibodies and antigen-binding fragments thereof that bind 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.

[0093] In some embodiments, with respect to antibody 216E7A9, VH CDR1 comprises the amino acid sequence of SEQ ID NO:79, VH CDR2 comprises the amino acid sequence of SEQ ID NO:80, VH CDR3 comprises the amino acid sequence of SEQ ID NO:81, VL CDR1 comprises the amino acid sequence of SEQ ID NO:82, VL CDR2 comprises the amino acid sequence of SEQ ID NO:83, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:84.

[0094] In one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO:15 and a VL of SEQ ID NO:16. In some embodiments, antibodies and antigen-binding fragments thereof that bind 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.

[0095] 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, 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] Sequence analysis revealed that the VH CDR2 sequence (SEQ ID NO:86) contains residues that may be modified post-translationally. To avoid the risk of post-translational modification (PTM) and thus simplify manufacturing, certain risk-reduced versions of VH CDR2 were designed and tested in this disclosure, including SEQ ID NO:87.

[0097] 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.

[0098] In one embodiment, the antibody or fragment thereof comprises VH of SEQ ID NO:17 and VL of SEQ ID NO:18. In some embodiments, antibodies and antigen-binding fragments thereof that bind 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.

[0099] In some embodiments, with respect to antibody 294A3C4, VH CDR1 comprises the amino acid sequence of SEQ ID NO:92, VH CDR2 comprises the amino acid sequence of SEQ ID NO:93, VH CDR3 comprises the amino acid sequence of SEQ ID NO:97, VL CDR1 comprises the amino acid sequence of SEQ ID NO:98, VL CDR2 comprises the amino acid sequence of SEQ ID NO:99, and VL CDR3 comprises the amino acid sequence of SEQ ID NO:101.

[0100] Sequence analysis revealed that the VH CDR2 sequence (SEQ ID NO:93) and VL CDR2 (SEQ ID NO:99) comprise residues that may be modified post-translationally. To avoid the risk of post-translational modification (PTM) and thus simplify manufacturing, 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, were designed and tested in this disclosure.

[0101] Accordingly, in some embodiments, antibodies and fragments having CDR sequences are provided, wherein 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.

[0102] In one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO:19 and a VL of SEQ ID NO:20. In some embodiments, antibodies and antigen-binding fragments thereof that bind 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.

[0103] In some embodiments, with respect to antibody 312E1E2, 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.

[0104] In one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO:21 and a VL of SEQ ID NO:22. In some embodiments, antibodies and antigen-binding fragments thereof that bind 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.

[0105] In some embodiments, with respect to antibody 429H9F10, 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, the VL CDR1 comprises the amino acid sequence of SEQ ID NO:113, 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.

[0106] Sequence analysis revealed that the VH CDR3 sequence (SEQ ID NO:110) and the VL CDR1 (SEQ ID NO:113) include residues that may be modified after translation. To avoid the risk of post-translational modification (PTM) and thus simplify manufacturing, 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, were designed and tested in this disclosure.

[0107] Accordingly, in some embodiments, antibodies and fragments having CDR sequences are provided, wherein VH CDR1 includes the amino acid sequence of SEQ ID NO:108, VH CDR2 includes the amino acid sequence of SEQ ID NO:109, VH CDR3 includes the amino acid sequence of SEQ ID NO:110, 111, or 112, VL CDR1 includes the amino acid sequence of SEQ ID NO:113, 114, or 115, VL CDR2 includes the amino acid sequence of SEQ ID NO:116, and VL CDR3 includes the amino acid sequence of SEQ ID NO:117.

[0108] In one embodiment, the antibody or fragment thereof includes VH of SEQ ID NO:23 and 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.

[0109] In some embodiments, with respect to antibody 43G8A9, VH CDR1 includes the amino acid sequence of SEQ ID NO:182; VH CDR2 includes the amino acid sequence of SEQ ID NO:183; VH CDR3 includes the amino acid sequence of SEQ ID NO:187; VL CDR1 includes the amino acid sequence of SEQ ID NO:188; VL CDR2 includes the amino acid sequence of SEQ ID NO:192; and VL CDR3 includes the amino acid sequence of SEQ ID NO:193.

[0110] Sequence analysis revealed that the VH CDR2 sequence (SEQ ID NO: 183) and the VL CDR1 (SEQ ID NO: 188) include residues that may be modified after translation. To avoid the risk of post-translational modification (PTM) and thus simplify manufacturing, certain risk-reduced versions of VH CDR2 were designed and tested in this disclosure, including SEQ ID NO: 184, 185, and 186, as well as risk-reduced versions of VL CDR1, including SEQ ID NO: 188, 189, 190, and 191.

[0111] Accordingly, in some embodiments, antibodies and fragments having CDR sequences are provided, wherein 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.

[0112] In one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO: 180 and a VL of SEQ ID NO: 181. In some embodiments, antibodies and antigen-binding fragments thereof that bind 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.

[0113] In some embodiments, with respect to antibody AHP05564, 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, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 129, 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.

[0114] Sequence analysis revealed that the VH CDR2 (SEQ ID NO: 127) and VH CDR3 sequences (SEQ ID NO: 129) include residues that may be modified after translation. To avoid the risk of post-translational modification (PTM) and thus simplify manufacturing, certain risk-reduced versions of VH CDR2, including SEQ ID NO: 128, and risk-reduced versions of VH CDR3, including SEQ ID NO: 130 and 131, were designed and tested in this disclosure.

[0115] Accordingly, in some embodiments, antibodies and fragments having CDR sequences are provided, wherein VH CDR1 includes the amino acid sequence of SEQ ID NO: 126, VH CDR2 includes the amino acid sequence of SEQ ID NO: 127 or 128, VH CDR3 includes the amino acid sequence of SEQ ID NO: 129, 130 or 131, VL CDR1 includes the amino acid sequence of SEQ ID NO: 132, VL CDR2 includes the amino acid sequence of SEQ ID NO: 133, and VL CDR3 includes the amino acid sequence of SEQ ID NO: 134.

[0116] In one embodiment, the antibody or fragment thereof includes VH of SEQ ID NO: 118 and 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.

[0117] In some embodiments, with respect to antibody AHP06331, VH CDR1 includes the amino acid sequence of SEQ ID NO: 135, VH CDR2 includes the amino acid sequence of SEQ ID NO: 136, VH CDR3 includes the amino acid sequence of SEQ ID NO: 138, VL CDR1 includes the amino acid sequence of SEQ ID NO: 139, VL CDR2 includes the amino acid sequence of SEQ ID NO: 140, and VL CDR3 includes the amino acid sequence of SEQ ID NO: 141.

[0118] Sequence analysis revealed that VH CDR2 (SEQ ID NO: 136) includes residues that may be modified after translation. To avoid the risk of post-translational modification (PTM) and thus simplify manufacturing, certain risk-reduced versions of VH CDR2, including SEQ ID NO: 137, were designed and tested in this disclosure.

[0119] Accordingly, 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.

[0120] In one embodiment, the antibody or fragment thereof comprises VH of SEQ ID NO:120 and 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.

