Nanobody targeting human b7h3 and use thereof

Abstract

The present disclosure relates to an antibody targeting human B7-H3, a polynucleotide encoding same, a host cell, an immunoconjugate and an immunofusion comprising the antibody, as well as a composition and use thereof.

Description

Nanobodies targeting human B7H3 and their applications Technical Field

[0001] This disclosure relates to the field of antibody technology, and more particularly to antibodies targeting human B7-H3, polynucleotides encoding the same, host cells, and immune conjugates and immune fusions containing said antibodies, as well as compositions and applications thereof. Background Technology

[0002] Nanobodies are the smallest antibody molecules currently known, with a molecular weight one-tenth that of ordinary antibodies. Initially discovered in camel blood by Belgian scientist R. Hamers, they are a highly anticipated class of engineered antibody products. In addition to possessing the antigenic reactivity of monoclonal antibodies, nanobodies exhibit unique functional properties such as small molecular weight, high stability, good solubility, easy expression, strong targeting, and simple humanization. They are particularly suitable for the development of bispecific / multispecific therapeutic antibodies and chimeric antigen receptor (CAR)-based cell immunotherapies (such as CAR-T cells, CAR-M cells, and CAR-NK cells).

[0003] B7-H3 (also known as CD276) is a type I transmembrane glycoprotein belonging to the B7 superfamily and is a newly discovered immune checkpoint. Human B7-H3 has two isoforms: 2IgB7-H3 and 4IgB7-H3. 2IgB7-H3 contains an extracellular region consisting of a pair of IgV-like and IgC-like immunoglobulin domains (IgV-IgC), a transmembrane region, and a short cytoplasmic tail. 4IgB7-H3 is the major expressed form of human B7-H3, possessing the same transmembrane and cytoplasmic regions as 2IgB7-H3, but its extracellular region contains tandemly repeated VC domains, exhibiting an IgV1-IgC1-IgV2-IgC2 structure from the N-terminus to the C-terminus. Studies have shown that B7-H3 participates in T cell suppression and is an important regulatory molecule for tumor progression.

[0004] In the field of cancer treatment, the heterogeneity and drug resistance of tumors necessitate the continuous development of new therapeutic approaches based on effective targeted molecules to expand treatment options, broaden the therapeutic window, and meet the clinical needs of cancer treatment.

[0005] Invention Overview

[0006] In a first aspect, this disclosure provides an anti-B7-H3 antibody comprising a VHH domain targeting B7-H3, or an antigen-binding fragment thereof. In some embodiments, the anti-B7-H3 antibody of the present invention has one or more of the following properties:

[0007] (1) High affinity binding to human B7-H3 antigen;

[0008] (2) It binds to B7-H3 on the cell surface;

[0009] (3) It exhibits cross-reactivity with monkey B7-H3; and

[0010] (4) It has B7-H3-mediated endocytosis activity.

[0011] In some embodiments, the anti-B7-H3 antibody of the present invention, or its antigen-binding fragment, comprises a VHH domain, wherein the VHH domain comprises three complementarity-determining regions (CDRs), CDR1, CDR2, and CDR3, selected from the heavy chain variable regions of SEQ ID NOs:3-5. Optionally, the CDRs are defined according to Kabat, AbM, IMGT, Chothia, or combinations thereof. In some embodiments, the CDRs are defined according to Kabat. In some embodiments, the anti-B7-H3 antibody of the present invention is a nanobody comprising, substantially comprising, or composed of the VHH domain. In other embodiments, the anti-B7-H3 antibody of the present invention is a heavy chain antibody comprising the VHH domain. In other embodiments, the anti-B7-H3 antibody of the present invention is a bispecific or multispecific antibody comprising the VHH domain.

[0012] In a second aspect, this disclosure provides a polynucleotide encoding the anti-B7-H3 antibody of the first aspect of the invention, a vector and a host comprising the polynucleotide, and a method for producing the anti-B7-H3 antibody of the invention.

[0013] In a third aspect, this disclosure provides immunoconjugates or immunofusions comprising the anti-B7-H3 antibody or its antigen-binding fragment of the first aspect of the invention. In some embodiments, the immunoconjugate comprises one or more therapeutic or diagnostic agents conjugated to the anti-B7-H3 antibody or its antigen-binding fragment. In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC). In some embodiments, the immunofusion comprises one or more heteropeptides or polypeptides fused to the anti-B7-H3 antibody or its antigen-binding fragment. In some embodiments, the immunofusion is a chimeric antigen receptor. In this aspect, this disclosure also provides the use of the anti-B7-H3 antibody or its antigen-binding fragment of the first aspect of the invention for preparing the immunoconjugates or immunofusions.

[0014] In a fourth aspect, this disclosure provides compositions comprising an antibody or antigen-binding fragment thereof from the first aspect of the invention and an immunoconjugate or immunofusion from the third aspect of the invention. In some embodiments, the compositions are pharmaceutical compositions.

[0015] In a fifth aspect, this disclosure provides the use of the antibody or antigen-binding fragment thereof of the first aspect of the invention and the immunoconjugate or immunofusion of the third aspect of the invention as a medicament or in the preparation of a medicament, optionally for the treatment or prevention of B7-H3-related diseases, particularly B7-H3-positive tumors. In this aspect, this disclosure also provides a method for treating or preventing B7-H3-related diseases, particularly B7-H3-positive tumors, comprising administering to a subject in need an effective amount of the antibody or antigen-binding fragment thereof of the first aspect of the invention or the immunoconjugate or immunofusion of the third aspect of the invention.

