WO2025157244A1 - Anti-b7h3 antibody and use thereof - Google Patents

Abstract

The present application discloses an antibody specifically binding to B7H3 or an antigen-binding fragment thereof, a nucleic acid encoding same, a recombinant vector, a host cell, a preparation method, a pharmaceutical composition, and a use in the treatment of tumor diseases, which can be used for the development of B7H3-targeted therapeutic drugs and the development of detection reagents.

Description

Anti-B7H3 antibodies and their applications

This disclosure claims priority to Chinese patent application number 202410110647.3, filed with the China Patent Office on January 25, 2024, entitled “Anti-B7H3 Antibodies and Their Applications,” the entire contents of which are incorporated herein by reference.

Technical Field

The present disclosure relates to the field of antibodies, and in particular, to anti-B7H3 antibodies.

Background Art

B7-H3 is a type I transmembrane protein, also known as CD276, and a member of the B7 ligand family. Early studies suggested that B7H3 is a T cell co-stimulatory factor, playing a regulatory role in T cell activation and IFNγ production. Other studies suggest that B7H3 plays an inhibitory role in adaptive immunity, possibly through different receptors, but the specific receptor for B7H3 is currently unclear. The human B7H3 protein is 534 amino acids long and exists in two isoforms: B7H3-4Ig and B7H3-2Ig. The former contains two pairs of immunoglobulin variable region (IgV) and immunoglobulin constant region (IgC)-like domains and is the predominant isoform in humans. The B7H3-2Ig isoform contains only one pair of IgV and IgC domains.

B7H3 is widely expressed in normal tissues, but expression levels are limited and relatively low, such as in the liver, colon, and prostate. However, it is highly expressed in tumor tissues and is closely associated with cancer progression, survival, and prognosis. Studies have found that B7H3 is highly expressed in a variety of tumor tissues, including small cell lung cancer, prostate cancer, renal cancer, colorectal cancer, and squamous cell lung carcinoma. Within tumor tissues, B7H3 is expressed both in tumor cells and in stromal cells, such as tumor vascular endothelial cells, pericytes, and fibroblasts. This suggests that B7H3 plays an important role in cancer progression, migration, invasion, anti-apoptosis, metabolism, and angiogenesis. Although the physiological functions of B7H3 remain largely undetermined, its high expression levels and high number of positive patients make it an attractive target for cancer therapy. Currently, therapeutic approaches targeting B7H3 are under development, including ADCC-enhancing monoclonal antibodies, CD3 bispecific antibodies, CARTs, and antibody-drug conjugates (ADCs). However, to date, no B7H3-targeting drugs have been approved for marketing.

Summary of the Invention

The present disclosure provides a new tumor treatment method by providing a new antibody or antigen-binding fragment thereof that specifically binds to B7H3.

In a first aspect of the present disclosure, an antibody or antigen-binding fragment thereof that specifically binds to B7H3 is provided, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

(1) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 are the HCDR1, HCDR2 and HCDR3 of the VH domain shown in SEQ ID NO. 12; and

(2) The light chain variable region comprises LCDR1, LCDR2 and LCDR3, and the LCDR1, LCDR2 and LCDR3 are the LCDR1, LCDR2 and LCDR3 of the VL domain shown in SEQ ID NO.13.

In some specific embodiments, the HCDR1, HCDR2, and HCDR3 are determined according to the Kabat, Chothia, or IMGT numbering systems, and have amino acid sequences as shown in SEQ ID NO.14-16, SEQ ID NO.20-22, and SEQ ID NO.26-28, or sequence combinations having 1, 2, 3 or more amino acid insertions, deletions, and/or substitutions compared to the amino acid sequences shown in SEQ ID NO.14-16, SEQ ID NO.20-22, and SEQ ID NO.26-28.

In some specific embodiments, the LCDR1, LCDR2 and LCDR3 are determined according to the Kabat, Chothia or IMGT numbering system, and have an amino acid sequence as shown in SEQ ID NO.17-19, SEQ ID NO.23-25, SEQ ID NO.29-31, or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared with the amino acid sequence shown in SEQ ID NO.17-19, SEQ ID NO.23-25, SEQ ID NO.29-31.

In some specific embodiments, the antibody or its antigen binding protein comprises the following LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2, HCDR3:

(1) According to the Kabat numbering system, LCDR1-3 and HCDR1-3 have the amino acid sequences shown in SEQ ID NOs. 14-19, or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the amino acid sequences shown in SEQ ID NOs. 14-19;

(2) According to the IMGT numbering system, LCDR1-3 and HCDR1-3 have the amino acid sequences set forth in SEQ ID NOs. 20-25, or a combination of sequences having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the amino acid sequences set forth in SEQ ID NOs. 20-25;

(3) According to the Chothia numbering system, LCDR1-3 and HCDR1-3 have the amino acid sequences shown in SEQ ID NO.26-31 or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the amino acid sequences shown in SEQ ID NO.26-31.

In some specific embodiments, the heavy chain variable region sequence comprises the sequence shown in SEQ ID NO.12, or a sequence with 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the sequence shown; the light chain variable region sequence comprises the sequence shown in SEQ ID NO.13, or a sequence with 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the sequence shown.

In some specific embodiments, the antibody or antigen-binding fragment thereof is fully human.

In some specific embodiments, the antibody or antigen-binding fragment thereof can specifically bind to human or monkey B7H3 protein.

In some specific embodiments, the antibody or antigen-binding fragment thereof may further comprise any constant region sequence of human or mouse antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably, the constant region sequence of human or mouse antibody IgG1, IgG2, IgG3 or IgG4, or the constant region sequence of human or mouse antibody IgG1, IgG2, IgG3 or IgG4 with mutation; further, the antibody or antigen-binding fragment thereof is further coupled with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from radioisotopes, chemotherapeutic drugs, cytotoxic agents or immunomodulators, and the tracer is selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers; more preferably, the cytotoxic agent is selected from alkaloids, methotrexate, anthracycline antibiotics (doxorubicin), pyrrolobenzodiazepines, (pyrrolobenzodiazepine, PBD), gemcitabine, cytarabine, tegafur, ifosfamide, dacarbazine and oxaliplatin; more preferably, the cytotoxic agent is a taxane.

In some specific embodiments, the antigen-binding fragment is selected from one or more of F(ab') ₂ , Fab', Fab, Fv, scFv, nanobody or affibody.

In a second aspect of the present disclosure, a multispecific antigen-binding molecule is provided, comprising the antibody or antigen-binding fragment thereof described in the first aspect; preferably, the multispecific antigen-binding molecule further comprises an antibody or antigen-binding fragment thereof that specifically binds to an antigen other than B7H3 or binds to a B7H3 epitope different from the B7H3 epitope bound by the antibody or antigen-binding fragment thereof described in the first aspect;

Preferably, the antigen other than B7H3 is selected from the following group: (1) tumor-specific antigen (TSA) or tumor-associated antigen (TAA); (2) immune checkpoint; (3) target for recruiting and/or activating immune cells;

Preferably, the multispecific antigen-binding molecule may be bispecific, trispecific or tetraspecific;

Preferably, the multispecific antigen-binding molecule may be bivalent, trivalent, tetravalent, pentavalent or hexavalent.