[0121] 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, 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.

[0122] Sequence analysis revealed that VL CDR3 (SEQ ID NO:147) comprises residues that may be modified post-translationally. To avoid the risk of post-translational modification (PTM) and thus simplify manufacturing, certain risk-reduced versions of VL CDR3, including SEQ ID NO:148, were designed and tested in this disclosure.

[0123] 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.

[0124] In one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO:122 and a VL of SEQ ID NO:123. In some embodiments, antibodies and antigen-binding fragments thereof that bind 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.

[0125] 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, 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.

[0126] Sequence analysis revealed that VH CDR2 (SEQ ID NO:150), VH CDR3 (SEQ ID NO:153), and VL CDR3 (SEQ ID NO:157) contain residues that may be modified post-translationally. To avoid the risk of post-translational modification (PTM) and thus simplify manufacturing, certain risk-reduced versions of VH CDR2, including SEQ ID NO:151 and 152, risk-reduced versions of VH CDR3, including SEQ ID NO:154, and risk-reduced versions of VL CDR3, including SEQ ID NO:158, were designed and tested in this disclosure.

[0127] Thus, 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.

[0128] In one embodiment, the antibody or fragment thereof comprises a VH of SEQ ID NO:124 and a VL of SEQ ID NO:125. In some embodiments, antibodies and antigen-binding fragments thereof that bind 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.

[0129] In some embodiments, antibodies and antigen-binding fragments are also provided that comprise CDR sequences derived from the CDR sequences disclosed herein and having one, two, or three amino acid substitutions, deletions, and / or additions.

[0130] In some embodiments, the anti-B7-H3 antibody is a modified mAb that comprises a modified heavy chain constant region, such as a defucosylated heavy chain, which binds to activating Fcγ receptors with higher affinity compared to an unmodified mAb, and the activating Fcγ receptors mediate enhanced ADCC. In some embodiments, the anti-B7-H3 antibody comprises a heavy chain of a human IgG1 variant that comprises single or combinations of L234Y, L235Q, G236W, S239D / M, F243L, H268D, D270E, R292P, S298A, Y300L, V305I, K326D, A330L / M, I332E, K334A / E, P396L (all EU numbering) that enhance ADCC function.

[0131] Antibody-drug conjugate

[0132] In vitro and in vivo data show that antibody-drug conjugates (ADCs) derived from the novel antibody have higher anti-tumor activity than the benchmark antibody MGA017, making it suitable for ADCs.

[0133] In some embodiments, the antibody or fragment can be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biologic response modifier, pharmaceutical reagent, or PEG.

[0134] In one embodiment, the antibody or fragment disclosed herein is covalently attached to a drug moiety. The drug moiety can be or be modified to include a group that reacts with the conjugation point 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 a hydroxyl and a carboxyl group, amidation of an amino or carboxyl group, and conjugation to a thiol.

[0135] In some embodiments, the average number p of drug moieties conjugated to each antibody molecule is from 1 to 8; from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, or from 1 to 2. In some embodiments, p is on average from 2 to 8, from 2 to 7, from 2 to 6, from 2 to 5, from 2 to 4, or from 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 from about 1 to about 20, from about 1 to about 10, from about 2 to about 10, from about 2 to about 9, from about 1 to about 8, from about 1 to about 7, from about 1 to about 6, from about 1 to about 5, from about 1 to about 4, from about 1 to about 3, or from about 1 to about 2. In some embodiments, p is from about 2 to about 8, from about 2 to about 7, from about 2 to about 6, from about 2 to about 5, from about 2 to about 4, or from about 2 to about 3.

[0136] For example, when chemical activation of a protein results in the formation of free thiol groups, the protein can be conjugated with a sulfhydryl-reactive agent. In one aspect, the reagent is substantially specific for free thiol groups. Such reagents include, for example, maleimides, haloacetamides (e.g., iodine, bromine, or chlorine), haloesters (e.g., iodine, bromine, or chlorine), halomethyl ketones (e.g., iodine, bromine, or chlorine), benzyl halides (e.g., iodide, bromide, or chloride), vinyl sulfones, and pyridyl disulfides.

[0137] The drug can be linked to the antibody or fragment via a linker. Suitable linkers include, for example, cleavable and non-cleavable linkers. Cleavable linkers are generally labile under intracellular conditions. Suitable cleavable linkers include, for example, peptide linkers cleavable 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 maleimidocaproyl-valine-citrulline-p-aminobenzylcarbonyl (mc-Val-Cit-PABA) linker. Another linker is sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (smcc). The sulfosuccinimidyl-smcc conjugation occurs through a maleimide group reactive with sulfhydryl groups (thiol, -SH), while its sulfosuccinimidyl-NHS ester is reactive towards primary amines (such as those present in lysine and at the N-terminus of proteins or peptides). Yet another linker is maleimidocaproyl (mc). Other suitable linkers include linkers hydrolyzable 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 intracellularly to release the drug, such as mc linker, etc.

[0138] The linker may include a group for attaching to an antibody. For example, the linker may include an amino, hydroxyl, carboxyl, or sulfhydryl-reactive group (e.g., maleimide, haloacetamide (e.g., iodine, bromine, or chlorine), haloester (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 pyridyl disulfide).

[0139] In some embodiments, the drug moiety is a cytotoxic or cytostatic agent, an immunosuppressive agent, a radioisotope, a toxin, etc. The conjugate can be used to inhibit the proliferation of tumor cells or cancer cells, induce apoptosis of tumor or cancer cells, or be used to treat cancer in a patient. Thus, the conjugate can be used in various scenarios for treating cancer in animals. The conjugate can be used to deliver the drug 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 into the tumor cells or cancer cells by receptor-mediated endocytosis.

[0140] Once inside the cell, one or more specific peptide sequences within the conjugate (e.g., in the linker) are proteolytically cleaved by one or more tumor cell- or cancer cell-associated proteases, resulting in the release of the drug. The released drug then migrates freely within the cell and induces cytotoxicity or cytostasis or other activities. In some embodiments, the drug is cleaved from the antibody outside the tumor cell or cancer cell, and the drug then penetrates the cell or acts on the cell surface.

[0141] 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-valyl-L-valyl-(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-valyl-N5-(aminocarbonyl)-L-ornithyl]amino]phenyl]methoxy]carbonyl]-N-methyl-L-valyl-N-[(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.

[0142] The antibody or fragment can be conjugated or fused to a therapeutic agent, which can include a detectable label (such as a radioactive label), an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic agent or diagnostic agent, a cytotoxic agent (which can be a drug or a toxin), an ultrasound enhancer, a non-radioactive label, combinations thereof, and other such reagents known in the art.

[0143] Antibodies can be detectably labeled by conjugating them to chemiluminescent compounds. The presence of the chemiluminescently labeled antigen-binding polypeptide is then determined by detecting the presence of luminescence produced during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, thermatic acridinium ester, imidazole, acridinium salts, and oxalates.

[0144] Antibodies can also use fluorescence-emitting 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, for example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985)); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future 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)).