[0016] Brief description of the attached diagram

[0017] Figure 1 shows the binding of the anti-B7-H3 chimeric antibody to recombinant human-mouse-monkey B7-H3 as detected by ELISA.

[0018] Figure 2 shows the binding epitopes of the anti-B7-H3 chimeric antibody detected by ELISA.

[0019] Figure 3 shows the binding of the anti-B7-H3 chimeric antibody to MDA-MB-231 cells as detected by FACS.

[0020] Figure 4 shows the binding of the anti-B7-H3 chimeric antibody to LS1034 cells as detected by FACS.

[0021] Figure 5 shows the binding of the anti-B7-H3 chimeric antibody to C2BBe1 cells as detected by FACS.

[0022] Figure 6 shows the internalization of anti-B7-H3 chimeric antibody on MDA-MB-231 cells as detected by FACS.

[0023] Figure 7 shows the internalization of anti-B7-H3 chimeric antibody on LS1034 cells as detected by FACS.

[0024] Figure 8 shows the internalization of anti-B7-H3 chimeric antibody on C2BBe1 cells as detected by FACS.

[0025] Figure 9 shows the binding of the anti-B7-H3 humanized antibody to MDA-MB-231 cells as detected by FACS.

[0026] Figure 10 shows the binding of the anti-B7-H3 humanized antibody to LS1034 cells as detected by FACS.

[0027] Figure 11 shows the binding of the anti-B7-H3 humanized antibody to NCI-H1975 cells as detected by FACS.

[0028] Figure 12 shows the internalization of anti-B7-H3 humanized antibody on MDA-MB-231 cells as detected by FACS.

[0029] Figure 13 shows the internalization of anti-B7-H3 humanized antibody on LS1034 cells as detected by FACS.

[0030] Figure 14 shows the internalization of anti-B7-H3 humanized antibody on NCI-H1975 cells as detected by FACS.

[0031] Figure 15 shows the binding of the anti-B7H3 humanized antibody to DLD-1 cells as detected by FACS.

[0032] Figure 16 shows the binding of the anti-B7H3 humanized antibody to MDA-MB-231 cells as detected by FACS.

[0033] Figure 17 shows the binding of the anti-B7H3 humanized antibody to DLD-1 cells as detected by FACS.

[0034] Figure 18 shows the binding of the anti-B7H3 humanized antibody to Calu-6 cells as detected by FACS.

[0035] Figure 19 shows the binding of the anti-B7H3 humanized antibody to A549 cells as detected by FACS.

[0036] Figure 20 shows the binding of the anti-B7H3 humanized antibody to NCI-H460 cells as detected by FACS.

[0037] Figure 21 shows the binding of the anti-B7H3 humanized antibody to MDA-MB-468 cells as detected by FACS.

[0038] Figure 22 shows the binding of the anti-B7H3 humanized antibody to A431 cells as detected by FACS.

[0039] Figure 23 shows the binding of anti-B7H3 humanized antibody to PANC-1 cells as detected by FACS.

[0040] Figure 24 shows the binding of anti-B7H3 humanized antibody to HeLa cells as detected by FACS.

[0041] Figure 25 shows the binding of the anti-B7H3 humanized antibody to RKO cells as detected by FACS.

[0042] Figure 26 shows the binding of the anti-B7H3 humanized antibody to EBC-1 cells as detected by FACS.

[0043] Figure 27 shows the internalization of anti-B7H3 humanization in DLD-1 cells as detected by FACS.

[0044] Figure 28 shows the internalization of anti-B7H3 humanization in DLD-1 cells as detected by FACS.

[0045] Figure 29 shows the internalization of anti-B7H3 humanization in Calu-6 cells as detected by FACS.

[0046] Figure 30 shows the internalization of anti-B7H3 humanized antibody on A549 cells as detected by FACS.

[0047] Figure 31 shows the internalization of anti-B7H3 humanized antibody on NCI-H460 cells as detected by FACS.

[0048] Figure 32 shows the internalization of anti-B7H3 humanized antibody on MDA-MB-468 cells as detected by FACS.

[0049] Figure 33 shows the internalization of anti-B7H3 humanized antibody on A431 cells as detected by FACS.

[0050] Figure 34 shows the internalization of anti-B7H3 humanized antibody on PANC-1 cells as detected by FACS.

[0051] Figure 35 shows the internalization of anti-B7H3 humanized antibody on HeLa cells as detected by FACS.

[0052] Figure 36 shows the internalization of anti-B7H3 humanized antibody on RKO cells as detected by FACS.

[0053] Figure 37 shows the internalization of anti-B7H3 humanized antibody on EBC-1 cells as detected by FACS.

[0054] Figure 38 shows the change in internalization of anti-B7H3 humanized antibody on HCC827 cells over time as detected by FACS.

[0055] Figure 39 shows the change in internalization of anti-B7H3 humanized antibody on DLD-1 cells over time as detected by FACS.

[0056] Figure 40 shows the change in internalization of anti-B7H3 humanized antibody on Aspc-1 cells over time as detected by FACS.

[0057] Figure 41 shows the killing activity of the antibody-drug conjugate targeting B7H3 against SKOV3 tumor cells.

[0058] Figure 42 shows the tumor growth curve of the Nude mouse model of human lung cancer Calu-6.

[0059] Figure 43 shows tumor weight data of the Nude mouse Calu-6 human lung cancer model.

[0060] Figure 44 shows the tumor growth curve of the Nude mouse model of human colorectal cancer DLD-1.