The third aspect of the present disclosure provides a chimeric antigen receptor (CAR), which comprises at least an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises the antibody or antigen-binding fragment thereof according to the first aspect.

The fourth aspect of the present disclosure provides an immune effector cell, which expresses the chimeric antigen receptor described in the third aspect, or contains a nucleic acid fragment encoding the chimeric antigen receptor; preferably, the immune effector cell is selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), DNT cells (double negative T cells), monocytes, macrophages, dendritic cells or mast cells, and the T cells are selected from cytotoxic T cells, regulatory T cells or helper T cells; preferably, the immune effector cell is an autologous immune effector cell or an allogeneic immune effector cell.

In a fifth aspect of the present disclosure, an isolated nucleic acid fragment is provided, which encodes the antibody or antigen-binding fragment thereof described in the first aspect, the multispecific antigen-binding molecule described in the second aspect, or the chimeric antigen receptor described in the third aspect.

In a sixth aspect of the present disclosure, a vector is provided, comprising the nucleic acid fragment.

The seventh aspect of the present disclosure provides a host cell comprising the vector; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungi (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line).

The eighth aspect of the present disclosure provides a method for preparing the antibody or antigen-binding fragment thereof or the multispecific antigen-binding molecule described in the first aspect, which comprises culturing the host cells, and isolating the antibody or antigen-binding fragment thereof expressed by the host cells, or isolating the multispecific antigen-binding molecule expressed by the host cells.

The ninth aspect of the present disclosure provides a method for preparing the immune effector cell, which comprises introducing a nucleic acid fragment encoding the CAR described in the third aspect into the immune effector cell, and optionally, further comprises initiating the immune effector cell to express the CAR.

In a tenth aspect of the present disclosure, a pharmaceutical composition is provided, comprising the antibody or antigen-binding fragment thereof described in the first aspect, or the multispecific antigen-binding molecule described in the second aspect, or the immune effector cell described in the fourth aspect, the nucleic acid fragment described in the fifth aspect, or the vector described in the sixth aspect, or the host cell described in the seventh aspect, or the antibody or antigen-binding fragment thereof or the multispecific antigen-binding molecule prepared by the method described in the eighth aspect, or the immune effector cell prepared by the method described in the ninth aspect; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, or adjuvant.

In an eleventh aspect of the present disclosure, there is provided use of the antibody or antigen-binding fragment thereof described in the first aspect, or the multispecific antigen-binding molecule described in the second aspect, or the immune effector cell described in the fourth aspect, or the nucleic acid fragment described in the fifth aspect, or the vector described in the sixth aspect, or the host cell described in the seventh aspect, or the antibody or antigen-binding fragment thereof or multispecific antigen-binding molecule prepared by the method described in the eighth aspect, or the immune effector cell prepared by the method described in the ninth aspect, or the pharmaceutical composition described in the tenth aspect, in preparing a medicament for preventing and/or treating tumors; the tumor is selected from a solid tumor, a hematological tumor, or a cancer infiltrating and expressing B7H3;

Preferably, the tumor is selected from melanoma, prostate cancer, renal cell carcinoma, lung cancer (eg non-small cell lung cancer (NSCLC)) and other solid tumors.

In a twelfth aspect of the present disclosure, a method for preventing and/or treating tumors is provided, comprising administering to a patient in need thereof an effective amount of the antibody or antigen-binding fragment thereof of the first aspect, or the multispecific antigen-binding molecule of the second aspect, or the immune effector cell of the fourth aspect, or the nucleic acid fragment of the fifth aspect, or the vector of the sixth aspect, or the host cell of the seventh aspect, or the antibody or antigen-binding fragment thereof or the multispecific antigen-binding molecule prepared by the method of the eighth aspect, or the immune effector cell prepared by the method of the ninth aspect, or the pharmaceutical composition of the tenth aspect; wherein the tumor is selected from a solid tumor, a hematological tumor, or a cancer infiltrating and expressing B7H3;

Preferably, the tumor is selected from melanoma, prostate cancer, renal cell carcinoma, lung cancer (eg non-small cell lung cancer (NSCLC)) and other solid tumors.

In a thirteenth aspect of the present disclosure, a kit is provided, comprising the antibody or antigen-binding fragment thereof described in the first aspect, or the multispecific antigen-binding molecule described in the second aspect, or the immune effector cell described in the fourth aspect, or the nucleic acid fragment described in the fifth aspect, or the vector described in the sixth aspect, or the host cell described in the seventh aspect, or the antibody or antigen-binding fragment thereof or the multispecific antigen-binding molecule prepared by the method described in the eighth aspect, or the immune effector cell prepared by the method described in the ninth aspect, or the pharmaceutical composition described in the tenth aspect.

A fourteenth aspect of the present disclosure provides an in vitro method for detecting B7H3 expression, wherein a sample to be detected is contacted with the antibody or antigen-binding fragment thereof described in the first aspect under conditions where a complex can be formed between the antibody or antigen-binding fragment thereof and B7H3; preferably, the method further comprises detecting the formation of the complex, indicating the presence or expression level of DLL3 in the sample.

A fifteenth aspect of the present disclosure provides use of the antibody or antigen-binding fragment thereof described in the first aspect in preparing a B7H3 detection reagent.

Definitions and Explanations of Terms

Unless otherwise defined herein, scientific and technical terms related to the present disclosure shall have the meanings that are understood by those of ordinary skill in the art.

Furthermore, unless otherwise indicated herein, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly indicated otherwise.

The terms "include," "comprising," and "having" are used interchangeably herein and are intended to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "include," "comprising," and "having" in this document also provides a "consisting of" solution.

The term "and/or" as used herein includes the meanings of "and," "or," and "all or any other combination of elements linked by the associated term."

The term "B7H3" herein refers to a member of the B7-CD28 superfamily, expressed on antigen-presenting cells. B7-H3 binds to T cells, but the B7-H3 counter-receptor on the surface of these T cells has not been fully characterized. The predominant human form of B7H3 contains two extracellular tandem IgV-IgC domains (i.e., IgV-IgC-IgV-IgC). Although initially believed to contain only two Ig domains (IgV-IgC), a tetra-immunoglobulin extracellular domain variant ("4Ig-B7-H3") has been identified and is found to be the more common human form of the protein. The native murine form (2Ig) and the human 4Ig form exhibit similar functions. The 4Ig-B7-H3 molecule inhibits NK cell-mediated lysis of cancer cells. B7H3 mRNA expression has been found in heart, kidney, testis, lung, liver, pancreas, prostate, colon, and osteoblasts.