[0145] Multifunctional molecule

[0146] A multifunctional molecule comprising an antibody or antigen-binding fragment specific for B7-H3, such as those disclosed herein, and one or more antibodies or antigen-binding fragments specific for a second antigen.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] Also provided are bifunctional molecules that include not only antibodies or antigen-binding fragments. As tumor antigen-targeting molecules, antibodies or antigen-binding fragments specific for B7-H3 (such as those described herein) can optionally be combined with immunocytokines or ligands via a peptide linker. The linked immunocytokines or ligands include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, GM-CSF, TNF-α, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT, and GITRL. These bifunctional molecules can combine immune checkpoint blockade with local immunomodulation at the tumor site.

[0151] Chimeric antigen receptor

[0152] In one embodiment, also provided is a chimeric antigen receptor (CAR) comprising an antibody or fragment thereof disclosed herein as a targeting unit. In some embodiments, the CAR comprises an antibody or fragment thereof disclosed herein, a transmembrane domain, a co-stimulatory domain, and a CD3ξ intracellular domain.

[0153] The transmembrane domain can be designed to be fused to an extracellular domain including an antibody or fragment, optionally via a hinge domain. It can equally be fused to an intracellular domain, such as a co-stimulatory domain. In some embodiments, the transmembrane domain can include the native transmembrane region of a co-stimulatory domain (e.g., the TM region of CD28T or 4-1BB as a co-stimulatory domain) or the native transmembrane domain of a hinge region (e.g., the TM region of CD8α or CD28T as a hinge domain).

[0154] In some embodiments, the transmembrane domain can include sequences that span the cell membrane but extend into the cytoplasm and / or extracellular space of the cell. For example, the transmembrane can include transmembrane sequences which themselves can further 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, the transmembrane domain includes a transmembrane region but can further contain one or more amino acids that extend beyond the inner or outer surface of the membrane itself; such sequences can still be considered a "transmembrane domain".

[0155] 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 triplets (GSG) or alanine-alanine-alanine triplets (AAA) provide suitable linkers.

[0156] In some embodiments, the CAR further includes a co-stimulatory domain. In some embodiments, the co-stimulatory domain is located between the transmembrane domain and the activation domain. Exemplary 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 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex associated alpha chain), CD79B (B cell antigen receptor complex associated beta chain), CD84 (SLAMF5), CD96 (Tactile), CD 100 (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 (TNFRSF14), 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 I molecule, MHC class II molecule, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, activating NK cell receptor, Toll ligand receptor and fragments or combinations thereof.

[0157] In some embodiments, the cytoplasmic portion of the CAR further includes a signaling / activation domain. In one embodiment, the signaling / activation domain is the CD3ξ domain, or its amino acid sequence has at least about 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the CD3ξ domain.

[0158] Polynucleotides, mRNAs and methods of expressing or preparing antibodies

[0159] The present disclosure also provides polynucleotides or nucleic acid molecules encoding the antibodies, their variants or derivatives, or CARs of the present disclosure. The polynucleotides of the present disclosure can encode the entire heavy and light chain variable regions of the antigen-binding polypeptide, its variants or derivatives on the same polynucleotide molecule or on separate polynucleotide molecules. In addition, the polynucleotides of the present disclosure can encode portions of the heavy and light chain variable regions of the antigen-binding polypeptide, its variants or derivatives on the same polynucleotide molecule or on separate polynucleotide molecules.

[0160] In some embodiments, the polynucleotide is an mRNA molecule. In some embodiments, the mRNA can be introduced into target cells to express an antibody or a fragment thereof.

[0161] The mRNA can be synthesized according to any of various known methods. For example, the mRNA can be synthesized by in vitro transcription (IVT). Briefly, IVT generally uses 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 (such as T3, T7 or SP6 RNA polymerase), DNase I, pyrophosphatase and / or RNase inhibitor. The exact conditions will vary according to the specific application.

[0162] In some embodiments, to prepare mRNA encoding an antibody, the 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 the desired nucleotide sequence encoding the mRNA of the desired antibody (e.g., encoding the heavy or light chain) and a termination signal.

[0163] Standard methods can be used to determine the mRNA sequences encoding the desired antibodies (e.g., encoding the heavy or light chains) and incorporate them into a DNA template. For example, starting from the desired amino acid sequence (e.g., the desired heavy or light chain sequence), virtual reverse translation is performed based on the degenerate genetic code. Then, an optimization algorithm can be used to select appropriate 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, tRNA frequencies can be taken into account as much as possible according to codon usage. For example, the optimized RNA sequence can be established and displayed with the aid of an appropriate display device and compared with the original (wild-type) sequence. The secondary structure can also be analyzed to calculate the stabilizing and destabilizing properties of the RNA or corresponding regions.

[0164] mRNA can be synthesized as unmodified or modified mRNA. Generally, mRNA is modified to enhance stability. Modifications of mRNA can include, for example, modifications of RNA nucleotides. Thus, modified mRNA can include, for example, backbone modifications, sugar modifications, or base modifications. 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-N6-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, pseudouridine (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, methyl N-uracil-5-oxyacetate, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyluracil, 5-methoxy-uracil, methyl uracil-5-oxyacetate, uracil-5-oxyacetic acid (v), 1-methyl-pseudouridine, queosine, 13-D-mannosyl-queosine, wybutosine, and phosphoramidates, thiophosphates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, and inosine. The preparation of such analogs is known to those skilled in the art, for example, from U.S. Patent 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.

[0165] In some embodiments, the mRNA (e.g., mRNA encoding a heavy chain and a light chain) may contain RNA backbone modifications. Generally, a backbone modification is a modification in which the phosphodiester of the nucleotide backbone contained in the RNA is chemically modified. Exemplary backbone modifications generally include, but are not limited to, modifications from the group consisting of methylphosphonate, methylaminophosphate, aminophosphate, phosphorothioate (e.g., cytidine 5'-O-(1-thiophosphate)), boranophosphate, positively charged guanidinium groups, etc., which means replacing the phosphodiester bond with other anionic, cationic, or neutral groups.

[0166] In some embodiments, the mRNA (e.g., mRNA encoding a heavy chain and a light chain) may contain sugar modifications. Typical sugar modifications are chemical modifications to 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'-deamino-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'-arabinosylcytidine 5'-triphosphate, 2'-arabinosyluridine 5'-triphosphate), or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0167] In some embodiments, mRNA (e.g., mRNA encoding a heavy chain and a light chain) can contain modifications of nucleobases (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 ribonucleoside 5'-triphosphate, 2-aminoadenosine 5'-triphosphate, 2-thiocytidine 5'-triphosphate, 2-thiouridine 5'-triphosphate, 4-thiouridine 5'-triphosphate, 5-aminoallyl cytidine 5'-triphosphate, 5-aminoallyl uridine 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 ribonucleoside 5'-triphosphate, 7-deazaguanosine 5'-triphosphate, 7-deazaadenosine 5'-triphosphate, 8-azaguanosine 5'-triphosphate, 8-azidoadenosine 5'-triphosphate, benzimidazole ribonucleoside 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.