[0061] Invention Details

[0062] Before describing the invention in detail, it should be understood that the invention is not limited to the specific methods and experimental conditions described herein, as these methods and conditions can be modified. Furthermore, the terminology used herein is for illustrative purposes only and is not intended to be restrictive.

[0063] definition

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of this invention, the following terms are defined.

[0065] The term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​that have a lower limit of 10% less than the specified numeric value and an upper limit of 10% greater than the specified numeric value.

[0066] When the term “and / or” is used to connect two or more options, it should be understood to mean any one of the options or any two or more of the options.

[0067] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations consisting of the mentioned elements, integers, or steps. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0068] In this document, the terms "optional" or "optionally" mean that an event or condition described below may or may not occur, and the description includes instances where the event or condition occurs as well as instances where the event or condition does not occur. For example, when it is mentioned that an antibody "optionally contains an immunoglobulin constant region," it means that the immunoglobulin constant region may or may not be present in the antibody.

[0069] As used herein, the “percentage of identity (%)” for an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to those in the specific amino acid sequence shown in this specification after the candidate sequence has been aligned with the specific amino acid sequence shown in this specification and, if necessary, vacancy has been introduced to achieve the maximum percentage of sequence identity, without regard to any conservative substitutions as part of the sequence identity.

[0070] As used herein, when referring to a “variant” of a polypeptide, it means that the variant is at least 80% identical to the polypeptide in terms of amino acid sequence.

[0071] In this document, "at least 80% identity" means any percentage of identity ≥80%, such as at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% identity.

[0072] The terms “B7-H3” and “B7H3” are used interchangeably and in this document refer to B7 homolog 3 (also known as CD276), a member of the B7 family of immunomodulatory proteins. Unless otherwise stated, this term encompasses any isoform of human B7-H3 (including 4Ig and 2Ig types) and its variants, including sequence variants, especially naturally occurring variants, allelic variants, and post-translational modification and conformational variants. An example of B7-H3 is the human B7-H3 protein containing the amino acid sequence UniProtKB-Q5ZPR3-1. In this document, unless explicitly specified that B7-H3 originates from a non-human species (e.g., “mouse B7-H3”, “monkey B7-H3”, etc.), the expression “B7-H3” refers to human B7-H3.

[0073] The term "antibody" is used in its broadest sense herein to refer to a polypeptide containing at least one variable region of a light or heavy chain immunoglobulin that specifically recognizes and binds to an antigen. This term encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, single-chain or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), single-domain or multi-domain antibodies, heavy chain antibodies, murine antibodies, chimeric or humanized antibodies, and antibody fragments, provided they exhibit the desired antigen-binding activity. The term also encompasses antibody structures with chemical modifications, including but not limited to covalent modifications such as glycosylation, amidation, acetylation, phosphorylation, lipid groups, and PEGylation.

[0074] As used herein, the terms “antibody fragment” and “antigen-binding fragment” in relation to antibodies are used interchangeably and refer to an incomplete portion of an antibody that contains the antibody’s antigen-binding domain and accordingly retains the ability to bind to the antigen to which the antibody is bound. Fab, Fv, Fab', and F(ab')2 fragments are examples of antigen-binding fragments of conventional four-chain antibodies; VHH fragments are examples of antigen-binding fragments of heavy-chain antibodies. In this disclosure, unless otherwise stated or explicitly contradicted by the context, reference to the term “antibody” is equivalent to reference to “antibody or an antigen-binding fragment thereof”.

[0075] The term "heavy chain antibody" herein refers to an antibody that possesses only a heavy chain and lacks a light chain. Typically, the heavy chain antibody according to the invention is a dimer comprising two monomers, each monomer containing a VHH domain linked to the immunoglobulin CH2 and CH3 domains via an immunoglobulin hinge region. Heavy chain antibodies from camel species (e.g., camels, alpacas, dromedaries, llamas, and guanacos) are examples of naturally occurring heavy chain antibodies.

[0076] The term "VHH antibody" is used in this document to refer to an antibody composed of heavy chain variable domains derived from heavy chain antibodies.

[0077] The term "nanobody" is used in this document to refer to an antibody containing, or consisting primarily of, a VHH domain, or composed of a VHH domain, with a molecular weight of less than 20 kDa (typically about 12–15 kDa).

[0078] The terms “single-domain antibody” and “sdAb” are used interchangeably in this document and refer to an antibody peptide that recognizes and binds to a target antigen through a single variable antibody domain, such as a single VHH or a single VH or a single VL.

[0079] As used herein, when referring to an antibody as containing a “constant domain,” the constant domain refers to a constant domain of the immunoglobulin heavy or light chain.

[0080] As used herein, the term "immunoglobulin" refers to a protein with a structure that contains naturally occurring antibodies. For example, IgG immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. Each immunoglobulin heavy chain has a heavy chain variable region (VH), also called a heavy chain variable domain, from the N-terminus to the C-terminus, followed by three to four heavy chain constant domains (CH1, CH2, and CH3, and optionally CH4), also called heavy chain constant regions. Similarly, each immunoglobulin light chain has a light chain variable region (VL), also called a light chain variable domain, from the N-terminus to the C-terminus, followed by a light chain constant domain (CL), also called a light chain constant region. Immunoglobulin heavy chains can be classified into one of five types based on the type of their constant domains, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM). Some of these categories can be further subdivided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Immunoglobulin light chains can also be classified into one of two types based on the amino acid sequence of their constant domains, called κ and λ.

[0081] In this document, unless otherwise explicitly stated, references to "immunoglobulin constant domain" or "constant domain" encompass both native sequence constant domains and variant sequence constant domains. Those skilled in the art can readily determine the type or subtype of a constant domain by comparing its amino acid sequence with the sequences of corresponding constant domains of different types or subtypes of native immunoglobulins.