The term "specific binding" herein refers to an antigen-binding molecule (e.g., an antibody) that specifically binds to an antigen and a substantially identical antigen, typically with high affinity, but does not bind to unrelated antigens with high affinity. Affinity is typically reflected in terms of the equilibrium dissociation constant (KD), where a lower KD indicates a higher affinity. Taking antibodies as an example, a high affinity typically refers to a KD of about ^10-6 M or less, ^10-7 M or less, about ^10-8 M or less, or about ^10-9 M or less. KD is calculated as follows: KD = Kd/Ka, where Kd represents the dissociation rate and Ka represents the association rate. The equilibrium dissociation constant, KD, can be measured using methods well known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis.

The term "antigen binding molecule" is used herein in the broadest sense to refer to a molecule that specifically binds to an antigen. Exemplarily, antigen binding molecules include, but are not limited to, antibodies or antibody mimetics. "Antibody mimetics" refer to organic compounds or binding domains that are capable of specifically binding to an antigen but are unrelated to the structure of an antibody. Exemplarily, antibody mimetics include, but are not limited to, affibodies, affitins, affilins, designed ankyrin repeat proteins (DARPins), nucleic acid aptamers, or Kunitz-type domain peptides.

The term "antibody" herein is used in the broadest sense and refers to a polypeptide or combination of polypeptides that comprises sufficient sequence from the variable region of the immunoglobulin heavy chain and/or sufficient sequence from the variable region of the immunoglobulin light chain to be able to specifically bind to an antigen. "Antibodies" herein encompass various forms and various structures, as long as they exhibit the desired antigen binding activity. "Antibodies" herein include alternative protein scaffolds or artificial scaffolds with transplanted complementary determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which include mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds comprising, for example, biocompatible polymers. See, for example, Korndorfer, I.P., Beste, G. & Skerra, A. (2003). Proteins, 53, 121-129.; Roque, A.C.A., Lowe, C.R. & Taipa, M.A. Antibodies and genetically engineered related molecules: production and purification. Biotechnol. Prog. 20, 639-654 (2004); the contents of which are incorporated herein in their entirety. Such scaffolds may also include non-antibody-derived scaffolds, such as scaffold proteins known in the art for grafting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.

The term "antibody" includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. "Antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further divided into hypervariable regions, called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, which are arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). Differences in the amino acid composition and order of arrangement of the constant region of immunoglobulins' heavy chains result in varying antigenicity. Consequently, "immunoglobulins" can be classified into five classes, or isotypes, herein: IgM, IgD, IgG, IgA, and IgE, corresponding to their corresponding heavy chains: μ, δ, γ, α, and ε. Within the same class, Ig can be further divided into subclasses based on differences in the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are classified as either kappa or lambda chains based on differences in the constant region. Each of the five Ig classes can have either kappa or lambda chains.

In this article, "antibodies" also include antibodies that do not contain light chains, for example, heavy-chain antibodies (HCAbs) produced by camelids such as dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanicoes (Lama guanicoe) and alpacas (Vicugna pacos), as well as immunoglobulin new antigen receptors (Ig new antigen receptors, IgNARs) found in cartilaginous fish such as sharks.

The term "antibody" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, cassowaries, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g. sharks).

The term "heavy chain antibody" herein refers to an antibody lacking the light chains of a conventional antibody. The term specifically includes, but is not limited to, a homodimeric antibody comprising a VH antigen binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.

As used herein, the term "nanoantibody" refers to a naturally occurring heavy chain antibody lacking a light chain that exists in camels. Cloning its variable region can produce a single-domain antibody consisting only of the heavy chain variable region, also known as VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.

In this article, the terms "nanobody" and "single-domain antibody" (sdAb) have the same meaning and are used interchangeably. They refer to the construction of a single-domain antibody (sdAb) consisting of only one heavy-chain variable region by cloning the variable region of a heavy-chain antibody. This is the smallest fully functional antigen-binding fragment. Typically, a heavy-chain antibody naturally lacking the light chain and heavy-chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy-chain variable region.

For further description of “heavy chain antibodies” and “nanobodies”, see: Hamers-Casterman et al., Nature. 1993; 363; 446-8; the review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001); and the following patent applications, which are mentioned as general background art: WO 94/04678, WO 95/04079 and WO 96/34103; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1 134231 and WO 02/48193; WO97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and W O 03/055527; WO 03/050531; WO 01/90190; WO03/025020; and WO 04/041867, WO 04/ 041862, WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825, and any other prior art referred to in these applications (the contents of which are incorporated herein in their entirety).

The term "multi-specificity" herein refers to the ability of an antibody or its antigen-binding fragment to bind to, for example, different antigens or at least two different epitopes on the same antigen. Therefore, terms such as "bispecific," "trispecific," and "tetraspecific" refer to the number of different epitopes that an antibody can bind to. For example, conventional monospecific IgG antibodies have two identical antigen-binding sites (paratopes) and can therefore only bind to the same epitope (rather than binding to different epitopes). In contrast, multispecific antibodies have at least two different types of paratopes/binding sites and can therefore bind to at least two different epitopes. As described herein, "complementarity determining region" refers to the antigen-binding site of an antibody. In addition, a single "specificity" can refer to one, two, three, or more than three identical complementary determining regions (the actual number of complementary determining regions/binding sites in a single antibody molecule is referred to as "valence") in a single antibody. For example, a single natural IgG antibody is monospecific and bivalent because it has two identical paratopes. Accordingly, a multispecific antibody comprises at least two (different) complementary determining regions/binding sites. Therefore, the term "multispecific antibody" refers to an antibody having more than one paratope and the ability to bind to two or more different epitopes. The term "multispecific antibody" particularly includes bispecific antibodies as defined above, but generally also includes proteins, e.g. antibodies that specifically bind three or more different epitopes, scaffolds, i.e. antibodies with three or more paratopes/binding sites.

The term "valent" herein refers to the presence of a specified number of binding sites in an antibody/antigen-binding molecule. Thus, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six binding sites, respectively, in an antibody/antigen-binding molecule.

[0014] "Full-length antibody," "intact antibody," and "intact antibody" are used interchangeably herein to refer to antibodies having a structure substantially similar to that of a native antibody.

"Antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not have the entire structure of an intact antibody, but only contain a portion or partial variant of an intact antibody, wherein the portion or partial variant has the ability to bind to an antigen. Exemplarily, "antigen-binding fragment" or "antibody fragment" herein include, but are not limited to, Fab, F(ab') ₂ , Fab', Fab'-SH, Fd, Fv, scFv, diabodies, and single-domain antibodies.

The term "chimeric antibody" herein refers to an antibody that has variable sequences of an immunoglobulin derived from one source organism (such as rat, mouse, rabbit or alpaca) and constant regions of an immunoglobulin derived from a different organism (such as human). Methods for producing chimeric antibodies are known in the art. See, for example, US Pat. No. 5,807,715A; Morrison, 1985, Science 229 (4719): 1202-1207. Transfectomas Provide Novel Chimeric Antibodies; Gillies et al., J Immunol Methods. 1989 Dec 20; 125 (1-2): 191-202; the entire contents are incorporated herein.