[0168] Generally, mRNA synthesis includes adding 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.

[0169] Thus, in some embodiments, mRNA (e.g., 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 a terminal phosphate group from the 5' nucleotide, leaving two terminal phosphates; then guanosine triphosphate (GTP) is added to the terminal phosphate by a guanylyltransferase, generating 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.

[0170] In some embodiments, the mRNA (e.g., mRNA encoding a heavy chain and a light chain) includes a 3' poly(A) tail structure. The poly-A tail at the 3' end of the mRNA typically 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, the mRNA encoding an antibody (e.g., mRNA encoding a heavy chain and a light chain) includes a 3' poly(C) tail structure. A suitable poly-C tail at the 3' end of the mRNA typically includes 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 can be added to the poly-A tail or can replace the poly-A tail.

[0171] In some embodiments, the mRNA (e.g., mRNA encoding a heavy chain and a light chain) 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., a length of about 50 to 400 nucleotides, a length of about 50 to 300 nucleotides, a length of about 50 to 200 nucleotides, or a length of about 50 to 100 nucleotides).

[0172] In some embodiments, the 5' region of the mRNA (e.g., mRNA encoding a heavy chain and a light chain) includes a sequence encoding a signal peptide, such as those described herein. In certain embodiments, a signal peptide derived from human growth hormone (hGH) is incorporated into the 5' region. Typically, the signal peptide coding sequence is directly or indirectly linked to the heavy chain or light chain coding sequence at the N-terminus.

[0173] This 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.

[0174] Methods for preparing antibodies are well known in the art and are described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptides of the present disclosure are fully human. Fully human antibodies can be prepared using techniques described in the art and herein. For example, fully human antibodies against a particular antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled. Exemplary techniques that can be used to prepare 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.

[0175] Therapy and Use

[0176] 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.

[0177] The present disclosure further relates to antibody-based therapies that involve administering to a patient such as an animal, mammal, and human the antibodies or fragments, antibody-drug conjugates, chimeric antigen receptors (CARs) and CAR cells, encoding polynucleotides or derivatives of the present disclosure to treat one or more disorders or conditions described herein. The therapeutic molecules or cells of the present disclosure include, but are not limited to, the antibodies of the present disclosure (including variants and derivatives thereof described herein), antibody-drug conjugates, chimeric antigen receptors (CARs) and CAR cells, and nucleic acids or polynucleotides encoding the antibodies of the present disclosure (including variants and derivatives thereof described herein).

[0178] The molecules or cells of the present disclosure 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 for treating tumors.

[0179] Accordingly, in some embodiments, methods for treating cancer in a patient in need thereof are provided. In one embodiment, the method involves administering to the patient an effective amount of the molecules or cells of the present disclosure. In some embodiments, at least one type of cancer cell (e.g., stromal cells) in the patient overexpresses B7-H3.

[0180] The present disclosure also provides cell therapies, such as chimeric antigen receptor (CAR) T cell therapy. Suitable cells can be used, which are transduced with a vector encoding a CAR or contacted with a CAR that comprises an anti-B7-H3 antibody of the present disclosure (or alternatively engineered to express an anti-B7-H3 antibody of the present disclosure). After such contact or engineering, the cells can then be introduced into a cancer patient in need of treatment. The cancer patient may have any type of cancer disclosed herein. The cells (e.g., 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.

[0181] In some embodiments, the cells are isolated from the cancer patient themselves. In some embodiments, the cells are provided by a donor or a cell bank. When the cells are isolated from the cancer patient, unwanted immune responses can be minimized.

[0182] Non-limiting examples of cancers 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.

[0183] Other diseases or conditions related to increased cell survival that can be treated, prevented, diagnosed, and / or prognosed with the antibodies or their variants or derivatives of the present disclosure include, but are not limited to, the progression and / or metastasis of malignancies and related disorders, such as leukemia (including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia (including myeloblasts, promyelocytes, myelomonocytes, monocytes, and erythroleukemia)) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, and solid tumors, including but not limited to sarcomas and carcinomas, such as fibrosarcoma, 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, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, 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 a variety of factors, including the specific antibody, its variant or derivative used, the patient's age, weight, general health, sex, and diet, as well as the time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated. The judgment of medical care providers regarding 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 pharmacological and pharmacokinetic principles well known in the art.

[0184] Methods of administering the antibody or fragment include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antigen-binding polypeptide or composition can be administered by any convenient route, such as by infusion or bolus injection, absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa, etc.), and can be co-administered with other bioactive agents. Thus, the pharmaceutical composition containing the antigen-binding polypeptide of the present disclosure can be administered orally, rectally, parenterally, intracisternally, vaginally, intraperitoneally, topically (such as by powder, ointment, drops, or transdermal patch), buccally, or as an oral or nasal spray.

[0185] As used herein, the term "parenteral" refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injection and infusion.

[0186] Administration can be systemic or local. Additionally, it is desirable to introduce the antibodies of the present disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection can be facilitated, for example, by an intraventricular catheter attached to a reservoir (such as an Ommaya reservoir). Pulmonary administration can also be employed, for example, by using an inhaler or nebulizer, and formulations containing an aerosolizing agent.

[0187] It is desirable to locally administer the antigen-binding polypeptides or compositions of the present disclosure 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 combination with a wound dressing after surgery), by injection, via a catheter, via a suppository, or via an implant, which is a porous, non-porous, or gelatinous material, including membranes, such as a sialastic membrane or fibers. Preferably, when administering the proteins (including antibodies) of the present disclosure, care must be taken to use materials that do not absorb the protein.

[0188] The amount of the antibodies or fragments of the present disclosure effective for treating, inhibiting, and preventing inflammatory, immune, or malignant diseases, disorders, or conditions can be determined by standard clinical techniques. Additionally, in vitro assays can optionally be employed to assist in determining the optimal dosage range. The precise 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 decided according to the judgment of the physician and the circumstances of each patient. The effective dose can be extrapolated from the dose-response curve derived from in vitro or animal model test systems.

[0189] As a general recommendation, the dose of the antibodies or fragments of the present disclosure administered to a patient is typically from 0.001 mg / kg to 100 mg / kg of patient body weight, between 0.01 mg / kg and 20 mg / kg of patient body weight, or between 0.5 mg / kg and 10 mg / kg of patient body weight. Generally, due to the immune response to foreign polypeptides, the half-life of human antibodies in the human body is longer than that of antibodies from other species. Therefore, the dose of human antibodies can generally be reduced and the frequency of administration decreased. Additionally, the dosage and frequency of administration of the antibodies of the present disclosure can be reduced by modifying (e.g., lipidating) to enhance the uptake and tissue penetration of the antibody (e.g., into the brain).

[0190] In additional embodiments, the compositions of the present disclosure are administered in combination with cytokines. Cytokines that can be co-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.

[0191] In additional embodiments, the compositions of the present disclosure are administered in combination with other therapeutic or prophylactic regimens (such as radiotherapy).