[0082] As used herein, the term "immunoglobulin Fc region" is used interchangeably with "Fc region" and "Fc domain" to define the constant region portion of the immunoglobulin heavy chain that contains the CH2 and CH3 domains. In some cases, this immunoglobulin portion may also contain a hinge region or a portion thereof, but typically does not include the variable region and the CH1 domain. When referring to the human IgG1 Fc region, the EU numbers of the amino acid residues can be found in the IMGT Scientific chart (https: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html), which is hereby incorporated for reference. According to this numbering, in the human IgG1 immunoglobulin heavy chain, amino acids 118-215 constitute the CH1 domain, amino acids 216-230 constitute the hinge region, amino acids 231-340 constitute the CH2 domain, and amino acids 341-447 constitute the CH3 domain. In some cases, the C-terminal lysine (Lys447) of the CH3 domain may or may not be present.

[0083] As used herein, the terms "Fc domain" or "Fc region" encompass both native and variant Fc regions. In this document, the term "native Fc region" encompasses naturally occurring Fc region sequences of various immunoglobulins, such as the Fc region sequences of various Ig isotypes and their allotypes. The term "variant Fc region" refers to a polypeptide containing a modified Fc region relative to the native Fc region sequence. The modification can be the addition, deletion, or substitution of amino acid residues. Substitution can include both naturally occurring and non-natural amino acids. The purpose of the modification may be to alter the physicochemical properties of the Fc region and / or the binding of the Fc region to its receptor and its effector functions.

[0084] The term "chimeric antibody" in this disclosure refers to an antibody in which (a) a constant region or a portion thereof is altered, replaced, or exchanged such that the antigen-binding site is linked to a constant region of different or altered class and / or species origin, or to a completely different polypeptide molecule (e.g., enzyme, toxin, hormone, growth factor, drug, etc.) that confers novel properties to the chimeric antibody; and / or (b) a variable region or a portion thereof is altered, replaced, or exchanged to a variable region or a portion thereof of different or altered antigen specificity and / or species origin.

[0085] The term "humanized antibody" as used herein refers to an antibody for which a CDR sequence derived from a non-human mammalian species (e.g., camels) has been grafted onto a human scaffold sequence. In some cases, additional scaffold region modifications may be made within the human scaffold sequence, and / or additional amino acid modifications may be made to the CDR sequence to maintain or modify the antigen-binding properties of the antibody. When a humanized antibody contains a constant region, said constant region is derived from human immunoglobulins.

[0086] As used herein, the terms "binding" or "specific binding" refer to the selective binding of an antibody to an antigenic epitope that is distinguishable from unwanted or nonspecific interactions. The binding ability or binding specificity of an antibody to a specific antigenic epitope can be determined by conventional binding assays known in the art, including but not limited to, detecting antibody-antigen binding by ELISA assays, detecting antibody binding to cells expressing antigens by FACS assays, or characterizing the binding affinity constant KD by surface plasmon resonance (SPR) or thin-layer interferometry (BLI) techniques.

[0087] As used herein, the term "affinity" or "binding affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigenic epitope). "Binding affinity" reflects the intrinsic binding affinity of a 1:1 interaction between members of a binding pair and is typically expressed using the binding dissociation equilibrium constant (K0). D) To characterize.

[0088] The term "K" D "(M)" in this paper refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Affinity and K D The values ​​are inversely correlated; that is, the higher the affinity, the higher the K value. D The smaller the value, the lower the affinity; conversely, the larger the value, the lower the affinity. D The higher the value, the better. Generally, K... D The value depends on the dissociation rate constant (K) between the interacting antibody-antigen pairs. d or K dis ,sec -1 ) and binding rate constant (K a M -1 x sec -1 An antibody that has binding specificity to a given antigen has an affinity K for that antigen. D The value is usually less than 1x10- 5 M, and preferably less than 1x10 -7 M.

[0089] The invention will now be described in detail in terms of various aspects.

[0090] I. Anti-B7-H3 antibody

[0091] The first aspect of this disclosure provides an anti-B7-H3 antibody or an antigen-binding fragment thereof. The anti-B7-H3 antibody according to the invention comprises a VHH domain that specifically binds to B7-H3 (also referred to herein as the anti-B7-H3 VHH domain).

[0092] The terms "VHH domain" and "VHH fragment" or "VHH region" are used interchangeably. It refers to a variable region fragment of a heavy chain antibody that lacks a light chain. It typically contains four conserved frame regions (FRs) and three complementarity-determining regions (CDRs), arranged from the N-terminus to the C-terminus in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The VHH domain does not require pairing with a light chain variable domain to recognize and bind to the antigen.

[0093] In the VHH domain, the CDR (complementarity-determining region) is the primary region responsible for antigen binding. It typically refers to a highly variable region within the variable chain that forms a structurally defined loop (“hypervariant loop”) and / or contains antigen contact residues (“antigen contact sites”). The CDRs in the VHH domain or the heavy chain variable region of a heavy chain antibody are sequentially numbered starting from the N-terminus and are commonly referred to as CDR1, CDR2, and CDR3. For any given VHH domain, as is known in the art, various known schemes can be used to determine its CDR sequence. The following are examples of CDR region ranges defined using the Kabat, AbM, Contact, and IMGT schemes.

