Recombinant antibodies, immunoconjugates comprising the same, and their use in the treatment of cancer

Abstract

This disclosure relates to a recombinant antibody or fragment thereof having binding affinity and specificity for B7-H3. According to certain embodiments of this disclosure, the recombinant antibody or fragment thereof comprises a heavy chain variation (VH) domain and a light chain variation (VL) domain having sequence numbers 7 and 8, respectively. This disclosure also relates to an immunoconjugate comprising the recombinant antibody or fragment thereof, and a method for treating cancer using the immunoconjugate of the present invention.

Description

Background of the Invention 1. Technical Field

[0002] This disclosure pertains to the field of treating diseases. More specifically, this disclosure relates to a novel antibody and its use in treating cancer. 2. Background Technology

[0004] Cancer is one of the leading causes of death worldwide. According to statistics from the International Agency for Research on Cancer (IARC), an estimated 10 million people died from cancer globally in 2020, with approximately 19.3 million new cancer cases. Despite global efforts to develop new cancer therapies and improve the effectiveness of existing treatments, cancer mortality rates remain extremely high. While various treatment methods are available, including chemotherapy, surgery, radiation therapy, hormone therapy, biotherapy, targeted therapy, and the latest immunotherapy and cell therapy, cancer continues to pose significant health risks and impose a substantial economic burden.

[0005] Tumor growth depends on its complex surrounding tissue environment, which not only sustains tumor cell growth but also provides a sufficient blood supply to tumor cells through angiogenesis. On the other hand, tumor growth and metastasis also depend on its ability to evade host immune surveillance and overcome host defenses. Most tumors are known to express antigens (also known as "tumor-associated antigens" (TAAs)). Due to their relatively weak immunogenicity, the host immune system can recognize these TAAs to varying degrees. However, in most cases, because cancer cell-derived escape mechanisms block the function of the host immune system, the tumor-induced immune response is often insufficient to inhibit tumor growth. These escape mechanisms include increasing the expression of programmed cell death ligand-1 (PD-L1) to modulate the immune system, releasing immunosuppressive factors, and attracting immunosuppressive cells to the tumor environment.

[0006] B7-H3, also known as CD276, is a type I transmembrane protein composed of an extracellular domain, a single transmembrane domain, and a short intracellular domain. B7-H3 belongs to the B7 family, a superfamily of immunoglobulins (Ig) possessing both an immunoglobulin V-like domain and an immunoglobulin C-like domain (e.g., IgV-IgC). Most human B7-H3 contains two extracellular tandem IgV-IgC domains (4Ig-B7-H3). Mouse and rat B7-H3 are currently known to possess only two Ig domains (IgV-IgC; 2Ig-B7-H3) and have functions similar to human 4Ig-B7-H3.

[0007] B7-H3 is known to be overexpressed in various types of cancer, including non-small cell lung cancer, pharyngeal cancer, oral cancer, kidney cancer, urothelial carcinoma, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, and pancreatic cancer. In particular, in prostate cancer, B7-H3 expression is positively correlated with the malignancy and progression of the cancer. Similarly, patients with head and neck cancers who have high B7-H3 expression typically have lower survival rates; while in pancreatic and ovarian cancers, B7-H3 expression is associated with lymph node metastasis and pathological progression. Furthermore, in B7-H3-positive cancer cell lines, administration of small interfering RNA (siRNA) that inhibits B7-H3 can reduce the metastatic and invasive abilities of cancer cells. Therefore, B7-H3 could serve as a potential drug target for the development of therapeutic antibodies or antibody-drug conjugates (ADCs) to treat cancer.

[0008] Several anti-B7-H3 antibodies have been developed in the pharmaceutical field; however, the safety and efficacy of these antibodies in patients still need to be confirmed through clinical trials. Furthermore, most anti-B7-H3 antibodies are mouse antibodies or humanized antibodies, which typically possess high immunogenicity and can have adverse effects on human individuals. Therefore, the field urgently needs a novel antibody with specific binding to B7-H3 to treat cancer more effectively and safely. Summary of the Invention

[0009] This summary is intended to provide a simplified overview of the present disclosure to enable the reader to gain a basic understanding of it. It is not a complete summary of the present disclosure and is not intended to identify key / critical components of the embodiments of the invention or to define the scope of the invention.

[0010] The first aspect of this disclosure relates to a B7-H3-targeting recombinant antibody (i.e., a recombinant antibody with binding affinity and specificity for B7-H3, capable of targeting B7-H3) or a fragment thereof (e.g., a single-chain variant fragment (scFv)). Structurally, the B7-H3-targeting recombinant antibody or antibody fragment of the present invention comprises a variable heavy chain (VH) domain and a light chain (VL) domain, wherein the VH domain comprises a first heavy chain complementarity-determining region (CDR) (CDR-H1), a second heavy chain CDR (CDR-H2), and a third heavy chain CDR (CDR-H3), and the VL domain comprises a first light chain CDR (CDR-L1), a second light chain CDR (CDR-L2), and a third light chain CDR (CDR-L3).

[0011] According to certain embodiments of this disclosure, CDR-H1, CDR-H2 and CDR-H3 respectively contain amino acid sequences with sequence numbers 1, 2 and 3, and CDR-L1, CDR-L2 and CDR-L3 respectively contain amino acid sequences with sequence numbers 4, 5 and 6.

[0012] According to certain embodiments of this disclosure, the VH and VL domains of the B7-H3-targeting recombinant antibody or antibody fragment respectively contain amino acid sequences having at least 85% sequence similarity to sequence numbers 7 and 8; preferably, they each contain amino acid sequences having at least 90% sequence similarity to sequence numbers 7 and 8; more preferably, they each contain amino acid sequences having at least 95% sequence similarity to sequence numbers 7 and 8. In an exemplary embodiment of this disclosure, the VH and VL domains of the B7-H3-targeting recombinant antibody or fragment thereof respectively contain amino acid sequences of sequence numbers 7 and 8 (i.e., they each contain amino acid sequences having 100% sequence similarity to sequence numbers 7 and 8).

[0013] The second aspect of this disclosure relates to the use of the B7-H3-targeting recombinant antibody or fragment thereof of the present invention in the preparation of immunoconjugates for treating cancer in individuals. According to embodiments of this disclosure, the immunoconjugate comprises the B7-H3-targeting recombinant antibody or antibody fragment of the present invention, a therapeutic agent, and a connector (also called a linker) for linking the therapeutic agent to the B7-H3-targeting recombinant antibody or antibody fragment. Depending on the intended purpose, the therapeutic agent may be a cytotoxic drug, a radioactive seed, a cytokine, a hormonal drug, an immunostimulant, or an immunotherapeutic agent.

[0014] According to certain preferred embodiments of this disclosure, the therapeutic agent is a cytotoxic drug, for example, aurestatin or a derivative thereof. In one exemplary embodiment, the therapeutic agent is monomethylaurestatin E (MMAE). In another embodiment, the therapeutic agent is monomethylaurestatin F (MMAF).

[0015] This disclosure also provides a pharmaceutical composition for treating cancer. The pharmaceutical composition comprises the immunoconjugate of the present invention, and, optionally, a pharmaceutically acceptable carrier.

[0016] Another aspect of this disclosure relates to a method for treating cancer in an individual. The method comprises administering to the individual an effective amount of an immunoconjugate or pharmaceutical composition of this disclosure.

[0017] Exemplary cancers treatable by the methods of the present invention include, but are not limited to, breast cancer, gastric cancer, colorectal cancer, gallbladder cancer, prostate cancer, cervical cancer, ovarian cancer, chronic or acute lymphoblastic leukemia, bladder cancer, kidney cancer, liver cancer, head and neck squamous cell carcinoma, glioblastoma, esophageal cancer, pancreatic cancer, oral cancer, lung cancer, melanoma, and lymphoma.

[0018] The individuals described are mammals; preferably humans.

[0019] After reading the following embodiments, those skilled in the art will easily understand the basic spirit and other inventive objectives of the present invention, as well as the technical means and implementation methods adopted by the present invention. Attached Figure Description

[0020] To make the above and other objects, features, advantages and embodiments of the present invention more apparent and understandable, the accompanying drawings are described below:

[0021] Figure 1 The analysis results of enzyme-conjugated immunosorbent assay (ELISA) illustrated in Example 1.1 of this disclosure are used to illustrate the binding activity of antibody 91H06 to a specific protein, wherein antibody 91H06 has binding specificity to B7-H3 and does not cross-react with other B7 family proteins.

[0022] Figure 2 The following diagrams, based on the flow cytometry analysis results illustrated in Example 1.2 of this disclosure, illustrate the binding affinity of ifenastatin and antibody 91H06 to B7-H3 positive cells; Inset (A): Binding affinity of ifenastatin to EMT6 cells; Inset (B): Binding affinity of antibody 91H06 to EMT6 cells; Inset (C): Binding affinity of ifenastatin to ID8 cells; Inset (D): Binding affinity of antibody 91H06 to ID8 cells; The cell binding activity (filled peak) of the antibodies was analyzed and tested using flow cytometry; The specificity of the staining was confirmed using an isotype control antibody (unfilled peak);

[0023] Figure 3 The linear graph shown in Example 3 of this disclosure illustrates the tumor volume in mice with Detroit 562 tumors after receiving specific treatment; 2-(N-morpholino)ethanesulfonic acid (MES) was administered to mice with tumors weekly for three weeks; 1.5 mpk: 91H06-MMAE (1.5 mg / kg) was administered to mice with tumors weekly for three weeks; 5 mpk: 91H06-MMAE (5 mg / kg) was administered to mice with tumors weekly for three weeks.