[0192] Composition

[0193] The present disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of an antibody or fragment, antibody-drug conjugate, chimeric antigen receptor (CAR) and CAR cells, encoding polynucleotide or derivative, and an acceptable carrier. In some embodiments, the composition further includes a second anti-cancer agent (e.g., an immune checkpoint inhibitor).

[0194] 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 pharmacopeias for use in animals and more particularly in humans. Additionally, a "pharmaceutically acceptable carrier" is generally a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or any type of formulation aid.

[0195] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates, or phosphates. Bacteriostatic agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for regulating tonicity such as sodium chloride or dextrose are also contemplated. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. 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, saccharin sodium, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in E.W. Martin in Remington’s Pharmaceutical Sciences, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, and a suitable amount of carrier to provide the form for proper administration to the patient. The formulation should be suitable for the mode of administration. Parenteral formulations can be enclosed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0196] In an embodiment, the composition is formulated into a pharmaceutical composition suitable for intravenous administration to humans according to conventional procedures. Generally, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition can also include solubilizing agents and local anesthetics such as lidocaine to relieve the pain at the injection site. Generally, the ingredients are provided individually or mixed in unit dosage forms, for example, as a dry freeze-dried powder or an anhydrous concentrate, in a sealed container (such as an ampoule or sachet) indicating the amount of the active agent. When the composition is administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, ampoules of sterile water for injection or saline can be provided to mix the ingredients before administration.

[0197] The compounds of the present disclosure can be formulated in neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2 - ethylaminoethanol, histidine, procaine, etc.

[0198] Examples

[0199] Example 1. Generation and Testing of Mouse Anti - Human B7 - H3 Antibodies

[0200] This example describes the generation of mouse anti - human B7 - H3 monoclonal antibodies using hybridoma technology.

[0201] Antigen: Human B7 - H3(4Ig) His - tagged protein. Protein immunization was performed by subcutaneous injection (s.c.) and intraperitoneal injection (i.p.).

[0202] 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 the immune response, the titrated sera of the mice were screened by ELISA and flow cytometry as described below. Sera were screened by antibodies that bind to human B7 - H3 protein and B7 - H3 - overexpressing CHO - K1 cell line as well as negative control CHO - K1 cell line that does not express B7 - H3. Before hybridoma fusion, animals with sufficient ELISA titers and a FACS binding ratio of positive cell line (B7 - H3 - overexpressing CHO - K1 cells) to negative cell line (vector - only expressing CHO - K1) were selected and given a final boost with B7 - H3 protein.

[0203] Cell fusion and hybridoma screening: As described above, spleens were isolated from the final boosted mice. Hybridomas were generated by cell fusion of the immortalized mouse myeloma cells with the spleen cells based on electric field - based electrofusion. The fused cells were plated in 96 - well flat - bottom microtiter plates for the selection of hybridomas. The supernatants were screened for epitope binning by ELISA and FACS using B7 - H3 - overexpressing CHO - K1 and negative control cell lines.

[0204] Sub - cloning and screening: Positive primary clones with different epitopes in each fusion were sub - cloned by limiting dilution to ensure that the sub - clones were derived from a single parental cell. The sub - clones were screened in the same method as the primary clones. In addition, to confirm the binding potency of the antibodies, the culture supernatants of the positive clones were tested by FACS on A375 cells.

[0205] Hybridoma clones were selected for further analysis. The amino acid sequences of the variable regions are provided in Table 1A below.

[0206] Table 1A. Antibody Variable Region Sequences

[0207]

[0208]

[0209] The CDR sequences (according to the Kabat system) of these antibodies are listed in Table 1B below. Their variants are also shown, where 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 preserve the biological activity of the antibody while preventing PTM to facilitate manufacturing.

[0210] Table 1B. CDR Sequences (with optional mutations to avoid PTM, Kabat numbering)

[0211]

[0212]

[0213] Bold / underlined residues indicate mutations to avoid PTM

[0214]

[0215] Example 2: Human Native Phage Library Panning and Screening

[0216] In this example, fully human anti-B7-H3 antibodies were screened from a human native phage library.

[0217] Antigens: Human B7-H3 (2Ig)-Avi-biotin, Human B7-H3 (4Ig) His-tag, Human B7-H3 (4Ig)-Avi-biotin-His.

[0218] Preparation of the human native phage library: The phage library was constructed using a phagemid vector consisting of antibody gene fragments amplified from PBMC of healthy human subjects. The library was in the form of a Fab phage library. The library size was 1.04×10 11 .

[0219] Solid-phase and solution panning of a phage library against human B7-H3 protein: For solution panning, the phage library was first negatively selected by incubation with BSA-coated streptavidin Dynabeads. The resulting phages were incubated with biotinylated B7-H3-His-tagged protein and washed with a Kingfisher magnetic bead system. The bound phages were eluted with trypsin. For solid-phase panning, the phage library was first blocked with PBS supplemented with 5% milk. The resulting phages were incubated with B7-H3 His-tagged protein and washed with PBST. The bound phages were eluted with trypsin.

[0220] Subsequently, the titer of the eluted phages binding to the antigen was tested and co-cultured with Escherichia coli. Four rounds of panning and screening were performed. The titers of output 3 and output 4 increased significantly.

[0221] Single clones were picked from outputs 3 and 4 and then cultured in 96-well deep plates. The IgG concentration and antigen-binding titer of the culture supernatants were evaluated. A total of 232 positive clones were selected and sequenced. Sequence post-analysis identified 65 unique sequences. ELISA binding analysis was performed on all these clones, and the sequences of the strong binders are shown in Table 2A below.

[0222] Table 2A. Antibody variable region sequences

[0223]

[0224]

[0225] The CDR sequences (according to the Kabat system) of these antibodies are listed in Table 2B below. Their variants are also shown, where 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.

[0226] Table 2B. CDR sequences (with optional mutations to avoid PTM, Kabat numbering)

[0227]

[0228] Bold / underlined residues indicate mutations to avoid PTM

[0229]

[0230] Example 3. Binding activity to human B7-H3 antigen

[0231] This example tested the binding activity of the antibodies to human B7-H3 antigen.

[0232] 3.1 Cell-based binding to human B7-H3

[0233] To evaluate the binding activity of the cloned antibodies, the test antibodies were subjected to FACS assay on A375 or A375.S2 cells.

[0234] Briefly, a total of 4×10 4 A375 cells were incubated with 3-fold or 5-fold serial dilutions of the antibodies in FACS buffer at 4 °C for 30 minutes. After washing with FACS buffer, a fluorescent dye-conjugated anti-human IgG antibody was added to each well and incubated at 4 °C for 30 minutes. The samples were washed twice with FACS buffer. The mean fluorescence intensity (MFI) was evaluated by MACSQuant Analyzer 16.

[0235] As Figure 1 shown, among all these hybridoma clones, 43G8A9 showed comparable binding to MGA017, and others showed better binding activity than the positive reference MGA017. Four antibodies derived from the human natural phage library showed weaker binding activity than MGA017.