The term "humanized antibody" herein refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and/or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, the ability to increase immune cell activity or the ability to enhance immune response, etc.

The term "fully human antibody" herein refers to an antibody having a variable region in which both FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, "fully human antibodies" herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted onto human framework sequences.

The term "variable region" herein refers to the region of an antibody heavy or light chain involved in antigen binding. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The variable domains of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007); the contents of which are incorporated herein in their entirety. A single VH or VL domain may be sufficient to confer antigen-binding specificity.

The terms "complementarity determining region" and "CDR" are used interchangeably herein and generally refer to the hypervariable regions (HVRs) found in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are referred to as framework regions (FRs). As understood in the art, the amino acid positions representing the hypervariable regions of an antibody can vary depending on the context and various definitions known in the art. Some positions within the variable domain can be considered as hybrid hypervariable positions because these positions can be considered to be within the hypervariable region under one set of standards (such as IMGT, Chothia or KABAT), while being considered to be outside the hypervariable region under different sets of standards (such as KABAT, Chothia or IMGT). One or more of these positions can also be found in an extended hypervariable region. The present disclosure includes antibodies comprising modifications in these hybrid hypervariable positions. The heavy chain variable region CDRs can be abbreviated as HCDRs, and the light chain variable region can be abbreviated as LCDRs. The variable domains of native heavy and light chains each contain four framework regions, primarily in a sheet configuration, connected by three CDRs (CDR1, CDR2, and CDR3), which form loops connecting, and in some cases forming part of, the sheet structure. The CDRs in each chain are held together by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and contribute to the formation of the antigen-binding site of antibodies with the CDRs from other antibody chains (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, Md. 1987; the contents of which are incorporated herein in their entirety).

For further description of CDRs, see Kabat et al., J. Biol. Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:9 27-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc M.P. et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001). "CDRs" herein may be annotated and defined using methods known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, using websites including but not limited to the AbRSA website (http://cao.labshare.cn/AbRSA/cdrs.php), the abYsis website (www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi), and the IMGT website (http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results). The CDRs herein include overlaps and subsets of amino acid residues defined using different methods. (The foregoing is incorporated herein in its entirety.)

The term "Kabat numbering system" in this article generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

The term "Chothia numbering system" herein generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classic rule for identifying CDR region boundaries based on the position of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).

The term "IMGT numbering system" herein generally refers to the numbering system based on the international Immunogenetics information system (IMGT) initiated by Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003 (incorporated herein in its entirety).

The term "heavy chain constant region" herein refers to the carboxyl-terminal portion of an antibody heavy chain, which is not directly involved in binding the antibody to an antigen, but exhibits effector functions, such as interactions with Fc receptors, and has a more conserved amino acid sequence than the variable domains of antibodies. A "heavy chain constant region" can be selected from the CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof. A "heavy chain constant region" includes a "full-length heavy chain constant region" and a "heavy chain constant region fragment," the former having a structure substantially similar to that of a native antibody constant region, while the latter only includes "a portion of a full-length heavy chain constant region." For example, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include a CH1 domain. For example, a typical "heavy chain constant region fragment" can be selected from an Fc or CH3 domain.

The term "light chain constant region" herein refers to the carboxyl terminal portion of the antibody light chain, which is not directly involved in binding the antibody to the antigen, and the light chain constant region can be selected from a constant kappa domain or a constant lambda domain.

The term "Fc region" as used herein is used to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. For example, the Fc region of a human IgG heavy chain may extend from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage, removing one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include a full-length heavy chain, or it may include a cleavage variant of the full-length heavy chain. This may be the case when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index). Thus, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may be present or absent. Typically, an IgG Fc region comprises an IgG CH2 and IgG CH3 domains, and optionally, may further comprise a complete or partial hinge region, but does not comprise a CH1 domain. The "CH2 domain" of a human IgG Fc region typically extends from the amino acid residue at about position 231 to the amino acid residue at about position 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain. The "CH3 domain" comprises the stretch of residues at the C-terminus of the CH2 domain in the Fc region (i.e., from the amino acid residue at about position 341 to the amino acid residue at about position 447 of IgG). The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having a "knob" introduced into one chain thereof and a corresponding "cavity" or "hole" introduced into the other chain thereof; see U.S. Patent No. 5,821,333, the contents of which are incorporated herein in their entirety). As described herein, such variant CH3 domains can be used to promote heterodimerization of two non-identical antibody heavy chains.

Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, ^5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991; the contents of which are incorporated herein in their entirety.

The term "Fc variant" herein refers to changes in Fc structure or function caused by one or more amino acid substitutions, insertions, or deletions at appropriate sites on the Fc protein. "Inter-Fc variant interactions" refer to interactions between Fc variants engineered to form space-filling effects, electrostatic interactions, hydrogen bonding, hydrophobic interactions, and other interactions. These interactions contribute to the formation of stable heterodimeric proteins. Preferred mutational designs are "knob-into-hole" mutational designs.

The mutation design technology of Fc variants has been widely used in the field to prepare bispecific antibodies or heterodimeric Fc fusion protein forms. Representative ones include the "Knob-into-Hole" form proposed by Cater et al. (Protein Engineering vol.9 no.7 pp.617-621, 1996); Amgen technicians used electrostatic steering to form Fc-containing heterodimers (US20100286374 A1); Jonathan H. Davis et al. (Protein Engineering, Design & Selection pp.1-8, 2010) proposed a heterodimer formed by IgG/Ig chain exchange (S EEDbodies); bispecific molecules formed by Genmab's DuoBody (Science, 2007.317(5844)) platform technology; Xencor's technical staff integrated structural calculations and Fc amino acid mutations to form heterodimeric protein forms based on different modes of action (mAbs 3:6,546-557; November/December 2011); Suzhou Alphamab's charge network-based Fc modification method (CN201110459100.7) to obtain heterodimeric protein forms; and other genetic engineering methods based on Fc amino acid changes or functional modification means to achieve the formation of heterodimeric functional proteins. The knob/hole structure on the Fc variant fragment described in the present disclosure refers to the mutation of each of the two Fc fragments, which can be combined in a "knob-into-hole" form after the mutation. Preferably, the "knob-into-hole" model of Cater et al. is used to perform site-specific mutations on the Fc region, so that the resulting first Fc variant and the second Fc variant can be combined in a "knob-into-hole" form to form a heterodimer. The selection of a specific immunoglobulin Fc region from a specific immunoglobulin class and subclass is within the skill of those skilled in the art. Preferably, the Fc region of human antibodies IgG1, IgG2, IgG3, or IgG4 is used, and more preferably, the Fc region of human antibody IgG1. Randomly select one of the first Fc variant or the second Fc variant to perform a knob mutation, and the other to perform a hole mutation. (The above content is incorporated herein in its entirety).