[0024] Figure 4 The graph shown in Example 3 of this disclosure illustrates the tumor volume in mice with FaDu tumors after receiving specific treatment; Carrier: a single dose of MES buffer administered to mice with tumors; 1.5 mpk: a single dose of 91H06-MMAE (1.5 mg / kg) administered to mice with tumors; 5 mpk: a single dose of 91H06-MMAE (5 mg / kg) administered to mice with tumors; and

[0025] Figure 5 The line graph is based on Example 3 of this disclosure and is used to illustrate the tumor volume of mice with Detroit 562 tumors after weekly administration of 91H06-MMAE (5 mg per kilogram) for three consecutive weeks. Detailed Implementation

[0026] To make the description of this disclosure more detailed and complete, illustrative descriptions of embodiments and specific examples of the present invention are provided below; however, these are not the only forms of implementing or utilizing the specific examples of the present invention. The embodiments cover features of multiple specific examples and methods and steps for constructing and operating these specific examples, as well as their order. However, other specific examples may also be used to achieve the same or equivalent functions and order of steps.

[0027] I. Definition

[0028] Unless otherwise defined in this specification, scientific and technical terms used herein have the same meaning as understood and commonly used by those skilled in the art. Furthermore, unless conflicting with the context, singular nouns used herein include their plural forms, and vice versa.

[0029] While the numerical ranges and parameters used to define the broader scope of this invention are approximate, the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any numerical value inevitably contains standard deviations due to individual testing methods. Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the term "approximately" may mean that the actual value falls within the acceptable standard error of the average, as determined by those skilled in the art. Except for experimental examples, or unless explicitly stated otherwise, it is understood that all ranges, quantities, values, and percentages used herein (e.g., to describe material usage, duration, temperature, operating conditions, quantity ratios, and the like) are modified with "approximately". Therefore, unless otherwise stated, the numerical parameters disclosed in this specification and the accompanying claims are approximate values ​​and are subject to change as needed. At a minimum, these numerical parameters should be understood as the indicated significant digits and values ​​obtained by applying general rounding. Here, a range of values ​​is expressed as a distance from one endpoint to another or between two endpoints; unless otherwise stated, all ranges of values ​​herein include the endpoints.

[0030] The term “antibody” has the broadest meaning in this disclosure, specifically including monoclonal antibodies (mAbs; including full-length monoclonal antibodies), multiclonal antibodies, pleiotropic antibodies (e.g., bifunctional antibodies), chimeric antibodies, humanized antibodies, or antibody fragments that produce specific biological activities. The term “antibody fragment” in this disclosure includes a portion of a full-length antibody, which is typically the site or variant region (e.g., VL and VH domains) within the antibody that binds to an antigen. Exemplary antibody fragments include antigen-binding fragments (Fab), Fab', F(ab')2, single-chain variant fragments (scFv), double-chain antibodies, linear antibodies, single-chain antibody molecules, and pleiotropic antibodies formed from antibody fragments. Based on the amino acid sequence of the constant domain of the antibody heavy chain, immunoglobulins can be classified into types such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), and immunoglobulin E (IgE). Each type can be further divided into different subtypes (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Known immunoglobulins contain α, γ, δ, ε, or μ heavy chain constant domains. Those skilled in the art are familiar with the subunit structures and three-dimensional conformations of different types of immunoglobulins; for example, see Abbas et al. (Cellular and Molecular Immunology, 4th Edition (2000)). Antibodies can be part of a larger fusion molecule (formed by covalent or non-covalent binding of an antibody to one or more other proteins or peptides).

[0031] In this disclosure, a “monoclonal antibody” (mAb) refers to an antibody derived from a substantially homogeneous group of antibodies. Unlike polyclonal antibodies, which contain different antibodies that can target different epitopes, a monoclonal antibody targets only a single antigenic determinant (i.e., epitope) of an antigen. Monoclonal antibodies can be prepared by fusing antibody-producing B cells with rapidly growing cells (e.g., immortalized cells). The resulting fusion cells (or fusion tumors) can proliferate rapidly to obtain selected strains (also known as clones) that produce large amounts of antibodies. Alternatively, monoclonal antibodies or antibody fragments possessing specific biological activities can be prepared using recombinant DNA methods, wherein a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence of an antibody derived from one species or belonging to one antibody type or subtype, while the remainder is identical or homologous to a corresponding sequence of an antibody derived from another species or another antibody type or subtype.

[0032] In this disclosure, the term "recombinant antibody" refers to an antibody expressed and isolated from cells or cell lines, wherein the cells or cell lines are transfected with an expression vector (one or more expression vectors, usually two expression vectors) containing the coding sequence of the antibody, and the coding sequence is not naturally associated with the cells.

[0033] In this specification, a "complementarity-determining region" (CDR) refers to a highly variable region of an antibody molecule that can form a complementary surface with the three-dimensional surface of the binding antigen. From the N-terminus to the C-terminus, each antibody's heavy and light chains each contain three CDRs (i.e., CDR-1, CDR-2, and CDR-3). Therefore, an antigen-binding site contains a total of six CDRs: three located in the heavy chain variable region (i.e., CDR-H1, CDR-H2, and CDR-H3) and three located in the light chain variable region (i.e., CDR-L1, CDR-L2, and CDR-L3).

[0034] The "variable domain" of an antibody refers to the amino-terminal domain of its heavy or light chain. These positions are the most variable parts of the antibody and contain antigen-binding sites. The term "variation" refers to the fact that certain parts of the variable domain exhibit significant sequence differences between antibodies and contribute to the binding and specificity of each particular antibody to its specific antigen. However, variability is not uniformly distributed throughout the entire variable domain of an antibody. It is mainly concentrated in the three CDRs or highly variable regions in the light and heavy chain variable domains. The more highly conserved parts of the variable domain are called framework regions (FRs). The variable domains of the natural heavy and light chains each contain four FRs, which mostly adopt a β-sheet configuration and are linked by three CDRs that form a loop and, in some cases, part of the β-sheet structure. The CDRs in each antibody chain are linked together very closely by FRs and together with the CDRs of the other chain, they contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).

[0035] This disclosure and the claimed inventive concept also include minor variations in the amino acid sequence of the antibody (particularly minor variations in the FR sequence of the antibody), provided that such variations maintain at least 85% sequence similarity, for example, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence similarity. The properties of the antibodies of this disclosure can be altered by specific modifications without affecting their physiological activity. For example, certain amino acid residues located in the framework region of the antibody can be changed and / or deleted without affecting the physiological activity of the antibodies of this invention. In particular, reserved amino acid substitutions are also included. Reserved substitutions are mutual substitutions between amino acids having similar / related side chains. Generally, amino acids encoded by genes can be divided into four main categories: (1) acidic amino acids, namely aspartic acid and glutamic acid; (2) basic amino acids, namely lysine, arginine, and histidine; (3) nonpolar amino acids, namely alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) nonpolar amino acids, namely glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. A better classification is: serine and threonine belong to the aliphatic hydroxyl group; asparagine and glutamine belong to the amide group; alanine, valine, leucine, and isoleucine belong to the aliphatic group; while phenylalanine, tryptophan, and tyrosine belong to the aromatic group. For example, it is conceivable that replacing leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or another amino acid with a structurally similar amino acid will not cause significant changes in molecular binding or protein properties, especially when the substitution site is not located at the antigen-binding site (e.g., CDR). Substitution between amino acids will not affect these properties. Whether a change in an amino acid can form a functional antibody can be determined by detecting the specific activity of the antibody derivative. Antibody fragments or analogs can be prepared using methods known to those skilled in the art. The preferred amino and carboxyl terms of the fragment or analog are the boundaries of adjacent functional domains.

[0036] The "sequence similarity percentage" mentioned in this disclosure refers to the percentage of amino acid residues in a candidate sequence that are identical to those in a reference sequence. When performing the above alignment, the candidate sequence and the reference sequence can be placed side-by-side, with gaps introduced if necessary, to achieve the highest possible sequence similarity. When calculating similarity, conserved substitutions of amino acid residues are considered distinct residues. Various methods exist in the art for performing this side-by-side alignment, such as publicly available software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR). Those skilled in the art can select appropriate parameters and calculation methods to obtain the optimal arrangement when performing the alignment. In this specification, the sequence alignment between two amino acid sequences is performed using the Blastp protein-protein BLAST analysis database provided by the National Center for Biotechnology Information (NCBI). The calculation method for the amino acid similarity (also referred to in this specification as the percentage (%) of amino acid similarity between sequence A and sequence B) of a candidate sequence A compared to a reference sequence B is as follows: % Where X is the number of identical amino acid residues obtained by arranging sequences A and B using the BLAST analysis database, and Y is the total number of amino acid residues in the shorter of sequences A and B.

[0037] In this disclosure, the terms “link,” “coupled,” and “connected” are interchangeable and are used to refer to a linking relationship between two components, which may be a direct link or an indirect link.