[0236] 3.2 Kinetic activity with human B7-H3-4Ig

[0237] Using the capture method, the binding of the test antibodies to human B7-H3(4Ig) protein (his-tag) was tested by Biacore. The mAb was captured by a Protein A chip. Serial dilutions of the human B7-H3 protein were injected onto the captured antibody at a flow rate of 30 μL / min for 3 minutes. The antigen was allowed to dissociate for 600 s. All experiments were performed on a Biacore T200. The binding traces were fitted according to a 1:1 model by the Biacore T200 evaluation software to calculate the association (ka), dissociation (kd) rate constants, and equilibrium constant (KD). The results are shown in Table 3 below.

[0238] Table 3. Kinetic results measured by Biacore

[0239]

[0240] 3.3 Binding epitope of the test antibodies

[0241] The binding epitopes of the test antibodies were characterized using Octet by epitope binning assay.

[0242] In short, capturing human B7-H3 protein at a concentration of 100 nM on the sensor generated a signal of 0.3 nm. Then, the sensor was transferred to 100 nM of competing antibodies (72D1D11, AHP05562, AHP06345, CA84D), which were injected into the sensor for antigen saturation and associated for 120 s. Then, the test antibody was injected for antigen competition testing for another 120 s. After the baseline step, the sensor was transferred to 100 nM of the test antibody for 120 s. Data analysis was performed using Octet Analysis Studio 12.2 software. The binding inhibition ratios between the test antibody and the competing antibodies are shown in Table 4A below. Based on the results, the binding epitopes of the test antibody can be classified into bins Aα, Aβ, Aγ, B, and C, as shown in Table 4B.

[0243] Table 4A. Inhibition ratios by Octet testing

[0244]

[0245]

[0246] Table 4B. Epitope binning results

[0247] 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

[0248] Example 4. Internalization of the test antibody

[0249] This example characterized the internalization of the test antibody in A375 cells.

[0250] The pHAb thiol dye is a pH-sensitive dye that has very low fluorescence at pH > 7 and its fluorescence increases sharply when internalized into endosomes or lysosomes with pH values of approximately 6.3 or 4.7, respectively. In short, the α-hIgG secondary antibody labeled with pHAb thiol dye (10 nM) was incubated with serial dilutions of the antibody starting from 5 - 50 nM for 30 min. And the mixture was added to a 96-well assay plate pre-seeded with 2×10 4 A375 cells per well. After culturing for 48 hours, the fluorescence signal was captured on a multimode plate reader ( 2105) or an Operetta.

[0251] As Figure 2As shown, almost all hybridoma clones showed better internalization than MGA017. Among the antibodies derived from the human natural phage library, AHP06331 and AHP06358 showed lower internalization than MGA017 at lower concentrations, but at higher concentrations, the internalization of AHP06331 and AHP06358 was much stronger than that of MGA017, as indicated by higher EC50 and higher top value in the assay. The internalization of AHP05564 and AHP05346 was lower than that of MGA017.

[0252] Example 5. In vitro cytotoxicity of B7-H3 ADC

[0253] This example characterized the killing efficacy of vc-MMAE-labeled B7-H3 ADC against RKO tumor cells.

[0254] The B7-H3 lead hit was labeled with vc-MMAE (L1-D1), where the drug-to-antibody ratio (DAR) was approximately 4.0. The cytotoxicity of 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 in a CO 2 incubator at 37 °C for 72 hours or 96 hours. At the end of the incubation, 100 μL of CellTiter-Glo reaction reagent was added, and the mixture was incubated at room temperature for 10 minutes. The luminescence signal was obtained by a multimode plate reader ( 2105).

[0255] As Figure 3 shown, compared with MGA017, 72D1D11, 81C8A1, 62H9H5, 106A5B3, 126C10B10, and 227E12D1 mediated stronger cytotoxicity against RKO cell killing. AHP06358 showed comparable cell killing to MGA017.

[0256] Example 6. Humanization of B7-H3 antibody

[0257] 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 the existing human Ig gene sequence database to find the overall best-matching human germline Ig sequences.

[0258] For the light chain of 72D1D11, IGKV2-40*01 and IGKV2-28*01 are the best-fitting germlines, and for the heavy chain of 72D1D11, IGHV7-4-1*02 is selected as the humanized framework. Then, a humanized 72D1D11 CDR-grafted antibody is designed, in which CDR-L1, L2, and L3 are grafted onto the framework sequences of IGKV2-40*01 and IGKV2-28*01, and CDR-H1, H2, and H3 are grafted onto the framework sequence of IGHV7-4-1*02. Then, a 3D model is generated to identify the amino acids in the original murine framework regions that are crucial for antibody binding and conformation. Based on the analysis, back mutations are introduced into the grafted antibody, resulting in 4 additional humanized heavy chains and 4 additional light chains.

[0259] For the light chain of 81C8A1, IGKV6-21*01 is the best-fitting germline, and for the heavy chain of 81C8A1, IGHV2-70*04 is selected as the humanized framework. Then, a humanized 81C8A1 CDR-grafted antibody is designed, in which CDR-L1, L2, and L3 are grafted onto the framework sequence of IGKV6-21*01, and CDR-H1, H2, and H3 are grafted onto the framework sequence of IGHV2-70*04. Then, a 3D model is generated to identify the amino acids in the original murine framework regions that are crucial for antibody binding and conformation. Based on the analysis, back mutations are introduced into the grafted antibody, resulting in 3 additional humanized heavy chains and 4 additional light chains.

[0260] The sequences of the resulting humanized sequences are listed in Table 5-6.

[0261] Table 5A. Humanization of 72D1D11

[0262]

[0263]

[0264] Table 5B. Humanized antibodies from 72D1D11

[0265]

[0266] Table 6A. Humanization of 81C8A1

[0267]

[0268]

[0269] Table 6B. Humanized antibodies from 81C8A1

[0270]

[0271] Example 7. Binding Activity of Humanized Antibodies

[0272] Flow cytometry was performed to confirm the binding activity of the humanized antibodies to A375 cells.

[0273] Briefly, a total of 4×10 4 A375 cells were incubated with antibodies serially diluted 3-fold starting from 30 nM in FACS buffer at 4 °C for 30 minutes. After washing with FACS buffer, a PE-conjugated anti-human IgG antibody (eBioscience TM , Invitrogen) was 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) of PE was evaluated by MACSQuant Analyzer 16.

[0274] As Figure 4 shown, all humanized 72D1D11 antibodies were comparable to the chimeric antibody in terms of human B7-H3 binding. For the humanized 81C8A1 antibody, 81C8A1-z3, 81C8A1-z4, 81C8A1-z7, 81C8A1-z8 showed comparable binding to the chimeric antibody.

[0275] Example 8. PTM Removal of Chimeric 72D1D11

[0276] 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, in this example, NG on VL CDR1 was mutated to QG or NA, and DG on VH CDR3 was mutated to EG to prevent PTM. The sequences of potential PTM removal sites are listed in Table 7.

[0277] Table 7. PTM Removal of 72D1D11

[0278]

[0279] As Figure 5 shown, 72D1D11-p1 / p2 showed comparable human B7-H3 binding to the corresponding parental antibody on A375 cells. 72D1D11-p3 showed a slight loss of human B7-H3 binding on A375 cells.