The term "conservative amino acid" herein generally refers to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). For example, the amino acids within each of the following groups are conservative amino acid residues of each other, and substitutions of amino acid residues within the group are substitutions of conservative amino acids:

1) Alanine (A), serine (S), threonine (T);

2) Aspartic acid (D), glutamic acid (E);

3) Asparagine (N), glutamine (Q);

4) Arginine (R), Lysine (K), Histidine (H);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

The term "identity" herein can be calculated in the following manner: to determine the "identity" percentage of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., spaces can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal comparison, or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between the two sequences varies as the number of identical positions shared by the sequences changes, taking into account the number of spaces that need to be introduced and the length of each space for optimal comparison of the two sequences.

The comparison of sequences and calculation of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm, which has been integrated into the GAP program in the GCG software package (available at www.gcg.com), is used with a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences. For another example, the GAP program in the GCG software package (available at www.gcg.com) is used with a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and one that should be used unless otherwise specified) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4. (The aforementioned contents are incorporated herein in their entirety.)

Additionally or alternatively, the nucleic acid sequences and protein sequences described in the present disclosure can be further used as "query sequences" to perform searches against public databases, for example to identify other family member sequences or related sequences. For example, such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215: 403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules of the present disclosure. In order to obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov (incorporated herein in their entirety).

The term "chimeric antigen receptor (CAR)" herein refers to an artificial cell surface receptor that is modified to be expressed on immune effector cells and specifically binds to an antigen, which comprises at least (1) an extracellular antigen binding domain, such as an antibody heavy chain variable region and/or light chain variable region, (2) a transmembrane domain that anchors CAR into immune effector cells, and (3) an intracellular signaling domain. CAR is able to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC restricted manner using the extracellular antigen binding domain.

The term "nucleic acid" herein includes any compound and/or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'. In this article, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, nucleic acid molecules as described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derived sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules that are suitable as vectors for directly expressing the antibodies of the present disclosure in vitro and/or in vivo, for example in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or expression of the encoded molecule so that the mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038/nm.4356 or EP2101823B1; the foregoing are incorporated herein in their entirety).

As used herein, an "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

The term "host cell" herein refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the original transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected for in the initially transformed cell are included herein.

The term "pharmaceutical composition" herein refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.

The term "pharmaceutically acceptable carrier" herein includes any and all solvents, dispersion media, coating materials, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and the like, and combinations thereof, which are known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th ed. Mack Printing Company, 1990, pp. 1289-1329; the contents of which are incorporated herein in their entirety). Except in cases where it is incompatible with the active ingredient, any conventional carrier is contemplated for use in therapeutic or pharmaceutical compositions.

The term "treatment" herein refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) unwanted physiological changes or pathologies, such as cancer and tumors, in the subject being treated. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. Subjects in need of treatment include those already suffering from the condition or disease, as well as those susceptible to the condition or disease or those for whom the condition or disease is to be prevented. When terms such as slow down, alleviate, weaken, alleviate, and relieve are mentioned, their meaning also includes elimination, disappearance, non-occurrence, and the like.

The term "subject" herein refers to an organism that is being treated for a particular disease or condition as described herein. Exemplarily, a "subject" includes a mammal, such as a human, primate (e.g., monkey), or non-primate mammal, being treated for a disease or condition.

As used herein, the term "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating a disease symptom or the progression of that disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

As used herein, the term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition. As used herein, the term "tumor" or "neoplasm" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when used herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1A is an ELISA assay for the binding activity of SCR-10437 to human B7H3(4Ig)-his protein;

FIG1B is an ELISA test showing the binding activity of SCR-10437 to human B7H3(2Ig)-his protein;

FIG1C is an ELISA test of the binding activity of SCR-10437 to monkey B7H3-his protein;

Figures 2A-2D show the expression levels of B7H3 in tumor cells A375, A549, 786-O, and NCI-H1975;

FIG3A is a FACS assay showing the binding reaction between SCR-10437 and the human endogenous tumor cell line A375;

FIG3B is a FACS assay showing the binding reaction between SCR-10437 and the human endogenous tumor cell line A549;

FIG4A is a FACS assay showing the endocytic activity of B7H3 antibodies on the tumor cell line 786-O;

FIG4B is a FACS assay showing the endocytic activity of B7H3 antibodies on the tumor cell line NCI-H1975.

DETAILED DESCRIPTION

The present disclosure is further described below with reference to specific examples, and the advantages and features of the present disclosure will become more apparent as the description proceeds. Where specific conditions are not specified in the examples, conventional conditions or those recommended by the manufacturer were used. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

The embodiments of the present disclosure are merely exemplary and do not constitute any limitation on the scope of the present disclosure. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present disclosure may be modified or replaced without departing from the spirit and scope of the present disclosure, but such modifications and replacements shall fall within the scope of protection of the present disclosure.

Example 1. Preparation and purification of recombinant protein and control antibody

1.1 Design and expression of recombinant proteins

Construction of B7H3-4Ig recombinant protein: Using human B7H3-4Ig protein (UniProt No.: Q5ZPR3-1) as a template sequence, a tagged fusion protein was designed and cloned into the pTT5 vector (Ubi Biotech, VT2202) to construct the B7H3-4Ig plasmid. Similarly, construction of B7H3-2Ig recombinant protein: Using human B7H3-2Ig protein (UniProt No.: Q5ZPR3-2) as a template sequence, a tagged fusion protein was designed and cloned into the pTT5 vector to construct the B7H3-2Ig plasmid. The antigens and detection proteins disclosed herein were transiently expressed in Expi 293F cells (Gibco, A14527). The preparation method for cynomolgus macaque B7H3 recombinant protein is similar to that for human recombinant protein. The cynomolgus macaque B7H3 sequence was obtained from UniProt No.: A0A2K5U2B3. The specific sequence information of the recombinant protein is shown in Table 1.

Purification of recombinant protein by nickel column: The cell expression supernatant sample was centrifuged at high speed to remove impurities, and the nickel column was equilibrated with 20mM PBS + 500mM NaCl solution and washed with 2-5 column volumes. The culture supernatant was loaded onto a Ni affinity chromatography column (purchased from GE Healthcare), and the changes in the UV absorbance value (A280nm) were monitored with a UV detector. The column was flushed with equilibration solution until the A280 reading dropped to the baseline, and then gradient eluted with equilibration solution containing 10mM, 20mM, 40mM, 90mM, 250mM, and 500mM imidazole, respectively. Each elution peak was collected, and the component where the target protein was located was determined based on the SDS-PAGE gel map. The collected elution product containing the target protein can be concentrated and further purified by gel chromatography Superdex200 (GE) with PBS as the mobile phase to remove aggregates and impurity protein peaks, and the elution peak of the target product was collected. The obtained protein was identified as correct by electrophoresis, peptide mapping, and LC-MS and then aliquoted for use. The proteins purified by this protocol include human B7H3 4Ig-His, human B7H3 2Ig-His, and monkey B7H3-His.