[0094] Furthermore, it is known in the art that although CDRs differ between antibodies, only a limited number of amino acid sites within a CDR directly participate in antigen binding. Minimal overlapping regions can be determined using at least two of the Kabat, IMGT, AbM, and Contact methods, thus providing a “minimum binding unit” for antigen binding. Such a minimum binding unit can be a sub-region of a CDR. The remaining residues of the CDR sequence, as will be apparent to those skilled in the art, can be determined through the antibody’s structure and protein folding.

[0095] Based on the defined CDR sequence, different framework regions can be selected to construct multiple VHH domains that bind B7-H3 with equivalent effectiveness. The framework regions may have the same amino acid sequence as exemplary heavy chain antibody VHH domains with known binding functions, but may also have different amino acid sequences, preferably with at least 80%, 85%, or 90% sequence identity, or more preferably more than 95%, 96%, 97%, 98%, or 99% sequence identity.

[0096] Furthermore, the VHH domain of exemplary antibodies can be engineered to obtain other VHH domain variants with equivalent binding efficacy to B7-H3. For example, modifications can be made to one or more CDR region residues and / or one or more framework region residues, particularly by substitution of conserved residues; or the VHH domain can be modified via CDR transplantation to construct variants that mimic the binding properties of known VHH domains. Following mutation or engineering modifications, the properties of the variants, such as target antigen binding properties or other desired functional properties, such as binding affinity, species cross-reactivity, and / or endocytic activity, can be examined, as appropriate, using methods known in the art and described herein, to identify functional variants with the desired properties.

[0097] In some embodiments, this disclosure therefore provides anti-B7-H3 antibodies or antigen-binding fragments thereof comprising an anti-B7-H3 VHH domain. In some embodiments, the anti-B7-H3 VHH domain according to the invention comprises three complementarity-determining regions (CDRs) selected from the heavy chain variable regions of SEQ ID NOs:3-5. The CDRs can be determined according to any CDR definition scheme known in the art, for example, according to AbM, Kabat, IMGT, Contact, or any combination thereof. In some embodiments, preferably, the CDRs are defined according to the Kabat scheme.

[0098] In some embodiments, the anti-B7-H3 VHH domain according to the present invention comprises CDR1, CDR2, and CDR3 sequences, wherein:

[0099] -CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:6;

[0100] -CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:7; and

[0101] -CDR3 contains or consists of the amino acid sequence shown in SEQ ID NO:8.

[0102] In some embodiments, the anti-B7-H3 VHH domain according to the present invention comprises CDR1, CDR2, and CDR3 sequences, wherein:

[0103] -CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:22;

[0104] -CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:7; and

[0105] -CDR3 contains or consists of the amino acid sequence shown in SEQ ID NO:8.

[0106] In some embodiments, the anti-B7-H3 VHH domain according to the present invention comprises CDR1, CDR2, and CDR3 sequences, wherein:

[0107] -CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:23;

[0108] -CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:7; and

[0109] -CDR3 contains or consists of the amino acid sequence shown in SEQ ID NO:8.

[0110] In some embodiments, the anti-B7-H3 VHH domain according to the present invention comprises CDR1, CDR2, and CDR3 sequences, wherein:

[0111] -CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:24;

[0112] -CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:7; and

[0113] -CDR3 contains or consists of the amino acid sequence shown in SEQ ID NO:8.

[0114] In some embodiments, the anti-B7-H3 VHH domain according to the present invention comprises CDR1, CDR2, and CDR3 sequences, wherein:

[0115] -CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:25;

[0116] -CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:7; and

[0117] -CDR3 contains or consists of the amino acid sequence shown in SEQ ID NO:8.

[0118] In some embodiments, the anti-B7-H3 VHH domain according to the present invention comprises CDR1, CDR2, and CDR3 sequences, wherein:

[0119] -CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:26;

[0120] -CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:7; and

[0121] -CDR3 contains or consists of the amino acid sequence shown in SEQ ID NO:8.

[0122] In some embodiments, the anti-B7-H3 VHH domain according to the present invention comprises CDR1, CDR2, and CDR3 sequences, wherein:

[0123] -CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:6;

[0124] -CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:7; and

[0125] -CDR3 contains or consists of the amino acid sequence shown in SEQ ID NO:27.

[0126] In some embodiments, the anti-B7-H3 VHH domain according to the present invention comprises CDR1, CDR2, and CDR3 sequences, wherein:

[0127] -CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:6;

[0128] -CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:7; and

[0129] -CDR3 contains or consists of the amino acid sequence shown in SEQ ID NO:28.

[0130] In some embodiments, the anti-B7-H3 VHH domain according to the present invention comprises CDR1, CDR2, and CDR3 sequences, wherein:

[0131] -CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:6;

[0132] -CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:7; and

[0133] -CDR3 contains or consists of the amino acid sequence shown in SEQ ID NO:29.

[0134] In some embodiments, the anti-B7-H3 VHH domain according to the present invention comprises CDR1, CDR2, and CDR3 sequences, wherein:

[0135] -CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:6;

[0136] -CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:7; and

[0137] -CDR3 contains or consists of the amino acid sequence shown in SEQ ID NO:30.

[0138] In some embodiments, the anti-B7-H3 VHH domain according to the present invention comprises CDR1, CDR2, and CDR3 sequences, wherein:

[0139] -CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:6;

[0140] -CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:7; and

[0141] -CDR3 contains or consists of the amino acid sequence shown in SEQ ID NO:31.

[0142] In some embodiments, the anti-B7-H3 VHH domain according to the present invention comprises CDR1, CDR2, and CDR3 sequences, wherein:

[0143] -CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:6;

[0144] -CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:7; and

[0145] -CDR3 contains or consists of the amino acid sequence shown in SEQ ID NO:32.