[0280] Example 9. Confirmation of PTM Removal of Humanized 72D1D11

[0281] Thus, 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 sequence is listed in Table 8.

[0282] Table 8A. Humanized 72D1D11 Antibody with Potential PTM Sites Removed

[0283]

[0284] Table 9. Humanization of 72D1D11 with Potential PTM Sites Removed

[0285]

[0286]

[0287] As Figure 6 shown in A, 72D1D11-z3p1 showed comparable human B7-H3 binding to the parental antibody 72D1D11-z3 on A375 cells. 72D1D11-z3p2 showed a slight loss of human B7-H3 binding on A375 cells. As Figure 6 shown in B, 72D1D11-z3p6, 72D1D11-z3p7, and 72D1D11-z3p8 lost human B7-H3 binding on A375 cells.

[0288] ***

[0289] The scope of the present disclosure is not limited by the specific embodiments described, which are intended to be illustrative of 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 can be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Accordingly, 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.

[0290] All publications and patent applications mentioned in this specification are incorporated herein 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 antigen-binding fragment thereof that is specific for human B7-H3 (CD276) protein and comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising VH CDR1, VH CDR2, and VH CDR3, and the light chain variable region comprising VL CDR1, VL CDR2, and VL CDR3, wherein: (a) the VH CDR1 is the amino acid sequence of SEQ ID NO:39; the VH CDR2 is the amino acid sequence of SEQ ID NO:40; the VH CDR3 is the amino acid sequence of SEQ ID NO:41, 42, 43, 44, 45, or 46; the VL CDR1 is the amino acid sequence of SEQ ID NO:47, 48, or 49; the VL CDR2 is the amino acid sequence of SEQ ID NO:50; and the VL CDR3 is the amino acid sequence of SEQ ID NO:51, (b) the VH CDR1 is the amino acid sequence of SEQ ID NO:52; the VH CDR2 is the amino acid sequence of SEQ ID NO:53; the VH CDR3 is the amino acid sequence of SEQ ID NO:54; the VL CDR1 is the amino acid sequence of SEQ ID NO:55; the VL CDR2 is the amino acid sequence of SEQ ID NO:56; and the VL CDR3 is the amino acid sequence of SEQ ID NO:57, (c) the VH CDR1 is the amino acid sequence of SEQ ID NO:33; the VH CDR2 is the amino acid sequence of SEQ ID NO:34; the VH CDR3 is the amino acid sequence of SEQ ID NO:35; the VL CDR1 is the amino acid sequence of SEQ ID NO:36; the VL CDR2 is the amino acid sequence of SEQ ID NO:37; and the VL CDR3 is the amino acid sequence of SEQ ID NO:38, (d) the VH CDR1 is the amino acid sequence of SEQ ID NO:25; the VH CDR2 is the amino acid sequence of SEQ ID NO:26, 27, or 28; the VH CDR3 is the amino acid sequence of SEQ ID NO:29; the VL CDR1 is the amino acid sequence of SEQ ID NO:30; the VL CDR2 is the amino acid sequence of SEQ ID NO:31; and the VL CDR3 is the amino acid sequence of SEQ ID NO:32, (e) the VH CDR1 is the amino acid sequence of SEQ ID NO:58; the VH CDR2 is the amino acid sequence of SEQ ID NO:59; the VH CDR3 is the amino acid sequence of SEQ ID NO:60; the VL CDR1 is the amino acid sequence of SEQ ID NO:61; The VL CDR2 is the amino acid sequence of SEQ ID NO:62; and the VL CDR3 is the amino acid sequence of SEQ ID NO:63, (f) the VH CDR1 is the amino acid sequence of SEQ ID NO:64; the VH CDR2 is the amino acid sequence of SEQ ID NO:65 or 66; the VH CDR3 is the amino acid sequence of SEQ ID NO:67; the VL CDR1 is the amino acid sequence of SEQ ID NO:68; the VL CDR2 is the amino acid sequence of SEQ ID NO:69; and the VL CDR3 is the amino acid sequence of SEQ ID NO:70, (g) the VH CDR1 is the amino acid sequence of SEQ ID NO:71; the VH CDR2 is the amino acid sequence of SEQ ID NO:72, 73 or 74; the VH CDR3 is the amino acid sequence of SEQ ID NO:75; the VL CDR1 is the amino acid sequence of SEQ ID NO:76; the VL CDR2 is the amino acid sequence of SEQ ID NO:77; and the VL CDR3 is the amino acid sequence of SEQ ID NO:78, (h) the VH CDR1 is the amino acid sequence of SEQ ID NO:79; the VH CDR2 is the amino acid sequence of SEQ ID NO:80; the VH CDR3 is the amino acid sequence of SEQ ID NO:81; the VL CDR1 is the amino acid sequence of SEQ ID NO:82; the VL CDR2 is the amino acid sequence of SEQ ID NO:83; and the VL CDR3 is the amino acid sequence of SEQ ID NO:84, (i) the VH CDR1 is the amino acid sequence of SEQ ID NO:85; the VH CDR2 is the amino acid sequence of SEQ ID NO:86 or 87; the VH CDR3 is the amino acid sequence of SEQ ID NO:88; the VL CDR1 is the amino acid sequence of SEQ ID NO:89; the VL CDR2 is the amino acid sequence of SEQ ID NO:90; and the VL CDR3 is the amino acid sequence of SEQ ID NO:91, (j) the VH CDR1 is the amino acid sequence of SEQ ID NO:92; the VH CDR2 is the amino acid sequence of SEQ ID NO:93, 94, 95 or 96; the VH CDR3 is the amino acid sequence of SEQ ID NO:97; the VL CDR1 is the amino acid sequence of SEQ ID NO:98; the VL CDR2 is the amino acid sequence of SEQ ID NO:99 or 100; and the VL CDR3 is the amino acid sequence of SEQ ID NO:101, (k) the VH CDR1 is the amino acid sequence of SEQ ID NO:102; The VH CDR2 is the amino acid sequence of SEQ ID NO: 103; The VH CDR3 is the amino acid sequence of SEQ ID NO: 104; The VL CDR1 is the amino acid sequence of SEQ ID NO: 105; The VL CDR2 is the amino acid sequence of SEQ ID NO: 106; and The VL CDR3 is the amino acid sequence of SEQ ID NO: 107, (l) The VH CDR1 is the amino acid sequence of SEQ ID NO: 108; The VH CDR2 is the amino acid sequence of SEQ ID NO: 109; The VH CDR3 is the amino acid sequence of SEQ ID NO: 110, 111 or 112; The VL CDR1 is the amino acid sequence of SEQ ID NO: 113, 114 or 115; The VL CDR2 is the amino acid sequence of SEQ ID NO: 116; and The VL CDR3 is the amino acid sequence of SEQ ID NO: 117, or (m) The VH CDR1 is the amino acid sequence of SEQ ID NO: 182; The VH CDR2 is the amino acid sequence of SEQ ID NO: 183, 184, 185 or 186; The VH CDR3 is the amino acid sequence of SEQ ID NO: 187; The VL CDR1 is the amino acid sequence of SEQ ID NO: 188, 189, 190 or 191; The VL CDR2 is the amino acid sequence of SEQ ID NO: 192; and The VL CDR3 is 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 is the amino acid sequence of SEQ ID NO: 39; The VH CDR2 is the amino acid sequence of SEQ ID NO: 40; The VH CDR3 is the amino acid sequence of SEQ ID NO: 41, 42, 43, 44, 45 or 46; The VL CDR1 is the amino acid sequence of SEQ ID NO: 47, 48 or 49; The VL CDR2 is the amino acid sequence of SEQ ID NO: 50; and The VL CDR3 is 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 is the amino acid sequence of SEQ ID NO: 39; The VH CDR2 is the amino acid sequence of SEQ ID NO: 40; The VH CDR3 is the amino acid sequence of SEQ ID NO: 41; The VL CDR1 is the amino acid sequence of SEQ ID NO: 47; The VL CDR2 is the amino acid sequence of SEQ ID NO: 50; and The VL CDR3 is 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 NO:5 and 159 - 162, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO:6 and 163 - 166.