Table 1 Sequences of human and monkey B7H3-His recombinant proteins

1.2 Design and expression of control antibodies

The control antibodies used in this disclosure are all from published patents. The DS7300 antibody is derived from the published patent CN103687945B, the MGC018 antibody is derived from the published patent WO2017180813A, and the enoblituzumab antibody is derived from the published patent WO2011109400A2. Unless otherwise specified, the DS7300, enoblituzumab, and MGC018 control antibodies were all recombinantly expressed using the human IgG1+κ subtype.

The expression and purification process of the control antibody is as follows: the antibody sequence gene is synthesized and cloned into the expression vector pTT5, and then transiently transfected into Expi293F cells (purchased from Gibco, A14527). After culturing on a shaker at 37°C for 7 days, the cell supernatant is collected for Protein A antibody purification. The Protein A affinity column is washed with 0.1M NaOH for 3-5 column volumes, and then washed with pure water for 3-5 column volumes. The chromatography column is equilibrated for 3-5 column volumes using a 1×PBS (pH 7.4) buffer system as an equilibrium buffer. The cell supernatant is loaded and bound at a low flow rate, and the flow rate is controlled so that the retention time is about 1 minute or longer. After binding is complete, the chromatography column is washed with 1×PBS (pH 7.4) for 3-5 column volumes until the UV absorbance returns to the baseline. The sample was eluted using 50mM citric acid/sodium citrate (pH 3.0-3.5) buffer, and the elution peak was collected according to ultraviolet detection. The eluted product was quickly adjusted to pH 5-6 using 1M Tris-HCl (pH 8.0) for temporary storage. The eluted product can be subjected to solution replacement using methods familiar to those skilled in the art, such as ultrafiltration concentration and solution replacement to the desired buffer system using an ultrafiltration tube, or molecular exclusion such as G-25 desalting to replace the desired buffer system, or high-resolution molecular exclusion columns such as Superdex 200 to remove aggregate components in the eluted product to improve sample purity. The obtained control antibodies were named DS7300-hIgG1, MGC018-hIgG1, and Enoblituzumab-hIgG1. The specific sequence information of the antibodies is shown in Table 2.

Table 2 Control antibody sequence list

Example 2: Preparation of antibodies against B7H3

2.1 Animal immunization

The monoclonal antibodies disclosed herein are produced by immunizing mice. The mice used in the experiment were female mice aged 6 to 8 weeks. The immunogen was the human B7H3 4Ig-his protein prepared in Example 1. During the initial immunization of the protein, the immunogen was emulsified with TiterMax (purchased from Sigma, T2684-1M) and injected subcutaneously (SC) and intraperitoneally (IP) at a dose of 0.1 mL, i.e., each mouse was injected with 50 μg of the immunogen; during the booster immunization, the immunogen was injected subcutaneously and intraperitoneally with Imject Alum (purchased from Thermo) at a dose of 0.1 mL, i.e., each mouse was injected with 25 μg of the immunogen. The immunization frequency was once a week, and blood was collected on days 3, 19, 47, and 61. The binding to the human B7H3 protein (His tag, coating concentration 2 μg/mL) was detected by ELISA, and the presence and antibody titer of antibodies recognizing human B7H3 in the serum of the immunized animals were tested. Based on the serum titer, mice with high antibody titer in serum and titer approaching a plateau were selected for booster immunization.

2.2 Antibody Screening and Sequencing

After the booster immunization, antigen-specific B cells were directly isolated from the immunized mice without fusion with myeloma cells. Plasma cells secreting antigen-specific monoclonal antibodies were screened using an Optofluidic System (Berkeley Lights Inc.). The antibody light and heavy chain variable region sequences were directly obtained from antigen-specific B cells by reverse transcription and PCR sequencing. The resulting humanized anti-B7H3 antibody, designated SCR10437, was screened using conventional methods. The amino acid sequences of its heavy chain variable region (HCVR) and light chain variable region (LCVR) are shown below:

SCR10437 HCVR (SEQ ID NO.12):

SCR10437 LCVR (SEQ ID NO.13):

The CDR regions of the B7H3 monoclonal antibody were analyzed. The CDR regions were identified and annotated using the Kabat numbering system, the Chothia numbering system, and the IMGT numbering system. The specific results are shown in Table 3.

Table 3 Antibody CDR sequences and numbers

2.3 Construction, expression and purification of fully human antibodies

Based on the sequencing results of the variable region genes of the SCR10437 antibody obtained in Section 2.2 above, primers were designed and PCR was used to construct the VH/VL gene segments of each antibody. The obtained VH/VL gene segments were homologously recombined with the expression vector pTT5 to construct a full-length expression vector plasmid for a fully human antibody. The SCR10437 antibody was expressed in the form of human IgG1 (the sequences of the heavy chain constant region and the light chain constant region are shown in Table 2). After the plasmid preparation was completed, it was transfected into Expi293F cells and cultured on a shaker at 37°C for 7 days. The supernatant was collected, centrifuged, and the antibody was purified according to the purification method described in Section 1.2 of Example 1. The resulting antibody was named SCR10437-hIgG1.

Example 3: Antibody affinity determination

The binding strength of antibodies and antigens was determined using a BIAcore 8K instrument and a Protein A capture assay. Protein A was first immobilized onto a CM4 chip (GE, BR-1005-34) using the amino coupling method. Following the instructions for the Amine Coupling Kit (GE, BR100633), HBS-EP (pH 7.4) was used as the mobile phase. NHS and EDC were mixed and the chip was activated for approximately 600 seconds. Protein A was then diluted to 50 μg/mL with 10 mM sodium acetate, pH 4.5, and injected for 600 seconds. Finally, any remaining active sites were blocked with ethanolamine. Then, the multi-cycle kinetic method was used to determine the affinity between the antibody and the antigen. In each cycle, the antibody to be tested was first captured by the Protein A chip, and then a single concentration of antigen protein was injected. The binding and dissociation processes of the antibody and antigen protein were recorded, and finally the chip was regenerated with Glycine pH 1.5. The mobile phase was HBS-EP+ (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20), with a flow rate of 30μL/min, a regeneration time of 30s, and a detection temperature of 25°C. Finally, the data were analyzed according to the 1:1 binding model, and the antibody-antigen binding kinetic parameters were fitted, including the association rate constant Ka, the dissociation rate constant Kd, the equilibrium dissociation constant KD, and the maximum binding signal Rmax.

The association rate (Ka), dissociation rate (Kd) and binding affinity (KD) of SCR10437-hIgG1 antibody to human and monkey B7H3 protein are shown in Table 4.