[0038] In this disclosure, the term "treatment" includes the partial or complete prevention, improvement, reduction, and / or management of cancer-related symptoms, secondary symptoms, or signs. The term "treatment" in this disclosure also refers to the application or administration of one or more of the antibody or immunoconjugate of this invention to an individual suffering from cancer-related symptoms, secondary symptoms, or signs, to achieve partial or complete reduction, slowing, cure of the disease, delay of onset, inhibition of disease progression, reduction of disease severity, and / or reduction of the occurrence of one or more cancer-related symptoms, signs, or secondary signs. Cancer-related symptoms, secondary symptoms, and / or signs include, but are not limited to, nausea, vomiting, loss of appetite, changes in bowel habits, constipation, fatigue, muscle weakness, fractures, swelling or lumps, bleeding, cough, fever, neurological problems (e.g., epilepsy, visual changes, hearing changes, or facial drooping), weight changes (i.e., weight gain or loss), coma, and pain. Here, "treatment" can also refer to administration to an individual with early signs or symptoms to reduce that individual's risk of developing cancer-related signs, secondary signs, and / or symptoms. Treatment is considered "effective" when it reduces one or more signs or clinical markers. Alternatively, treatment is considered "effective" when it reduces, slows, or stops the progression of disease, signs, or symptoms.

[0039] "Effective dose" here refers to an amount of an ingredient sufficient to produce the desired therapeutic response. An effective dose also means that the therapeutic benefit of an ingredient outweighs its toxic or harmful effects. An effective dose of a drug does not necessarily cure a disease or symptom, but rather provides treatment for the disease or symptom, delays, suppresses, or prevents the onset of the disease or symptom, or improves the associated symptoms. Effective doses can be administered in appropriate forms as one, two, or multiple doses to an individual over a specific period of time. The specific effective dose depends on various factors, such as the specific condition to be treated, the patient's physiological condition (e.g., patient weight, age, or sex), the type of mammal or animal receiving treatment, the duration of treatment, the nature of the current therapy (if any), and the specific formulation and structure of the compound or its derivatives used. For example, an effective dose can be expressed as the total weight of the drug (e.g., in grams, milligrams, or micrograms), or as the ratio of the drug's weight to body weight (in milligrams per kilogram (mg / kg)). Alternatively, the effective amount can be expressed as the concentration of the active ingredient (e.g., the immunoconjugate of this disclosure), such as molar concentration, weight concentration, volume concentration, weight-molar concentration, mole fraction, weight fraction, and mixing ratio. Those skilled in the art can calculate the human equivalent dose (HED) of the drug (e.g., the immunoconjugate of this invention) based on animal model dosages. For example, those skilled in the art can estimate the maximum safe starting dose for human use based on the "Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers" published by the U.S. Food and Drug Administration (FDA).

[0040] The term “subject” refers to an animal, including humans, that can receive treatment with the recombinant antibodies, antibody fragments, immunoconjugates, and / or methods of the present invention. Unless otherwise specified, the term “subject” also means both male and female.

[0041] II. Detailed Description of the Invention

[0042] (i) Recombinant antibodies

[0043] The first embodiment of this disclosure provides an anti-B7-H3 monoclonal antibody (mAb) named "91H06". According to the embodiments of this disclosure, the technical feature of the present invention's mAb is that it has binding affinity and specificity to human B7-H3 protein, and can effectively induce the internalization reaction of surface B7-H3 protein within cells.

[0044] According to certain embodiments of this disclosure, the mAb 91H06 of the present invention is prepared using a phage-expressed scFv antibody library. It is conceivable that the mAb 91H06 of the present invention can also be prepared by conventional immunochemical methods (i.e., immunizing animals with specific peptides to induce antigen-specific antibodies) or recombinant DNA technology (also known as "DNA selection technology"; i.e., constructing recombinant DNA encoding specific antibodies and then transducing it into host cells to produce antibodies).

[0045] Structurally, mAb 91H06 contains three CDRs located in its VH domain (i.e., CDR-H1, CDR-H2 and CDR-H3) and three CDRs located in its VL domain (i.e., CDR-L1, CDR-L2 and CDR-L3).

[0046] According to certain embodiments of this disclosure, CDR-H1, CDR-H2, and CDR-H3 of mAb 91H06 respectively contain the amino acid sequences “GFTFSDYGMG” (sequence number: 1), “SISWDSSSKEYADSVKG” (sequence number: 2), and “AWIAIIGGGAHFDY” (sequence number: 3), and CDR-L1, CDR-L2, and CDR-L3 of mAb 91H06 respectively contain the amino acid sequences “RASQSVSSHLA” (sequence number: 4), “LTSSLQS” (sequence number: 5), and “MQSKSLPFT” (sequence number: 6).

[0047] The amino acid sequences of the VH and VL domains of mAb 91H06 are illustrated by the following sequence numbers: 7 and 8, wherein each CDR is indicated in bold in sequence (i.e., CDR-H1, CDR-H2 and CDR-H3 of the VH domain, and CDR-L1, CDR-L2 and CDR-L3 of the VL domain).

[0048] Serial number: 7 (VH field of mAb 91H06)

[0049] Serial number: 8 (VL field of mAb 91H06)

[0050] Since the binding affinity and specificity of an antibody depend primarily on its CDR sequence, it is conceivable that the FR sequences of the VL and VH domains can be varied (e.g., substituted with retainable or non-retainable amino acid residues) without affecting the binding affinity and / or specificity of the antibody of the present invention. Preferably, the FR sequence is retained by substituting one or more amino acid residues with similar properties; for example, leucine (another nonpolar amino acid residue) is replaced by isoleucine, alanine, valine, proline, phenylalanine, or tryptophan (a nonpolar amino acid residue); aspartic acid (another acidic amino acid residue) is replaced by glutamic acid (an acidic amino acid residue); or lysine (another basic amino acid residue) is replaced by arginine or histidine (a basic amino acid residue).

[0051] Accordingly, those skilled in the art can substitute one or more amino acids in the FR sequence of the VH and VL domains of mAb 91H06 without affecting its activity and / or function (i.e., binding to B7-H3 and / or initiating an internalization reaction of B7-H3). Therefore, the scope of protection of this disclosure also covers antibodies with substituted amino acids in the FR sequences of VH and VL. According to some embodiments, the VH domain of mAb 91H06 comprises an amino acid sequence having at least 85% (i.e., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence similarity to sequence number 7, and the VL domain of mAb 91H06 comprises an amino acid sequence having at least 85% (i.e., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence similarity to sequence number 8. According to some preferred embodiments, the VH and VL domains of mAb 91H06 respectively comprise amino acid sequences having at least 90% sequence similarity to sequences numbered 7 and 8. More preferably, the VH and VL domains of mAb 91H06 contain amino acid sequences with at least 95% sequence similarity to sequence numbers 7 and 8, respectively.

[0052] Depending on the intended purpose, the mAb 91H06 of the present invention can be prepared in the form of IgG, IgA, IgM, IgD, or IgE. According to certain exemplary embodiments, the mAb of the present invention is prepared in the form of IgG, comprising a pair of Fc regions and a pair of Fab regions respectively connected to the N-terminus of the pair of Fc regions.

[0053] According to certain embodiments of this disclosure, mAb 91H06 is a whole-human anti-B7-H3 antibody that does not contain the mouse antibody portion, and therefore has low immunogenicity in human individuals.

[0054] This disclosure also relates to fragments of the mAb of the present invention, including scFv, Fab, Fab', F(ab')2 and double-stranded antibodies.

[0055] According to certain embodiments, the antibody of the present invention (comprising mAb 91H06 and its fragments) exhibits specific binding to B7-H3 without cross-reactivity with other B7 family members. In one embodiment, the antibody of the present invention recognizes and binds to human B7-H3 (Sequence Number: 9). In one embodiment, the antibody of the present invention recognizes and binds to monkey B7-H3 (Sequence Number: 10). In another embodiment, the antibody of the present invention recognizes and binds to rat B7-H3 (Sequence Number: 11). In another embodiment, the antibody of the present invention recognizes and binds to mouse B7-H3 (Sequence Number: 12). According to a particular embodiment, the antibody of the present invention binds to the IgV2 and / or IgC2 domains of human B7-H3, wherein the IgV2 and IgC2 domains comprise the amino acid sequence of Sequence Number: 13.

[0056] (ii) Immunoconjugates comprising the recombinant antibodies of the present invention

[0057] According to certain embodiments of this disclosure, the antibody of the present invention has a binding affinity for B7-H3 molecules expressed on the surface of cancer cells and can effectively induce the internalization reaction of B7-H3 on the cell surface. Based on the characteristics of target targeting and internalization reaction, the antibody of the present invention can serve as a targeting module to deliver therapeutic agents linked to it to an individual's cancer cells.

[0058] Therefore, the second aspect of this disclosure relates to an immunoconjugate. Structurally, the immunoconjugate of the present invention comprises mAb 91H06 or a fragment thereof (e.g., scFv), a therapeutic agent, and a connector for linking the therapeutic agent to the mAb / antibody fragment.

[0059] Depending on the purpose of implementation, the therapeutic agent may be a cytotoxic drug, a radioactive nucleus, a cytokine, a hormone drug, an immunostimulant, or an immunotherapy drug.