[0146] In some embodiments, the B7-H3 VHH domain of the present invention preferably includes the following CDR1, CDR2, and CDR3:

[0147] - Contains SEQ ID NOs:6, 7 and 8 or CDR1, CDR2 and CDR3 composed of them respectively;

[0148] - Containing SEQ ID NOs:22, 7 and 8 or CDR1, CDR2 and CDR3 composed of them respectively;

[0149] - Containing SEQ ID NOs:26, 7 and 8 or CDR1, CDR2 and CDR3 composed of them respectively;

[0150] - Each of the following contains SEQ ID NOs:6, 7 and 27 or CDR1, CDR2 and CDR3 composed of them;

[0151] - Each of the following contains SEQ ID NOs:6, 7 and 28 or CDR1, CDR2 and CDR3 composed of them;

[0152] - Each of the following contains SEQ ID NOs:6, 7 and 29 or CDR1, CDR2 and CDR3 composed thereof;

[0153] - Containing SEQ ID NOs:6, 7, and 31, or CDR1, CDR2, and CDR3 thereof; or

[0154] - Contains SEQ ID NOs:6, 7 and 32 or CDR1, CDR2 and CDR3 composed of them respectively.

[0155] In some embodiments, the anti-B7-H3 VHH domain according to the present invention comprises or is composed of the following amino acid sequence:

[0156] (i) an amino acid sequence selected from SEQ ID NOs:3-5 or 11-21; or

[0157] (ii) an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same amino acid sequence as (i); or

[0158] (iii) An amino acid sequence having at least 1-30 (e.g., 1-20, 1-15, 1-10, or 1-5) amino acid additions, deletions, and / or substitutions relative to the amino acid sequence of (i).

[0159] In some preferred embodiments, the anti-B7-H3 VHH domain according to the present invention comprises or is composed of an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or more of the same identity as SEQ ID NO:4. In other preferred embodiments, the anti-B7-H3 VHH domain according to the present invention comprises or is composed of an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or more of the same identity as SEQ ID NO:5. In other preferred embodiments, the anti-B7-H3 VHH domain according to the present invention comprises or is composed of an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or more of the same identity as SEQ ID NO:11. In other preferred embodiments, the anti-B7-H3 VHH domain according to the present invention comprises or is composed of an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or more of the same identity as SEQ ID NO:15. In some preferred embodiments, the anti-B7-H3 VHH domain according to the invention comprises or is composed of an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or more of the same identity as SEQ ID NO:16. In some preferred embodiments, the anti-B7-H3 VHH domain according to the invention comprises or is composed of an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or more of the same identity as SEQ ID NO:17. In some preferred embodiments, the anti-B7-H3 VHH domain according to the invention comprises or is composed of an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or more of the same identity as SEQ ID NO:18. In some preferred embodiments, the anti-B7-H3 VHH domain according to the invention comprises or is composed of an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or more of the same identity as SEQ ID NO:20. In some other preferred embodiments, the anti-B7-H3 VHH domain according to the invention comprises or is composed of an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or more of the same identity as SEQ ID NO:21.

[0160] In some preferred embodiments, the anti-B7-H3 VHH domain according to the present invention comprises or is composed of the amino acid sequence of SEQ ID NO:4. In other preferred embodiments, the anti-B7-H3 VHH domain according to the present invention comprises or is composed of the amino acid sequence of SEQ ID NO:5. In other preferred embodiments, the anti-B7-H3 VHH domain according to the present invention comprises or is composed of the amino acid sequence of SEQ ID NO:11. In other preferred embodiments, the anti-B7-H3 VHH domain according to the present invention comprises or is composed of the amino acid sequence of SEQ ID NO:15. In other preferred embodiments, the anti-B7-H3 VHH domain according to the present invention comprises or is composed of the amino acid sequence of SEQ ID NO:16. In other preferred embodiments, the anti-B7-H3 VHH domain according to the present invention comprises or is composed of the amino acid sequence of SEQ ID NO:17. In other preferred embodiments, the anti-B7-H3 VHH domain according to the present invention comprises or is composed of the amino acid sequence of SEQ ID NO:18. In some other preferred embodiments, the anti-B7-H3 VHH domain according to the invention comprises or is composed of the amino acid sequence of SEQ ID NO:20. In some other preferred embodiments, the anti-B7-H3 VHH domain according to the invention comprises or is composed of the amino acid sequence of SEQ ID NO:21.

[0161] Depending on the specific application, the anti-B7-H3 antibody of the present invention may include other antibody components in addition to the anti-B7-H3 VHH domain according to the present invention. Examples of other components that may be mentioned include, but are not limited to, immunoglobulin constant regions and antibody fragments selected from: VHH domain, Fab, scFab, disulfide-linked scFab, Fab', F(ab')2, Fab'-SH, Fv, scFv, disulfide-linked scFv, linear antibodies, diabody, triabody, tetrabody, and minibody.

[0162] In some embodiments, the anti-B7-H3 antibody according to the invention further comprises an immunoglobulin constant region. In some embodiments, the anti-B7-H3 antibody according to the invention comprises an immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is directly or via a hinge region connected to the anti-B7-H3 VHH domain according to the invention. In some embodiments, the immunoglobulin Fc region is a human IgG isotype Fc region, such as a human IgG1 or IgG4 isotype Fc region. By including the immunoglobulin Fc constant region, in some cases, the serum half-life of the anti-B7-H3 antibody according to the invention can be increased, effector functions such as ADCC or CDC activity can be conferred, and / or a drug conjugation site can be provided.