Table 4 Affinity of antibodies and B7H3 protein detected by SPR (biacore)

Example 4: Identification of Antibody Binding Activity

4.1 ELISA detection of binding of fully human antibodies to human and monkey B7H3 proteins

Human B7H3 (4Ig) -his protein was diluted with PBS to a final concentration of 1 μg/mL, then 50 μl was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The next day, the plate was washed twice with PBST and blocked with blocking solution [PBS + 2% (w/w) BSA] for 2 hours at room temperature. The blocking solution was discarded, and 50 μl of the antibody starting at 50 nM and serially diluted 4 times, as well as positive and negative control antibodies, were added to each well. After incubation at 37°C for 1 hour, the plate was washed three times with PBST. HRP (horseradish peroxidase)-conjugated secondary antibody (purchased from Merck, Cat. No.: AP113P) was added, incubated at 37°C for 1 hour, and the plate was washed five times with PBST. TMB substrate (50 μl) was added to each well, incubated at room temperature for 10 minutes, and then stop solution (1.0 M HCl) was added to each well. OD _{450 nm} values were read using an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer). The binding activity of the antibodies to human B7H3(4Ig)-his protein is shown in Figure 1A. The results showed that the negative control anti-FITC-hIgG1 (derived from the literature J Biol Chem. 1990 Jan 5; 265(1): 133-8) did not bind to B7H3, while the SCR10437-hIgG1 antibody effectively bound to human B7H3(4Ig)-his protein, with binding activity comparable to that of the positive control antibody DS7300-hIgG1.

Human B7H3(2Ig)-his protein was diluted with PBS to a final concentration of 2 μg/mL, and 50 μl was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The binding activity of the B7H3 antibody to human B7H3(2Ig)-his protein was analyzed using the same ELISA assay described above. The results showed that the SCR10437-hIgG1 antibody effectively bound to human B7H3(2Ig)-his protein, with binding activity comparable to that of the positive control antibody DS7300-hIgG1 (Figure 1B).

Monkey cynoB7H3-his protein was diluted with PBS to a final concentration of 2 μg/mL, and 50 μl was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The binding activity of the B7H3 antibody to the cynoB7H3-his protein was analyzed using the same assay described above. The results showed that the SCR10437-hIgG1 antibody effectively bound to the cynoB7H3-his protein, with binding activity comparable to that of the positive control antibody DS7300-hIgG1 (Figure 1C).

4.2 Flow cytometry (FACS) detection of B7H3 antibody binding to tumor cells

Endogenous tumor cells A375 (Nanjing Kebai, CBP60329, B7H3 expression is shown in Figure 2A) were expanded and cultured in T-175 culture flasks to the logarithmic growth phase, the culture medium was removed, washed twice with PBS buffer, digested with trypsin, and then digested with complete culture medium, and the cells were pipetted to a single cell suspension. After cell counting, centrifugation was performed, and the cell pellet was resuspended to 2×10 ⁶ cells per milliliter with FACS buffer (PBS + 10% fetal bovine serum), and 100 μl was added to a 96-well FACS reaction plate per well. Centrifugation was performed, and the supernatant was discarded. The antibody sample to be tested (200nM as the starting concentration, 4-fold serial dilution) was added to each well at 50 μl, mixed with the cells, and incubated at 4°C for 1 hour. Washed 3 times with PBS buffer by centrifugation, 50 μl of Alexa Incubate with 647AffiniPure Goat Anti-Human IgG, Fcγ fragment-specific secondary antibody (purchased from Jackson, Cat. No. 109-605-098) at 4°C for 1 hour. Wash cells three times with PBS buffer by centrifugation, resuspend in 100 μl of PBS, and analyze using FACS (FACS Canto™, purchased from BD Biosciences). Data were analyzed using FlowJo software to obtain the mean fluorescence intensity (MFI) of the cells. GraphPad Prism 8 software was then used for data fitting and EC50 calculation. As shown in Figure 3A and Table 5, SCR10437-hIgG1 antibody specifically binds to A375 cells, with stronger binding than DS7300-hIgG1 and Enoblituzumab-hIgG1.

Table 5 Binding of B7H3 antibody to A375 cells

The same method was used to detect the binding of SCR10437-hIgG1 antibody to the lung cancer cell line A549 (Nanjing Kebai, CBP60084, B7H3 expression level is shown in Figure 2B), which has moderate B7H3 expression. The results are shown in Figure 3B and Table 6. SCR10437-hIgG1 has good binding activity with A549, and its binding ability is stronger than DS7300-hIgG1 and MGC018-hIgG1.

Table 6 Binding of B7H3 antibody to A549 cells

Example 5: Antibody endocytosis activity detection

To evaluate the endocytic activity of the anti-B7H3 antibody SCR10437, 786-O (Nanjing Kebai, CBP60442; B7H3 expression levels are shown in Figure 2C) and NCI-H1975 (Nanjing Kebai, CBP60121; B7H3 expression levels are shown in Figure 2D) cells were cultured to 80% confluence. The cells were harvested and stained with the anti-B7H3 antibody to be tested (100 nM) and a secondary antibody, Aleax Flour488-conjugated anti-human IgG, Fcγ fragment-specific antibody (2.2 μg/ml, Jackson, 109-545-098), at 4°C. The cells were washed and incubated at 37°C for 0 h (pre-incubation) and 4 h. Cells were washed and pre-incubated or incubated with an anti-Alexa 488 antibody (Invitrogen, A11094) at a concentration of 10 μg/ml to quench cell surface fluorescence, or unquenched to detect the entire signal, and analyzed by flow cytometry. The internalization rate (%) was calculated using the following formula: [1-(Na-Qa)/(Na-NaxQi/Ni)]x100, and the internalization amount (MFI value) was calculated using the following formula: Qa-Qi; Na: mean fluorescence intensity (MFI) of samples at each incubation time under unquenched conditions, Qa: MFI of samples at each incubation time under quenched conditions, Ni: MFI of samples pre-incubated under unquenched conditions, Qi: MFI of samples pre-incubated after quenching. The internalization amount represents the total amount of antibody internalized into the cell within 4 hours. The results are shown in Figures 4A and 4B. All antibodies were able to be internalized, and SCR10437-hIgG1 was internalized better than the positive control antibody.

Claims (23)