[0060] Exemplary cytotoxic drugs suitable for preparing the immunoconjugates of the present invention include, but are not limited to, taxanes (e.g., paclitaxel, docetaxel, and cabazitaxel); alkylating agents (e.g., dichloromethyldiethylamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide, busulfan, N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fomomustine, streptozotocin, etc.). Dacarbazine, mitozoprolol, temozolomide, thiotepa, mitomycin, diazinon (AZQ), cisplatin, carboplatin, oxaliplatin, carbazine, and hexamethylmelamine; antimetabolites (e.g., methotrexate, pemetrexed, fluorouracil, capecitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelabine, cladribine, clofarabine, pentostatin, thioguanine, and thiopurines); antimicrotubule agents (e.g., tubulin inhibitors, auristatin or its derivatives, maytansine, vincristine). Alkaloids, vinblastine, vinorelbine, vindesine, vinflunine, etoposide, and teniposide); topoisomerase inhibitors (e.g., irinotecan, toponotecan, etoposide, imitoxantrone, teniposide, neomycin, mercaptoside, and aclarubicin); cytotoxic antibiotics (e.g., chachiin, pyraclostrobin, pyraclostrobin, imitoxantrone, aclarubicin, donomycin, epirubicin, edamame, pirarubicin, aclarubicin, imitoxantrone, bleomycin, mitomycin C, and actinomycin); plant toxins ( Examples include ricin, absinthecin, saponins, senna root toxin, modidesin, leucopicrin, and quercetin; exotoxins (e.g., Pseudomonas aeruginosa exotoxin); botulinum toxins (e.g., botulinum toxin A, botulinum toxin B, botulinum toxin C, botulinum toxin D, botulinum toxin E, and botulinum toxin F); and endotoxins (e.g., diphtheria toxin). According to one exemplary embodiment of this disclosure, the cytotoxic agent is auristatin or a derivative thereof, such as MMAE or MMAF.

[0061] Radioactive nuclides, also known as radioactive isotopes or radioactive nuclides, can be yttrium-90 (Yttrium-90). 90 Y), Indium-111 111 In), Iodine-131 131 I), Samarium-153 ( 153 Sm), Lutetium-177 ( 177 Lu), Glue-211 ( 211 At), Bismuth-212 ( 212 Bi), Actinium-225 ( 225 Ac), Radium-223 ( 223 R) or Thorium-227 ( 227Th). Preferably, the radionuclides are complexed with a chelate, such as ethylenediaminetetramethylenephosphonic acid (EDTMP), 1,4,7,10-tetraazacyclododecanetetramethylenephosphonic acid (DOTMP), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane-1,4-diacetic acid (NODA), or diethylenetriaminepentaacetic acid (DTPA).

[0062] Cytokines can be any cytokines known to be used to stimulate an immune response, for example, interleukin (IL)-2, IL-10, IL-12, IL-15, IL-21, tumor necrosis factor (TNF)-α, interferon (IFN)-α, IFN-γ, or granulocyte-macrophage community-stimulating factor (GM-CSF).

[0063] Examples of hormonal drugs suitable for preparing the immunoconjugates of the present invention include, but are not limited to, aromatase inhibitors (e.g., anastrozole, exemestane, and letrozole); selective estrogen receptor modulators (SERMs; e.g., tamoxifen and raloxifene); estrogen receptor antagonists (e.g., fulvestrant and toremifene); luteinizing hormone-releasing hormone (LHRH) agonists (e.g., goserelin, leuprorelin, and triptorelin); antiandrogens (e.g., aperutamide, enzalutamide, dalostutamide, bicalutamide, flutamamide, and nilumid); CYP17 inhibitors (e.g., abiraterone and clotrimazole); progestins (e.g., methylprogesterone acetate and megastryce acetate); and adrenaline depressants (e.g., mitotane).

[0064] Exemplary immunostimulants include, but are not limited to, acetylmuramic acid, chlorpheniramine, burdock, deoxycholic acid (DCA), echinacea, PEGylated recombinant adenosine deaminase, flavonoids (e.g., rutin, isoliquiritigenin and glycyrrhizin), glatiramer acetate, olpresphingolipid, adenosine deaminase, plexafor, prolactin, triacetyl, terpenes (e.g., triterpenes), TLR7 / TLR8 agonists, TLR9 agonists, Sting agonists and NLRP agonists.

[0065] As for immunotherapy drugs, they can be immune checkpoint inhibitors (e.g., inhibitors of cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed cell death-1 (PD-1), or PD-L1) or immunomodulators (e.g., thalidomide and lenalidomide).

[0066] The connectors used to link the mAb / antibody fragments and therapeutic agents of the present invention can be cleavable or non-cleavable connectors. Exemplary cleavable connectors include, but are not limited to, protease-sensitive connectors (e.g., valine-citrulline (VC) dipeptide, valine-alanine (VA) dipeptide, valine-lysine (VL) dipeptide, valine-arginine (VR) dipeptide, and glutamate-valine-citrulline (EVC) tripeptide), pH-sensitive connectors (e.g., hydrazone connectors, ester connectors, and amide connectors), and glutamylsulfonate-sensitive connectors (e.g., N-succinimino-4-(2-pyridinedithio)butyrate (SPDB) and N-succinimino-4-(2-pyridinedithio)valerate (SPP)). Exemplary non-cleavage linkers include, but are not limited to, maleimide hexanoyl (MC), maleimide methylcyclohexane-1-carboxylic acid ester (MCC), and 4-[N-maleimide methyl]cyclohexane-1-carboxylic acid succinimide ester (SMCC). Alternatively, the linker can be any linker known for linking a bifunctional motif (e.g., the antibody and loaded drug of an ADC) in an immunoconjugate. Those skilled in the art can select appropriate linkers for preparing the immunoconjugates of the present invention according to the purpose of implementation. According to an exemplary embodiment, the linker for linking the mAb / antibody fragment and the therapeutic agent of the present invention comprises a polyethylene glycol (PEG) chain and a protease-sensitive linker linked to the PEG chain; preferably, the PEG chain has 1 to 10 EG repeating units. According to an embodiment of the present disclosure, the linker comprises a PEG chain and a VC dipeptide linked to the PEG chain; in this embodiment, the immunoconjugate is prepared in the form of “mAb / antibody fragment-PEG chain-VC dipeptide-therapeutic agent”. In another embodiment of this disclosure, the connector comprises a PEG chain and an EVC tripeptide linked to the PEG chain; in this embodiment, the immunoconjugate is prepared in the form of “mAb / antibody fragment-PEG chain-EVC tripeptide-therapeutic agent”.

[0067] As described above, the mAb of the present invention can be prepared in the form of IgG, IgA, IgM, IgD, or IgE, depending on the intended use. According to certain preferred embodiments of this disclosure, the mAb of the present invention is prepared in the form of IgG. In these embodiments, the therapeutic agent is attached to the Fc region of the mAb.

[0068] According to certain embodiments of this disclosure, the immunoconjugates of the present invention are prepared using trimannosyl ADC technology, which is a platform for site-specifically linking a loaded drug to a targeting antibody (for example, see WO 2018 / 126092 A1). In these embodiments, a recombinant antibody or antibody fragment is first modified to be coupled to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more) azide groups; the azide-modified antibody can be linked by a copper-free click reaction to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more) connectors having dibenzocyclooctynyl (DBCO) groups—the loaded drug (each comprising a connector, a DBCO group attached to one end of the connector, and a therapeutic agent attached to the other end of the connector)—wherein the copper-free click reaction occurs between the azide group and the DBCO. It is conceivable that other groups suitable for click chemistry reactions can be used to replace the azide group or DBCO, for example, alkynyl, tetraazine, or transcyclooctynyl (TCO). Depending on the purpose, other methods for synthesizing ADCs can be used to prepare the immunoconjugates of the present invention, for example, cysteine ​​conjugation, lysine conjugation, and disulfide bridging. Methods for synthesizing ADCs are well known to those skilled in the art; for the sake of simplicity, they will not be described in detail here.

[0069] According to certain exemplary embodiments of this disclosure, the recombinant antibody / antibody fragment has four azide groups, thus allowing it to be linked to four therapeutic agents (e.g., four MMAE or MMAF molecules). In these embodiments, the resulting immunoconjugate has a drug-to-antibody ratio (DAR) of approximately 4.

[0070] (iii) Pharmaceutical compositions comprising the immunoconjugates of the present invention

[0071] A third aspect of this disclosure relates to a pharmaceutical composition for treating cancer. According to certain embodiments, the pharmaceutical composition comprises the immunoconjugate of the present invention, and, optionally, a pharmaceutically acceptable carrier.

[0072] Generally, the immunoconjugate of the present invention accounts for approximately 0.1% to 99% of the total weight of the pharmaceutical composition. In some embodiments, the immunoconjugate of the present invention accounts for at least 1% of the total weight of the pharmaceutical composition. In some embodiments, the immunoconjugate of the present invention accounts for at least 5% of the total weight of the pharmaceutical composition. In some embodiments, the immunoconjugate of the present invention accounts for at least 10% of the total weight of the pharmaceutical composition. In other embodiments, the immunoconjugate of the present invention accounts for at least 25% of the total weight of the pharmaceutical composition.

[0073] Pharmaceutically acceptable carriers can be any pharmaceutically acceptable material or medium, such as liquid or solid fillers, diluents, excipients, solvents, or coating materials, for carrying the active ingredient (e.g., the mAb or immunoconjugate of the present invention) or for delivering the active ingredient (e.g., the mAb or immunoconjugate of the present invention) from one organ or site of the body to another organ or site of the body. The carrier must be "acceptable," meaning compatible with other components in the formulation; while minimizing degradation of the active ingredient and reducing individual side effects to a minimum. Depending on the intended purpose, the pharmaceutical compositions of this disclosure may further comprise one or more pharmaceutically acceptable additives, including surfactants, buffers, diluents, stabilizers, emulsifiers, dispersants, suspending agents, and preservatives.

[0074] The pharmaceutically acceptable carrier suitable for use in conjunction with the immunoconjugate of the present invention can be selected based on the route of administration of the pharmaceutical composition. The pharmaceutical composition of the present invention can be administered to an individual via subcutaneous, intravenous, intra-arterial, intratumoral, intraperitoneal, or intramuscular injection.