[0163] In some embodiments, the anti-B7-H3 antibody according to the present invention further comprises one or more antibody fragments with different antigen-binding specificities. In some embodiments, the antibody fragments are selected from the VHH domain, Fab, and scFv.

[0164] The anti-B7-H3 antibody according to the present invention can be any suitable antibody structure capable of binding to antigen B7-H3, including but not limited to heavy chain antibodies, VHH antibodies, nanobodies, single-domain antibodies, monospecific antibodies, bispecific antibodies, multispecific antibodies, monovalent or multivalent antibodies, murine antibodies, chimeric antibodies or humanized antibodies.

[0165] In some embodiments, the anti-B7-H3 antibody according to the invention is a heavy chain antibody comprising the anti-B7-H3 VHH domain according to the invention. In some embodiments, the heavy chain antibody further comprises the Fc region derived from human IgG immunoglobulin.

[0166] In some embodiments, the anti-B7-H3 antibody according to the invention is a VHH antibody composed of the anti-B7-H3 VHH domain according to the invention.

[0167] In some embodiments, the anti-B7-H3 antibody according to the invention is a nanobody. In some embodiments, the nanobody comprises an anti-B7-H3 VHH domain according to the invention. In other embodiments, the nanobody consists of or is substantially composed of the anti-B7-H3 VHH domain according to the invention. In some cases, if desired, the half-life of the nanobody according to the invention can be extended, for example by chemical modification with polyethylene glycol (PEG), by fusing the nanobody to serum albumin nanoparticles, or by binding it to serum albumin.

[0168] In some embodiments, the anti-B7-H3 antibody according to the present invention is a single-domain antibody containing the anti-B7-H3 VHH domain according to the present invention.

[0169] In some embodiments, the anti-B7-H3 antibody according to the invention is a monovalent or multivalent antibody. As used herein, the antibody-related term "valence" or "valence number" refers to the total number of antigen-binding sites in the antibody molecule, or the number of antigen-binding sites having the same antigen-binding specificity. For example, a bivalent antibody means that the antibody molecule contains a total of 2 antigen-binding sites; the antibody molecule may be monospecific and thus have 2 identical antigen-binding sites; or the antibody molecule may be bispecific and thus have 2 distinct antigen-binding sites.

[0170] In some embodiments, the anti-B7-H3 antibody of the present invention is a monospecific antibody. In other embodiments, the anti-B7-H3 antibody of the present invention is a multispecific antibody. The term "monospecific" means that the antibody has one or more antigen-binding sites, each of which binds to the same epitope of the same antigen. The term "multispecific" means that the antibody has at least two antigen-binding sites that bind to different epitopes (different epitopes on the same antigen or different epitopes on different antigens).

[0171] In some embodiments, the anti-B7-H3 antibody of the present invention is a monospecific monovalent antibody. In some embodiments, the anti-B7-H3 antibody of the present invention is a monospecific multivalent antibody, such as a bivalent, trivalent, or quadrivalent antibody.

[0172] In some embodiments, the anti-B7-H3 antibody of the present invention is a bispecific antibody, such as a bispecific bivalent, trivalent, or quadrivalent antibody. In other embodiments, the anti-B7-H3 antibody of the present invention is a multispecific antibody, such as a trispecific bivalent, trivalent, or quadrivalent antibody. Other antigen-binding specificities that can be combined with the anti-B7-H3 VHH domain according to the present invention include, but are not limited to, antigen-binding domains targeting other tumor-associated antigens, or antigen-binding domains targeting immune cell membrane surface antigens (e.g., PD-L1 and CD3). See, for example, Life (Basel). 2022 Feb; 12(2):157, "ANovel Anti-B7-H3×Anti-CD3 Bispecific Antibody with Potent Antitumor Activity", doi:10.3390 / life12020157; Li, Hy. et al., Bispecific antibody targeting both B7-H3 and PD-L1 exhibits superior antitumor activities. Acta Pharmacol Sin 44, 2322–2330(2023). https: / / doi.org / 10.1038 / s41401-023-01118-2; Zachary T. Rosenkrans et al., Targeting both GD2 and B7-H3 using bispecific antibody improves tumor selectivity for GD2-positive tumors, doi:10.1101 / 2024.05.23.595624.

[0173] In some embodiments, the anti-B7-H3 antibody of the present invention is a chimeric antibody. In some specific embodiments, the anti-B7-H3 chimeric antibody of the present invention comprises the anti-B7-H3 VHH domain according to the present invention and comprises one or more constant regions derived from humans. In other specific embodiments, the anti-B7-H3 chimeric antibody of the present invention comprises the B7-H3 VHH domain according to the present invention and comprises variable regions with different antigen binding specificities (e.g., VH / VL pairs targeting different antigens).

[0174] In some embodiments, the anti-B7-H3 antibody of the present invention is a humanized antibody. In some cases, the anti-B7-H3 humanized antibody according to the present invention has a framework region sequence “derived” from a specific human ancestral sequence, and thereby has at least 90%, more preferably at least 95%, even more preferably at least 96%, 97%, 98% or 99% identity with said human ancestral framework region sequence, and retains antigen-binding activity.