An antibody or antigen-binding fragment thereof that specifically binds to B7H3, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (1) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 are the HCDR1, HCDR2 and HCDR3 of the VH domain shown in SEQ ID NO. 12; and (2) The light chain variable region comprises LCDR1, LCDR2 and LCDR3, and the LCDR1, LCDR2 and LCDR3 are the LCDR1, LCDR2 and LCDR3 of the VL domain shown in SEQ ID NO.13. The antibody or antigen-binding fragment thereof according to claim 1, wherein the HCDR1, HCDR2, and HCDR3 are determined according to the Kabat, Chothia, or IMGT numbering systems, and have an amino acid sequence as shown in SEQ ID NO.14-16, SEQ ID NO.20-22, or SEQ ID NO.26-28, or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions, and/or substitutions compared to the amino acid sequences shown in SEQ ID NO.14-16, SEQ ID NO.20-22, or SEQ ID NO.26-28. The antibody or antigen-binding fragment thereof according to claim 1, wherein the LCDR1, LCDR2 and LCDR3 are determined according to the Kabat, Chothia or IMGT numbering system, and have an amino acid sequence as shown in SEQ ID NO.17-19, SEQ ID NO.23-25, SEQ ID NO.29-31, or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the amino acid sequence shown in SEQ ID NO.17-19, SEQ ID NO.23-25, SEQ ID NO.29-31. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof comprises the following LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2, HCDR3: (1) According to the Kabat numbering system, LCDR1-3 and HCDR1-3 have the amino acid sequences shown in SEQ ID NOs. 14-19, or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the amino acid sequences shown in SEQ ID NOs. 14-19; (2) According to the IMGT numbering system, LCDR1-3 and HCDR1-3 have the amino acid sequences set forth in SEQ ID NOs. 20-25, or a combination of sequences having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the amino acid sequences set forth in SEQ ID NOs. 20-25; (3) According to the Chothia numbering system, LCDR1-3 and HCDR1-3 have the amino acid sequences shown in SEQ ID NO.26-31 or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the amino acid sequences shown in SEQ ID NO.26-31. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the heavy chain variable region sequence comprises the sequence shown in SEQ ID NO. 12, or a sequence with 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the sequence shown; and the light chain variable region sequence comprises the sequence shown in SEQ ID NO. 13, or a sequence with 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the sequence shown. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof is fully human. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof can specifically bind to human or monkey B7H3 protein. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof may further comprise any constant region sequence of human or mouse antibody IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably, the constant region sequence of human or mouse antibody IgG1, IgG2, IgG3 or IgG4, or the constant region sequence of human or mouse antibody IgG1, IgG2, IgG3 or IgG4 with a mutation; further, the antibody or antigen-binding fragment thereof is further coupled to a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from a radioisotope, a chemotherapeutic drug, a cytotoxic agent or an immunomodulator, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent and a photosensitizer; more preferably, the cytotoxic agent is selected from alkaloids, methotrexate, anthracycline antibiotics (doxorubicin), pyrrolobenzodiazepines (pyrrolobenzodiazepine, PBD), gemcitabine, cytarabine, tegafur, ifosfamide, dacarbazine and oxaliplatin; more preferably, the cytotoxic agent is a taxane. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antigen-binding fragment is selected from one or more of F(ab') ₂ , Fab', Fab, Fv, scFv, nanobody or affibody. A multispecific antigen-binding molecule comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 9; preferably, the multispecific antigen-binding molecule further comprises an antibody or antigen-binding fragment thereof that specifically binds to an antigen other than B7H3 or to a B7H3 epitope different from the B7H3 epitope bound by the antibody or antigen-binding fragment thereof of any one of claims 1 to 9; Preferably, the antigen other than B7H3 is selected from the following group: (1) tumor-specific antigen (TSA) or tumor-associated antigen (TAA); (2) immune checkpoint; (3) target for recruiting and/or activating immune cells; Preferably, the multispecific antigen-binding molecule may be bispecific, trispecific or tetraspecific; Preferably, the multispecific antigen-binding molecule may be bivalent, trivalent, tetravalent, pentavalent or hexavalent. A chimeric antigen receptor (CAR), comprising at least an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9. An immune effector cell, which expresses the chimeric antigen receptor according to claim 11, or contains a nucleic acid fragment encoding the chimeric antigen receptor according to claim 11; preferably, the immune effector cell is selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), DNT cells (double negative T cells), monocytes, macrophages, dendritic cells or mast cells, and the T cells are selected from cytotoxic T cells, regulatory T cells or helper T cells; preferably, the immune effector cell is an autologous immune effector cell or an allogeneic immune effector cell. An isolated nucleic acid fragment encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the multispecific antigen-binding molecule according to claim 10, or the chimeric antigen receptor according to claim 11. A vector comprising the nucleic acid fragment according to claim 13. A host cell comprising the vector of claim 14; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungi (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line). A method for preparing the antibody or antigen-binding fragment thereof of any one of claims 1 to 9 or the multispecific antigen-binding molecule of claim 10, comprising culturing the host cell of claim 15, and isolating the antibody or antigen-binding fragment thereof expressed by the host cell, or isolating the multispecific antigen-binding molecule expressed by the host cell. A method for preparing the immune effector cell of claim 12, comprising introducing a nucleic acid fragment encoding the CAR of claim 11 into the immune effector cell, optionally further comprising initiating the immune effector cell to express the CAR of claim 11. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the multispecific antigen-binding molecule according to claim 10, or the immune effector cell according to claim 12, or the nucleic acid fragment according to claim 13, or the vector according to claim 14, or the host cell according to claim 15, or the antibody or antigen-binding fragment thereof or the multispecific antigen-binding molecule prepared by the method according to claim 16, or the immune effector cell prepared by the method according to claim 17; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, or adjuvant. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the multispecific antigen-binding molecule according to claim 10, or the immune effector cell according to claim 12, or the nucleic acid fragment according to claim 13, or the vector according to claim 14, or the host cell according to claim 15, or the antibody or antigen-binding fragment thereof or multispecific antigen-binding molecule prepared by the method according to claim 16, or the immune effector cell prepared by the method according to claim 17; or the pharmaceutical composition according to claim 18 in the preparation of a medicament for preventing and/or treating tumors; wherein the tumor is selected from a solid tumor, a hematological tumor, or a cancer infiltrating and expressing B7H3; Preferably, the tumor is selected from melanoma, prostate cancer, renal cell carcinoma, lung cancer (eg non-small cell lung cancer (NSCLC)) and other solid tumors. A method for preventing and/or treating tumors, comprising administering to a patient in need thereof an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the multispecific antigen-binding molecule according to claim 10, or the immune effector cell according to claim 12, or the nucleic acid fragment according to claim 13, or the vector according to claim 14, or the host cell according to claim 15, or the antibody or antigen-binding fragment thereof or the multispecific antigen-binding molecule prepared by the method according to claim 16, or the immune effector cell prepared by the method according to claim 17, or the pharmaceutical composition according to claim 18; wherein the tumor is selected from a solid tumor, a hematological tumor, or a cancer infiltrating and expressing B7H3; Preferably, the tumor is selected from melanoma, prostate cancer, renal cell carcinoma, lung cancer (eg non-small cell lung cancer (NSCLC)) and other solid tumors. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the multispecific antigen-binding molecule according to claim 10, or the immune effector cell according to claim 12, or the nucleic acid fragment according to claim 13, or the vector according to claim 14, or the host cell according to claim 15, or the antibody or antigen-binding fragment thereof or the multispecific antigen-binding molecule prepared according to the method of claim 16, or the immune effector cell prepared according to the method of claim 17, or the pharmaceutical composition according to claim 18. A method for detecting B7H3 expression in vitro, comprising contacting a sample to be detected with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 under conditions where a complex can be formed between the antibody or antigen-binding fragment thereof and B7H3; preferably, the method further comprises detecting the formation of the complex, indicating the presence or expression level of DLL3 in the sample. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 in the preparation of a B7H3 detection reagent.