[0075] Pharmaceutical compositions intended for injection can be prepared as sterile water-soluble, sterile non-aqueous solutions, suspensions, or emulsions. Exemplary non-aqueous solutions include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Exemplary aqueous solutions include water, emulsions, and suspensions, such as physiological saline or buffer media. Common drug carriers suitable for non-gastrointestinal administration include sodium chloride solution, Ringer's glucose, glucose, lactated Ringer's injection, or fixed oils, while drug carriers suitable for intravenous administration typically include liquids, nutritional supplements, and electrolytes (e.g., electrolytes associated with Ringer's glucose).

[0076] (iv) Uses of the recombinant antibodies, immunoconjugates, and pharmaceutical compositions of the present invention

[0077] A fourth embodiment of this disclosure provides a method for treating cancer in an individual. The method comprises administering to the individual an effective amount of an immunoconjugate or pharmaceutical composition of this disclosure.

[0078] According to certain embodiments, the method of the present invention includes administering the immunoconjugate of the present disclosure to an individual. In these embodiments, the individual is a mouse, wherein the immunoconjugate is administered at a dose of about 0.01 to 100 mg per kilogram (e.g., about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 3... per kilogram). The immunoconjugate of the present invention (5, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 mg) is administered to an individual. Preferably, about 0.1 to 50 mg of the immunoconjugate of the present invention per kilogram is administered to an individual. More preferably, about 1 to 10 mg of the immunoconjugate of the present invention per kilogram is administered to an individual. According to one embodiment, about 5 mg of the immunoconjugate of the present invention per kilogram is administered to an individual. In one embodiment, administering about 1.5 mg of the immunoconjugate of the present invention per kilogram is sufficient to produce a therapeutic effect (i.e., inhibition of tumor growth) in the individual.

[0079] Those skilled in the art can determine the human equivalent dose (HED) of the immunoconjugate of the present invention based on the dosage determined by animal experiments of the embodiments of the present disclosure. Accordingly, the effective dose of the immunoconjugate of the present invention for human individuals can be from 1 microgram per kilogram of human body weight to 10 milligrams per kilogram of human body weight; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 45 0, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 76 0, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, or 990 micrograms, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 milligrams per kilogram. Dosage can be given as a single dose or divided into multiple equal doses. Skilled practitioners or clinicians may adjust the dosage or course of treatment according to the patient's condition or the severity of the disease.

[0080] Depending on the desired outcome, the immunoconjugate and / or pharmaceutical composition of the present invention may be administered to an individual once or twice a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, or for an extended period. Known techniques and methods can be used to monitor the progress of treatment. It is conceivable that the treatment duration may vary over time. According to certain exemplary embodiments of this disclosure, the immunoconjugate of the present invention is administered to an individual once a week. According to another exemplary embodiment, a single dose of the immunoconjugate of the present invention is sufficient to produce a therapeutic effect (i.e., inhibition of tumor growth) in an individual.

[0081] The immunoconjugates or pharmaceutical compositions of the present invention can be administered to an individual via appropriate routes, wherein the routes are selected from the group consisting of nasal, topical, mucosal, and non-gastrointestinal administration (e.g., subcutaneous, intratumoral, intramuscular, intravenous, intra-arterial, or intraperitoneal injection). According to certain illustrative embodiments of this disclosure, the immunoconjugates or pharmaceutical compositions of the present invention are administered intravenously to an individual.

[0082] It is conceivable that the method of the present invention can be administered to an individual alone, or in combination with other additional therapies beneficial to cancer prevention or treatment (e.g., surgery, chemotherapy, and / or radiation therapy). Depending on the desired / treatment purpose, the method of the present invention can be administered to the individual before, simultaneously with, or after the administration of additional treatment.

[0083] Cancers that can be treated with the methods of the present invention can be any B7-H3 positive cancer (i.e., cancer that expresses or overexpresses B7-H3 compared to normal cells), such as breast cancer, gastric cancer, colorectal cancer, gallbladder cancer, prostate cancer, cervical cancer, ovarian cancer, chronic or acute lymphoblastic leukemia, bladder cancer, kidney cancer, liver cancer, head and neck squamous cell carcinoma, glioblastoma, esophageal cancer, pancreatic cancer, oral cancer, lung cancer, melanoma, or lymphoma.

[0084] Basically, the individual who can receive treatment using the method of the present invention is a mammal, for example, a human, mouse, rat, guinea pig, hamster, monkey, pig, dog, cat, horse, sheep, goat, cow, and rabbit. Preferably, the individual is a human.

[0085] Based on the binding affinity and / or specificity to the B7-H3 protein, the mAb or antibody fragment of the present invention can serve as a detection antibody to detect cancer / cancer cells expressing or overexpressing B7-H3. Therefore, the fifth aspect of this disclosure relates to a method for diagnosing cancer in an individual using the mAb of the present invention. In this aspect, the mAb of the present invention is preferably linked to a reporter molecule, for example, a fluorophore or an enzyme. The method of the present invention comprises: (a) isolating a biological sample from an individual; (b) mixing the biological sample with the mAb of the present invention; (c) detecting the expression of the reporter molecule; and (d) diagnosing whether the individual has cancer based on the result of step (c), wherein the detection of the reporter molecule in step (c) indicates that the individual has cancer.

[0086] Alternatively, the mAb of the present invention can be used as a capture antibody to capture cancer / cancer cells that express or overexpress B7-H3, and then be detected using a secondary antibody (i.e., a detection antibody) conjugated to a reporter molecule, wherein the secondary antibody can identify the mAb of the present invention (e.g., the Fc region of the mAb) or cancer cells.

[0087] Depending on the purpose of implementation, the mAb of this invention can be used for different detection tests, such as immunohistochemistry (IHC), immunofluorescence, ELISA, flow cytometry, and Western ink dot method.

[0088] The following examples illustrate certain aspects of the invention to facilitate implementation by those skilled in the art, and should not be construed as limiting the scope of the invention. It is believed that those skilled in the art, upon reading the description herein, can fully utilize and practice the invention without undue interpretation. All publicly available documents cited herein are considered an integral part of this specification.

[0089] Example

[0090] Materials and Methods

[0091] Preparation of B7-H3 protein

[0092] The nucleotide sequence encoding the human (amino acid residues 29-466) or mouse or rat (amino acid residues 29-244) B7-H3 extracellular domain was fused with the mouse Fc domain and then constructed into the pcDNA3.4 vector. (FreeStyle) TM Recombinant human, rat, and mouse B7-H3-mFc proteins were expressed and purified in 293 cells. (Source: R&Dsystems) ® Purchase recombinant stone crab macaque B7-H3 protein.

[0093] Screening for B7-H3 mAb from a synthetic human antibody library

[0094] (A) Preparation of biopanaged scFv phages

[0095] The phage-expressed scFv antibody library (containing approximately 10) 11(Antibody variant) and TG1 cells were cultured in 2x yeast trypsin (YT) medium containing 100 μg ampicillin and 2% glucose per mL, and then incubated at 37°C with shaking until the OD at 600 nm reached 0.5. After adding helper phages, the cells were incubated in a 37°C water bath for another 30 minutes. After centrifuging the infected cells at 4,000 rpm for 15 minutes, the cell pellet was gently resuspended in 2x YTAK medium (YT medium containing 100 μg ampicillin and 25 μg benzoycin per mL), and then incubated at 37°C with shaking until the next day. After centrifuging the product at 10,000 rpm for 20 minutes, 1 / 5 volume of PEG / NaCl (20% polyethylene glycol 8000, 2.5 M NaCl) was added to the supernatant, and the cells were incubated at 4°C for at least one hour. After centrifugation at 10,000 rpm for 20 minutes, the precipitate was washed with sterile water to remove bacterial debris. 1 / 5 volume of PEG / NaCl was added to the supernatant, and the mixture was incubated at 4°C for at least one hour. The precipitate was then washed with phosphate-buffered saline (PBS) to remove bacterial debris.

[0096] (B) Screening bacteriophages

[0097] After coating the ELISA plate with human B7-H3-mFc protein, the reaction was carried out at 4°C until the next day. 300 μL of 5% skim milk (diluted in PBS) was added, and the plate was incubated at 37°C for 90 minutes. After washing three times with PBS, 100 μL of phage diluted in 5% MPBS (PBS containing 2% skim milk) was added (approximately 10 μL of phage). 11 Up to 10 12 Add one phage to an ELISA plate and incubate at 37°C for 90 minutes. Use PBST (containing Tween) ® After washing the ELISA dish three times with 20 μL of PBS, wash once with PBS. Add 100 μL of 100 mM trimethylamine (TEA) to the ELISA dish to extract the phage, then add 1 M Tris (pH 7.4) to neutralize the extraction product. Mix the obtained phage with TG1 cells and incubate at 37°C for 30 minutes. After centrifuging at 4,000 rpm for 15 minutes, suspend the TG1 cells in 2x TY medium and then inoculate them into 2x YTAG (YT medium containing ampicillin and glucose) culture dishes to expand the infected cells.

[0098] (C) Preparation of the next round of phages

[0099] Add 5-6 mL of 2x YT culture medium containing 15% glycerol to a bacterial dish, scraping the bacteria off the dish. Incubate 50-100 μL of the scraped bacteria in 100 mL of 2x YTAG at 37°C with shaking until the OD at 600 nm reaches 0.5. Add helper phages and incubate at 37°C for 30 minutes. Centrifuge the infected cells at 4,000 rpm for 15 minutes, gently resuspend the cell pellet in 2x YTAK culture medium, and incubate at 37°C with shaking until the next day. Centrifuge the product at 10,000 rpm for 20 minutes, add 1 / 5 volume (8 mL) of PEG / NaCl to the supernatant, and incubate at 4°C for at least one hour. Centrifuge at 10,000 rpm for 20 minutes, and wash the pellet with PBS to remove bacterial debris. A phage antibody library was injected into porous microdiscs coated with human B7-H3-mFc protein. ELISA was used to screen phage strains expressing anti-B7-H3 scFv.