5. The antibody or antigen-binding fragment 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 is the amino acid sequence of SEQ ID NO:39; the VH CDR2 is the amino acid sequence of SEQ ID NO:40; the VH CDR3 is the amino acid sequence of SEQ ID NO:41; the VL CDR1 is the amino acid sequence of SEQ ID NO:48 or 49; the VL CDR2 is the amino acid sequence of SEQ ID NO:50; and the VL CDR3 is 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 is the amino acid sequence of SEQ ID NO:52; the VH CDR2 is the amino acid sequence of SEQ ID NO:53; the VH CDR3 is the amino acid sequence of SEQ ID NO:54; the VL CDR1 is the amino acid sequence of SEQ ID NO:55; the VL CDR2 is the amino acid sequence of SEQ ID NO:56; and the VL CDR3 is 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 NO:7 and 167 - 169, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8 and 170 - 173.

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

38.

12. The antibody or antigen-binding fragment thereof according to claim 11, 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 for human B7-H3 (CD276) protein and comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising VH CDR1, VH CDR2 and VH CDR3, and the light chain variable region comprising VL CDR1, VL CDR2 and VL CDR3, wherein: (a) The VH CDR1 is the amino acid sequence of SEQ ID NO:149; the VH CDR2 is the amino acid sequence of SEQ ID NO:150, 151 or 152; the VH CDR3 is the amino acid sequence of SEQ ID NO:153 or 154; the VL CDR1 is the amino acid sequence of SEQ ID NO:155; the VL CDR2 is the amino acid sequence of SEQ ID NO:156; and the VL CDR3 is the amino acid sequence of SEQ ID NO:157 or 158, (b) The VH CDR1 is the amino acid sequence of SEQ ID NO:126; the VH CDR2 is the amino acid sequence of SEQ ID NO:127 or 128; the VH CDR3 is the amino acid sequence of SEQ ID NO:129, 130 or 131; the VL CDR1 is the amino acid sequence of SEQ ID NO:132; the VL CDR2 is the amino acid sequence of SEQ ID NO:133; and the VL CDR3 is the amino acid sequence of SEQ ID NO:134, (c) The VH CDR1 is the amino acid sequence of SEQ ID NO:135; the VH CDR2 is the amino acid sequence of SEQ ID NO:136 or 137; the VH CDR3 is the amino acid sequence of SEQ ID NO:138; the VL CDR1 is the amino acid sequence of SEQ ID NO:139; the VL CDR2 is the amino acid sequence of SEQ ID NO:140; and the VL CDR3 is the amino acid sequence of SEQ ID NO:141, or (d) The VH CDR1 is the amino acid sequence of SEQ ID NO:142; the VH CDR2 is the amino acid sequence of SEQ ID NO:143; the VH CDR3 is the amino acid sequence of SEQ ID NO:144; the VL CDR1 is the amino acid sequence of SEQ ID NO:145; the VL CDR2 is the amino acid sequence of SEQ ID NO:146; and the VL CDR3 is 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 is the amino acid sequence of SEQ ID NO: 149; the VH CDR2 is the amino acid sequence of SEQ ID NO: 150, 151 or 152; the VH CDR3 is the amino acid sequence of SEQ ID NO: 153 or 154; the VL CDR1 is the amino acid sequence of SEQ ID NO: 155; the VL CDR2 is the amino acid sequence of SEQ ID NO: 156; and the VL CDR3 is 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 is the amino acid sequence of SEQ ID NO: 149; the VH CDR2 is the amino acid sequence of SEQ ID NO: 150; the VH CDR3 is the amino acid sequence of SEQ ID NO: 153; the VL CDR1 is the amino acid sequence of SEQ ID NO: 155; the VL CDR2 is the amino acid sequence of SEQ ID NO: 156; and the VL CDR3 is 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 is the amino acid sequence of SEQ ID NO: 126; the VH CDR2 is the amino acid sequence of SEQ ID NO: 127 or 128; the VH CDR3 is the amino acid sequence of SEQ ID NO: 129, 130 or 131; the VL CDR1 is the amino acid sequence of SEQ ID NO: 132; the VL CDR2 is the amino acid sequence of SEQ ID NO: 133; and the VL CDR3 is 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 is the amino acid sequence of SEQ ID NO: 135; the VH CDR2 is the amino acid sequence of SEQ ID NO: 136 or 137; the VH CDR3 is the amino acid sequence of SEQ ID NO: 138; the VL CDR1 is the amino acid sequence of SEQ ID NO: 139; the VL CDR2 is the amino acid sequence of SEQ ID NO: 140; and This VL CDR3 is 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 is the amino acid sequence of SEQ ID NO:142; the VH CDR2 is the amino acid sequence of SEQ ID NO:143; the VH CDR3 is the amino acid sequence of SEQ ID NO:144; the VL CDR1 is the amino acid sequence of SEQ ID NO:145; the VL CDR2 is the amino acid sequence of SEQ ID NO:146; and the VL CDR3 is 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 antigen-binding fragment thereof according to any one of claims 1-22, wherein the antibody or antigen-binding fragment is a bivalent Fab antibody or a fragment selected from the group consisting of F(ab’)2, F(ab)2, Fab’, Fab, Fv, and scFv.

24. A multispecific antibody comprising the antigen-binding fragment 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 the 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 1-23, the multispecific antibody according to claim 24, or the CAR according to claim 25.

27. The one or more polynucleotides according to claim 26, which are one or more mRNAs.

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

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

30. An antibody-drug conjugate comprising: (a) the antibody or antigen-binding fragment thereof according to any one of claims 1-23 or the multispecific antibody according to claim 24; and (b) a conjugate moiety conjugated to the antibody or antigen-binding fragment, 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 1-23, a multispecific antibody according to claim 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. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-23, a multispecific antibody according to claim 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 for the preparation of a medicament for the treatment of cancer; the cancer being non-small cell lung cancer, renal cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, and / or pancreatic cancer.

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