[0100] (D) Using ELISA to screen for B7-H3 positive phages

[0101] The phage strain confirmed in step (C) was cultured in 200 mL of 2x YTAG medium and reacted at 37°C for the next day. 50 μL of the culture product was transferred to a 96-well dish containing 200 μL of 2x YTAG per well. The dish was incubated at 37°C with shaking for 2 hours. Add 10... 9 PFU helper phages were added and then cultured at 37°C for 90 minutes. After centrifugation at 4,000 rpm for 30 minutes, the precipitate was resuspended in 300 μL of 2x YTAG medium and reacted at 30°C until the next day. Cells were centrifuged at 4,000 rpm for 30 minutes, and 100 μL of supernatant was transferred to an ELISA plate (coated with human B7-H3-mFc protein and pretreated with 300 μL of 2% MPBS). After 90 minutes, the ELISA plate was washed three times with PBST. Appropriately diluted anti-M13 antibody bound to HRP (diluted in 2% MPBS) was added to the ELISA plate, followed by the addition of TMB acceptor solution (3,3',5,5'-tetramethylbenzidine) for colorimetric reaction. The reaction was stopped by adding 50 μL of 1 M sulfuric acid. OD was measured at 450 nm and 650 nm. 450 Measured value minus OD 650 The measured value.

[0102] After three to four rounds of biopanning, 60 scFv phage strains with significant B7-H3 affinity were obtained from the phage expression antibody library. The VH and VL chains of these strains were then inserted into expression vectors containing CH and CL chains, respectively, to construct the coding sequences. The constructed vectors were then transfected into FreeStyle... TM 293 cells. Using protein A Sepharose... ® Fast-flow antibody purification. After purification, OD was measured. 280 The antibodies were quantified using nanoparticles and analyzed by reducing and non-reducing polyacrylamide gel electrophoresis (PAGE).

[0103] ELISA combination assay

[0104] The binding affinity of the full-length antibody to human, monkey, mouse, or rat B7-H3 was assessed using ELISA. In short, B7-H3 protein (i.e., human B7-H3-mFc protein, rhesus macaque B7-H3 protein, mouse B7-H3Fc chimeric protein, or rat B7-H3 Fc chimeric protein) dissolved in coating buffer was coated onto 96-well plates and incubated at 4°C for the next day. After washing three times with PBS, the 96-well plates were inverted and gently tapped on clean absorbent paper to remove excess liquid. Blocking buffer (PBS containing 1% bovine serum albumin (BSA)) was added, followed by incubation at 37°C with shaking for 1 hour. After washing three times with PBS, serially diluted anti-B7-H3 antibody (dissolved in PBS containing 1% BSA) was added. The ELISA plates were incubated at 37°C with shaking for 1 hour. After washing three times with PBS, secondary antibody (AffiniPure antibody bound to peroxidase) was added. TM The F(ab')2 fragment of goat anti-human IgG (H+L) was incubated at 37°C with shaking for 1 hour. After washing three times with PBS, TMB receptor was added, and the reaction was carried out at room temperature (avoiding light) for 5 minutes. 1 N HCl was added to stop the reaction, and then the absorbance at 450 / 650 nm was measured. The binding affinity (EC50) of the antibody was determined using software.

[0105] Surface plasma resonance (SPR)

[0106] Leveraging Biacore TMThe binding kinetics of the antibody of this invention with human B7-H3 were determined through experiments. The carboxymethylated dextran biosensor chip (CM5) was activated according to the user manual. Anti-human IgG (Fc) was diluted with immobilization buffer and injected into the chip at a flow rate of 10 μL / min to generate approximately 10,000 response units (RU) of conjugate protein, followed by injection of 1 M ethanolamine to block unreacted groups. The human anti-B7-H3 antibody was captured onto the anti-human IgG chip. To measure binding kinetics, serially diluted human B7-H3-mFc protein (from 40 nM to 0.3125 nM) was injected into the manufacturer's HBS-EP. + Biacore TM Run the buffer at a flow rate of 30 μL / min. Compensate for the results using a reference subtraction method to determine the binding affinity of the antibody of this invention to the human B7-H3-mFc protein.

[0107] Flow cytometer

[0108] The binding affinity of the antibody of this invention to B7-H3-expressing cells was determined by indirect immunofluorescence staining. In simple terms, 100,000 head and neck squamous cell carcinoma (HNSCC), prostate carcinoma, non-small cell carcinoma (NSCLC), or hepatocellular carcinoma (HCC) cells were suspended in FACS buffer (PBS containing 0.5-1% BSA or 5-10% FBS, and 0.1% NaN3), and mixed with the antibody of this invention (1 μg per well) in a 96-well U-shaped microdisc, then incubated at 4°C for 60 minutes. After washing with PBS, a secondary antibody (dissolved in FACS buffer and Alexa Fluor) was added. ® Anti-human IgG (647-linked) was synthesized and reacted at 4°C for 60 minutes. Cell fluorescence intensity was measured using flow cytometry, and the results were then analyzed using software.

[0109] The internalization reaction of the antibodies in this invention was determined by indirect immunofluorescence staining. Simply put, cancer cells (6x10⁻¹²) were subjected to this process. 5 The antibody (containing HNSCC cells and prostate cancer cells) was suspended in FACS buffer and mixed with the antibody of this invention (6 μg per well) in a 96-well U-shaped microdisc, and then reacted at 4°C for 60 minutes. After washing with PBS to remove excess antibody, 1.5 x 10 μg of the antibody was added to the microdisc. 5 Cells were divided into four aliquots and incubated at 37°C for 30, 60, 120, or 180 minutes. The cells were then placed on ice and stained with a secondary antibody. After washing with PBS, the cells were stained with a secondary antibody (dissolved in FACS buffer and Alexa Fluor). ®Anti-human IgG (647-linked) was synthesized and reacted at 4°C for 60 minutes. Cell fluorescence intensity was measured using flow cytometry, and the results were then analyzed using software.

[0110] Cell culture

[0111] Detroit 562 cells (human laryngeal squamous cell carcinoma cell line) were cultured in minimum essential medium (MEM) containing 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 0.1% whey protein hydrolysate and 10% fetal bovine serum (FBS).

[0112] FaDu cells (human HNSCC cell line), DU145 cells (human cell line derived from brain metastases of prostate cancer), and HepG2 cells (human HCC cell line) were cultured in MEM containing 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, and 10% FBS. B7-H3 knockout FaDu (FaDu / B7-H3 KO) cells were prepared using CRISPR / Casp9 technology.

[0113] CAL 27 cells (human oral adenosquamous cell carcinoma cell line), Huh7 cells (human HCC cell line), and A549 cells (human NSCLC cell line) were cultured in DMEM containing 10% FBS.

[0114] OECM-1 cells (human oral squamous cell carcinoma cell line), LNCaP cells (human prostate cancer cell line derived from metastatic lymph node lesions), H1299 cells (human NSCLC cell line), and NCI-H520 cells (human lung squamous cell carcinoma cell line) were cultured in RPMI containing 10% FBS.

[0115] PC-3 cells (human prostate cancer cell line) were cultured in Ham's F12K medium containing 2 mM L-glutamine, 1.5 g sodium bicarbonate per liter, and 7% FBS.

[0116] HA59T cells (human HCC cell line) were cultured in DMEM containing 4 mM L-glutamine, 4.5 g glucose per liter, 0.1 mM non-essential amino acids and 10% FBS.

[0117] HuT78 cells (human skin T lymphocyte cell line) were cultured in IMDM (Iscove's Modified Dulbecco's Medium) containing 20% ​​FBS.

[0118] Ba / F3 cells (mouse interleukin-3 related pre-B cell line) were cultured in RPMI containing 10% FBS and 10 ng / mL of mouse IL-3.

[0119] EMT6 cells (mouse breast cancer cell line) were cultured in Waymouth's medium (MB 752 / 1) containing 2 mM L-glutamine and 15% FBS.

[0120] ID8 cells (mouse ovarian epithelial cell line) were cultured in DMEM containing 4% FBS and 1X ITS (a mixture of recombinant human insulin, human transferrin and sodium selenite).

[0121] Antibody-drug conjugates (ADCs)

[0122] The antibody-drug conjugate (ADC) of this invention was prepared using a trimannose antibody-drug conjugation platform. Specifically, 0.04 mL of the linker-loaded drug (10 mM, dissolved in dimethyl thiosulfate (DMSO) or dimethyl acetamide (DMA)) and 0.12 mL of DMSO or DMA were slowly added to MES buffer (pH 6.5) containing mAb-4 azide (Az) (0.4 mL, 5 mg per mL). The reaction mixture was stirred at 37°C for 18 hours. The resulting antibody-drug conjugate was centrifuged in MES buffer (pH 6.5) through a centrifuge filter with a nominal molecular weight limit (NMWL) of 30 kDa to remove salts and concentrate, yielding 91H06-MMAE and 91H06-MMAF, respectively. In the experiments of this invention, the connector used to link mAb 91H06 and the loaded drug (MMAE or MMAF) includes a PEG connector and a protease-sensitive connector (valine-citrulline dipeptide or glutamate-valine-citrulline tripeptide) linked to the PEG connector. The drug-to-antibody ratio (DAR) of the ADC was measured using liquid chromatography-mass spectrometry. The DAR of 91H06-MMAE and 91H06-MMAF was approximately between 3.5 and 3.9.

[0123] Cell toxicity test

[0124] Cells were seeded in triplicate in 96-well plates and left to stand for the next day. Serial dilutions of the ADC of this invention (final concentration: 500 nM to 0.025 nM) were added to each well. After incubation at 37°C for 120 hours, CellTiter-Glo was prepared in a dark environment according to the user manual. ® Add the reagents to each well. Place the culture dish on a shaker and shake for 2 minutes to mix the solution and induce cell lysis. After reacting at room temperature for 10 minutes, record the luminescence value and calculate the results using software.

[0125] animal testing

[0126] Detroit 562 and FaDu xenograft models were established in NOD SCID mice to evaluate the in vivo antitumor efficacy of 91H06-MMAE. NOD SCID mice aged 6-8 weeks were used in this study. Five mice were housed per cage. All animals were housed in a 12-hour light / 12-hour dark environment at 19-25°C. Animals had free access to rodent pellets and drinking water.

[0127] Detroit 562 tumor cells (3×10⁻⁶) 6 (cells) or FaDu tumor cells (1×10) 6 (cells) and Matrigel ® The mixture was prepared at a 1:1, v / v ratio and administered at a dose volume of 0.1 mL. The mixture was then subcutaneously (SC) injected into the right anterior side of the mouse. The tumor volume was measured to be approximately 160 mm³. 3 In the Detroit 562 tumor model or approximately 100 m³ (in the FaDu tumor model), mice with tumors were randomly assigned to three groups of five mice each. 91H06-MMAE or a carrier solution (25 mM MES buffer, pH 6.5) was administered intravenously to the tumor-bearing mice. The day of administration was designated as day 0. Animals receiving the carrier solution served as the disease (carrier) control group in this study to calculate tumor growth inhibition (TGI) rates. In the Detroit 562 tumor model, experimental group animals were administered 1.5 mg / kg (1.5 mpk) or 5 mg / kg (5 mpk) of 91H06-MMAE weekly for three weeks; in the FaDu tumor model, experimental group animals were administered a single dose of 1.5 mg / kg (1.5 mpk) or 5 mg / kg (5 mpk) of 91H06-MMAE.

[0128] In the experiment evaluating the antitumor efficacy of 91H06-MMAE, Detroit 562 tumor cells (3 × 10⁻⁶) were used. 6 (cells) and Matrigel ® The mixture was prepared in a 1:1, v / v ratio and administered at a dose volume of 0.1 mL. The mixture was then subcutaneously injected into the right anterior side of the mice. When the average tumor volume reached approximately 1,500 cubic millimeters, the mice with tumors were intravenously administered 5 mg (5 mpk) of 91H06-MMAE per kilogram of body weight once a week for three weeks.

[0129] Tumor volume, weight, mortality rate, and signs of significant toxicity were monitored and recorded three times a week. Measurements were taken using a digital ruler and the formula: TV = (W 2 The tumor volume (in cubic millimeters) is calculated as W = (W × L) / 2, where W is the width of the tumor (in millimeters) and L is the diameter of the tumor (in millimeters). The TGI percentage is calculated using the formula: GI = [1 - (Tx - T0 / Cx - C0)] × 100%, where Tx and Cx represent the average tumor volume on day X in the treatment group and the control group, respectively.

[0130] Example 1: Confirmation of mAb 91H06

[0131] 1.1 Protein binding affinity

[0132] This embodiment will evaluate the binding activity of mAb 91H06 or ifenastatin (an antibody targeting B7-H3, serving as a positive control) to B7-H3 in humans, monkeys, mice, or rats. As described in the “Materials and Methods” section of this disclosure, mAb 91H06 was added to culture dishes coated with chimeric proteins of human B7-H3-mFc, rhesus macaque B7-H3, mouse B7-H3 mFc, or rat B7-H3 mFc, and the binding affinity between the two proteins was then determined using ELISA.

[0133] The results indicated that mAb 91H06 of the present invention has EC50% activity against human, monkey, mouse, or rat B7-H3. 50 <10 -10 The binding affinity of phenastatin is shown in Table 1. In contrast, phenastatin only recognizes B7-H3 in humans and monkeys (Table 1).

[0134] Table 1. Binding affinity of ifenastatin or mAb 91H06 to specific B7-H3 proteins.

[0135] Further Biacore TM Experimental analysis of the binding kinetics of mAb 91H06. (Biacore) TM The results of the experiment confirmed the cross-reactivity of mAb 91H06 with B7-H3 in humans, monkeys, mice, or rats (Table 2).

[0136] Table 2. Binding affinity of mAb 91H06 to specific B7-H3 proteins.

[0137] It is known that human B7-H3 and B7-H4 (31%) and other members of the B7 family (24% to 31%) share sequence homology. Therefore, this embodiment also evaluates whether the mAb of the present invention cross-reacts with other B7 proteins. The results indicate that the mAb 91H06 of the present invention selectively binds to B7-H3, but not to other members of the B7 family (…). Figure 1 ).

[0138] 1.2 Cell binding affinity

[0139] This embodiment examines the binding activity of the mAb of the present invention to different human cancer cells. The results indicate that mAb 91H06 can recognize and bind to various HNSCC cell lines (including Detroit 562, CAL 27, FaDu, and OECM-1 cells) and various prostate cancer cell lines (including DU 145, LNCaP, and PC-3 cells) (Table 3). In addition to HNSCC and HCC cell lines, mAb 91H06 also exhibits binding affinity to different HCC cell lines (including HepG2, HA59T, and Huh7 cells) and different NSCLC cell lines (including A549, H1299, and NCI-H520 cells) (Table 3). Notably, the binding activity of mAb 91H06 to the tested cancer cells is comparable to that of the control antibody (ixenastatin), confirming that mAb 91H06 can effectively target cancer cells expressing B7-H3.

[0140] Table 3 Cell-binding activity of mAb 91H06 against specific cancer cells

[0141] Furthermore, this embodiment also evaluated the binding activity of mAb 91H06 to cancer cell lines with low B7-H3 expression (i.e., HuT78 cells) and B7-H3 knockout cancer cell lines (i.e., B7-H3 knockout FaDu cells). The results showed that mAb 91H06 did not bind to B7-H3 negative cancer cells (Table 3), demonstrating the binding specificity of mAb 91H06 to cancer cells with high B7-H3 expression.

[0142] To confirm the cross-species reactivity of the mAb of this invention, this embodiment further analyzed the binding activity of mAb 91H06 to rodent cells. Based on the flow cytometry analysis results, compared to the benchmark antibody ifenastatin, it showed no binding affinity to rodent cells. Figure 2In the insets (A) and (C) of Figure 2, mAb 91H06 can recognize and bind to rodent cell lines expressing B7-H3 (including EMT6 and ID8 cells; insets (B) and (D) of Figure 2), but not to B7-H3-negative Ba / F3 cells (results not shown). These data confirm that mAb 91H06 of the present invention has cross-species responsiveness to B7-H3-positive rodent cells.

[0143] 1.3 Competitive Combination Experiment

[0144] It is known that antibodies binding to different epitopes of the same target may produce different biological activities (e.g., binding-mediated internalization). Therefore, a competitive binding assay was used to determine the relative epitope positions of the present invention's mAb 91H06 and ifenastatin to the B7-H3 protein. The results in Table 4 indicate that the presence of ifenastatin (4 μg / mL) alters the binding affinity of ifenastatin-biotin to the B7-H3 protein; in contrast, the present invention's mAb 91H06 does not affect the binding of ifenastatin-biotin to the B7-H3 protein. The results indicate that mAb 91H06 does not compete with ifenastatin for binding to the B7-H3 protein, and the B7-H3 epitope recognized by mAb 91H06 is different from that recognized by ifenastatin.

[0145] Table 4. Binding affinity of ifenastatumab-biotin to B7-H3 protein in the presence or absence of ifenastatumab or mAb 91H06.

[0146] *Ifenatatumab-Biotin: Ifenatatumab bound to the biotin molecule.

[0147] 1.4 Cellular internalization reaction

[0148] Antibody-adjuvant drugs (ADCs) are a promising cancer treatment approach, in which a drug-loaded substance (e.g., a cytotoxic drug) is conjugated to an antibody (e.g., anti-H7-H3 mAb). The antibody's binding properties target and deliver the loaded drug to cancer cells expressing the corresponding antigen (e.g., B7-H3), thereby minimizing off-target toxicity and / or side effects in the individual. A key aspect of ADCs is the antibody's ability to bind to and internalize the antigen on cancer cells. The internalized ADC reacts in lysosomes or late endosomes, releasing the active cytotoxic substance within the cancer cells. Since cellular internalization is a prerequisite for ADC function, relevant research should extensively evaluate antibody-induced internalization responses to ensure appropriate subcellular function. Overall, selecting antibodies with high affinity and specificity for the target antigen is crucial in ADC preparation. Furthermore, the target antigen must be primarily expressed on the surface of target cells (e.g., cancer cells), with minimal expression on healthy cells. Additionally, an ideal ADC should be rapidly internalized to drive the delivery and release of the cytotoxic loaded drug into cancer cells. Therefore, this embodiment will evaluate whether the mAb of the present invention can induce an internalization reaction in cells.

[0149] Administration of the present invention's mAb 91H06 induced the internalization of B7-H3 on the surface of Detroit 562 cells, with the internalization rate being significantly faster than that of the control antibody ifenastatin (Table 5). Similar results were observed in CAL27 and FaDu cells (Tables 6 and 7).

[0150] Table 5 Internalization reaction of mAb 91H06 in Detroit 562 cells

[0151] Table 6. Internalization reaction of mAb 91H06 in Cal27 cells

[0152] Table 7 Internalization reaction of mAb 91H06 in FaDu cells

[0153] Furthermore, administration of the present invention's mAb 91H06 can also induce the internalization reaction of B7-H3 on the surface of DU 145 and LNCaP cells (Tables 8 and 9), and the degree of internalization caused by it is significantly better than that caused by the control antibody ifenastatin.

[0154] Table 8. Internalization response of mAb 91H06 in DU145 cells

[0155] Table 9. Internalization response of mAb 91H06 in LNCaP cells.

[0156] These results suggest that the present invention’s mAb 91H06 can effectively induce the internalization of antigens in cancer cells, and therefore can serve as a targeting module for constructing an ADC for cancer treatment.

[0157] Example 2 confirms the ADC of the present invention

[0158] mAb was linked to MMAE according to the procedure described in "Materials and Methods" of this disclosure. Based on the analytical results, the obtained 91H06-MMAE had high purity (>95%) and consistent DAR (DAR approximately 4) (results not shown).

[0159] Data from SPR indicates that both mAb 91H06 and 91H06-MMAE exhibit high binding affinity for the B7-H3 protein, with mAb 91H06 showing a higher binding rate constant (kΩ). a ), dissociation rate constant (k d The kinetic dissociation constants (KD) for 91H06-MMAE are 6.86E+5, 1.56E-4, and 2.27E-10, respectively, while the kD of 91H06-MMAE is... a k d The KD values ​​were 6.43E+5, 1.55E-4, and 2.4E-10, respectively. Flow cytometry data further confirmed that the 91H06-MMAE of this invention maintained high cell binding activity and specificity against CAL 27 and Detroit 562 cells (results not shown).

[0160] Next, the cytotoxic activity of the ADC of the present invention (i.e., 91H06-MMAE) was analyzed. As shown in Table 10, 91H06-MMAE exhibited cytotoxic activity against cancer cells with high B7-H3 expression (including oral adenosquamous cell carcinoma CAL 27 cells, pharyngeal squamous cell carcinoma Detroit 562 cells, pharyngeal carcinoma FaDu cells, and prostate cancer LNCaP cells); while it did not show significant cytotoxicity against cancer cells with low B7-H3 expression (i.e., HuT78 cells) and B7-H3 knockout cancer cells (i.e., FaDu / B7-H3 KO cells). The results of the cytotoxicity assay were consistent with the expression level of B7-H3 on the surface of the cancer cell lines measured in Example 1.2.

[0161] Table 10 Cytotoxic activity of 91H06-MMAE against B7-H3 positive cancer cell lines (IC50) 50 )

[0162] In addition to MMAE, the mAb of this invention was also linked to different cytotoxic loading drugs (i.e., MMAFs with higher cytotoxicity than MMAE). As shown in Table 11, administration of the test ADC (i.e., 91H06-MMAF) significantly induced death in oral adenosquamous cell carcinoma CAL 27 cells, pharyngeal squamous cell carcinoma Detroit 562 cells, pharyngeal carcinoma FaDu cells, and prostate cancer LNCaP cells, without producing definite cytotoxicity in HuT78 and FaDu / B7-H3 KO cells. Furthermore, the cytotoxicity of 91H06-MMAF was higher than that of 91H06-MMAE. These results are consistent not only with the expression level of B7-H3 on the cell surface but also with the efficacy of different cytotoxic loading drugs. Overall, the high efficacy and selective cytotoxicity of the ADC containing 91H06 suggest that 91H06 of this invention could be an ideal monoclonal antibody for the development of ADC drugs to treat B7-H3 positive cancers.

[0163] Table 11 Cytotoxic activity (IC50) of 91H06-MMAF to B7-H3 positive cancer cell lines 50 )

[0164] The above results confirm that mAb 91H06 can be linked to drugs with different toxicities and effectively kill different types of cancer cells.

[0165] Example 3: In vivo efficacy of 91H06-MMAE in mouse xenograft model

[0166] This embodiment will analyze the antitumor efficacy of the ADC of the present invention in animal models. Figure 3 and Figure 4 The results indicated that, compared to the control group, administration of 1.5 mpk and 5 mpk of 91H06-MMAE significantly inhibited the Detroit 562 xenograft modality. Figure 3 ) and FaDu xenotransplantation model ( Figure 4 Tumor growth was inhibited. Tumor growth inhibition (TGI) rates induced by administration of 1.5 mpk and 5 mpk doses of the present invention 91H06-MMAE were greater than 80% and 100%, respectively, in the Detroit 562 xenograft model (Table 12), while they were greater than 100% in the FaDu xenograft model (Table 13). No weight loss or abnormalities were observed in animals treated with 91H06-MMAE (results not shown).

[0167] Table 12 Antitumor activity of specific therapies in Detroit 562 xenograft modalities

[0168] *1.5 mpk: Mice were given 1.5 mg of 91H06-MMAE per kilogram of body weight once a week for three weeks.

[0169] **5 mpk: Mice were given 5 mg of 91H06-MMAE per kilogram of body weight once a week for three weeks.

[0170] Table 13 Antitumor activity of specific therapies in FaDu xenograft model

[0171] *1.5 mpk: Mice were given a single dose of 1.5 mg of 91H06-MMAE per kilogram of body weight.

[0172] **5 mpk: Mice were given a single dose of 5 mg of 91H06-MMAE per kilogram.

[0173] Notably, in mice with large tumors averaging over 1500 mm³, administration of 5 mg / kg of 91H06-MMAE significantly inhibited tumor growth. Figure 5 No weight loss or abnormal conditions were observed in the animals during the experiment (results not shown).

[0174] In summary, this disclosure provides a novel antibody named mAb 91H06. According to embodiments of this disclosure, mAb 91H06 exhibits high binding affinity and specificity for B7-H3 and can be used to prepare immunoconjugates (i.e., ADCs) for treating B7-H3 positive cancers (even in the case of large tumors).

[0175] It is understood that the above embodiments are merely illustrative, and those skilled in the art can make different modifications. The foregoing specification, embodiments, and data provide a complete description of exemplary embodiments of the present invention. Although specific embodiments of the present invention are disclosed in the foregoing embodiments, they are not intended to limit the present invention. Those skilled in the art can make various modifications and alterations without departing from the principles and spirit of the present invention. Therefore, the scope of protection of the present invention shall be defined by the appended claims.

Claims

1. A recombinant antibody or fragment thereof targeting B7-H3, comprising a variable heavy chain (VH) domain and a light chain (VL) domain, wherein the VH domain comprises a first heavy chain complementarity-determining region (CDR) (CDR-H1), a second heavy chain CDR (CDR-H2), and a third heavy chain CDR (CDR-H3), and the VL domain comprises a first light chain CDR (CDR-L1), a second light chain CDR (CDR-L2), and a third light chain CDR (CDR-L3), wherein The CDR-H1, CDR-H2, and CDR-H3 respectively contain amino acid sequences with sequence numbers 1, 2, and 3, and the CDR-L1, CDR-L2, and CDR-L3 respectively contain amino acid sequences with sequence numbers 4, 5, and 6.

2. The B7-H3-targeting recombinant antibody or fragment thereof as described in claim 1, wherein the VH and VL domains respectively contain amino acid sequences having at least 85% sequence similarity to sequence numbers 7 and 8.

3. The B7-H3-targeting recombinant antibody or fragment thereof as described in claim 2, wherein the VH and VL domains respectively comprise amino acid sequences having 100% sequence similarity to sequence numbers 7 and 8.

4. An immunoconjugate comprising a B7-H3-targeting recombinant antibody or a fragment thereof as claimed in claim 1, a therapeutic agent, and a connector for linking the therapeutic agent and the B7-H3-targeting recombinant antibody or fragment thereof.

5. The immunoconjugate of claim 4, wherein the therapeutic agent is a cytotoxic drug, a radioactive nucleus, a cytokine, a hormonal drug, an immunotherapy drug, or a combination thereof.

6. The immunoconjugate of claim 5, wherein the cytotoxic drug is selected from the group consisting of microtubule inhibitors, auristatin, maytansin, cazithromycin, pyroxine, pyroxine, Pseudomonas aeruginosa exotoxin, diphtheria toxin, botulinum toxin A, botulinum toxin B, botulinum toxin C, botulinum toxin D, botulinum toxin E, botulinum toxin F, lysine, abrinogenin, saponins, or derivatives thereof.

7. The immunoconjugate of claim 6, wherein the cytotoxic drug is auristatin or a derivative thereof.

8. The immunoconjugate of claim 7, wherein the cytotoxic drug is monomethylauratestatin E (MMAE).

9. A method of treating cancer in an individual, comprising administering to the individual an effective amount of the immunoconjugate as described in claim 4.

10. The method of claim 9, wherein the cancer is breast cancer, gastric cancer, colorectal cancer, gallbladder cancer, prostate cancer, cervical cancer, ovarian cancer, chronic or acute lymphoblastic leukemia, bladder cancer, kidney cancer, liver cancer, head and neck squamous cell carcinoma, glioblastoma, esophageal cancer, pancreatic cancer, oral cancer, lung cancer, melanoma, or lymphoma.

11. The method of claim 10, wherein the individual is a human being.

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