[0175] Properties of the antibodies of this invention

[0176] The anti-B7-H3 antibody according to the invention is isolated. "Isolated" antibody refers to artificial antibodies, recombinant antibodies, and antibodies that have been at least partially separated from components in the natural environment in which they originated. In some embodiments, the isolated antibody according to the invention has a purity of more than 90%, 95%, or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0177] The anti-B7-H3 antibody according to the present invention specifically binds to B7-H3. In some embodiments, the antibody of the present invention, which specifically binds to B7-H3, is also capable of binding to human B7-H3. In some embodiments, the antibody according to the present invention is capable of binding to human 4IgB7-H3 protein. In some embodiments, the antibody according to the present invention is capable of binding to the extracellular domain of human B7-H3 protein. The extracellular domain of human 4IgB7-H3 protein comprises, from the N-terminus to the C-terminus, the following domains: IgV-IgC-IgV-IgC, wherein the first IgV is also referred to as IgV1 in this disclosure, and the second IgV is referred to as IgV2; similarly, the first IgC is referred to as IgC1, and the second IgC is referred to as IgC2. In some embodiments, the antibodies of the present invention, capable of binding to the extracellular domain of human B7-H3 protein, can bind to the IgV1-IgC1 segment, but essentially cannot bind to the isolated IgV1 or IgV2 segment. The binding affinity constant K between the antibody according to the present invention and human B7-H3 can be determined using biofilm layer optical interferometry (BLI) as described in Embodiments 3 or 9 of this disclosure. D The binding specificity and binding activity of the antibody according to the present invention to different segments of the human B7-H3 extracellular domain epitopes can be characterized by ELISA assay, as described in Example 5 of this disclosure.

[0178] In some embodiments, the antibodies of the present invention that specifically bind to B7-H3 exhibit immune cross-reactivity with monkey B7-H3. In this document, the terms "immune cross-reactivity" or "cross-reactivity" are used interchangeably, meaning that an antibody that specifically binds to a specific antigen from one species can also specifically recognize and bind to that antigen from other species. Therefore, the anti-human B7-H3 antibody of the present invention that exhibits immune cross-reactivity with monkey B7-H3 not only specifically recognizes and binds to human B7-H3 but can also specifically recognize and bind to B7-H3 from monkeys. The presence of human-monkey species cross-reactivity in the antibody is advantageous, as this property can facilitate preclinical drug development of the antibody, such as toxicological assays of antigen-binding molecules composed of the antibody. Methods for determining cross-reactivity include the method described in Example 4 and standard assays known in the art, such as flow cytometry or cell ELISA assays.

[0179] In some embodiments, the antibody of the present invention, which specifically binds to B7-H3, is capable of binding to B7H3 expressed on the cell surface. B7-H3 is a transmembrane protein composed of an extracellular domain, a transmembrane domain, and a cytoplasmic domain. The expression level of B7-H3 on the cell surface can be determined by any conventional method known in the art for determining the expression level of cell surface antigens, such as FACS detection methods or immunofluorescence staining methods. It has been demonstrated that B7-H3 has significantly higher expression levels on a variety of tumor cells than on normal tissues / cells, including but not limited to human breast cancer cells, human colon adenocarcinoma cells, human colon cancer cells, and human lung adenocarcinoma cells. Such tumor cells that are positive for B7-H3 expression on their cell surface are also referred to herein as "B7-H3-positive tumor cells". In some embodiments, the antibody of the present invention, which specifically binds to B7-H3, is capable of binding not only to isolated B7-H3 protein but also exhibits the property of binding to B7-H3-positive tumor cells. The binding activity of the antibody according to the invention with B7-H3 positive tumor cells can be detected by the FACS assay described in Examples 6 and 10 of this disclosure or by standard assays known in the art.

[0180] In some embodiments, the antibodies of the present invention that specifically bind to B7-H3 possess B7-H3-mediated endocytic activity. The terms "endocytosis" and "internalization" are used interchangeably; in this disclosure, it refers to the process by which the antibody / B7-H3 complex is internalized and delivered into the cytoplasm, triggered by the binding of the antibody to B7-H3 on the cell surface. As an example method for detecting internalization activity, one could employ... Fabfluor-pH antibody labeling reagent. Antibody labeling reagents allow for the rapid and easy labeling of test antibodies containing human IgG Fc using a pH-sensitive fluorophore conjugated to the Fab fragment. Subsequently, this pH-sensitive dye-based system allows for the quantification of antibody internalization using the acidic environment of the lysosome. Specifically, when Fabfluor-labeled antibodies present in neutral extracellular solution (e.g., approximately pH 7.2–7.4) are internalized through interaction with cell surface-specific antigens, they enter the acidic environment of the lysosome (pH 4.5–5.5), resulting in a significant increase in fluorescence. Without internalization, the fluorescence intensity of the labeled antibody remains low. Integrated analysis software minimizes background fluorescence, allowing for real-time, kinetic assessment of antibody internalization. In some implementations, this is achieved through methods such as those described in the examples. The endocytosis rate was determined using the Fabfluor-pH antibody labeling method, and the anti-B7-H3 antibody according to the present invention exhibits endocytic activity on B7-H3 positive tumor cells. In some embodiments, the B7-H3 tumor cells are selected from human breast cancer cells, human colon adenocarcinoma cells, human colon cancer cells, and human lung adenocarcinoma cells. The anti-B7-H3 antibody of the present invention, exhibiting endocytic activity, can be used as a tool for delivering antitumor drugs into cancer cells in the ADC of the present invention.

[0181] In some preferred embodiments, the anti-B7-H3 antibody according to the present invention has one or more of the following properties:

[0182] (a) Binds to human B7-H3, optionally, with affinity K D Value less than 1x10 -9 M, preferably about 0.1-10x10 -10 M; The affinity determination is preferably performed according to the BLI determination method of Example 3 or 9;

[0183] (b) Binding to tumor cells expressing human B7-H3, preferably by FACS detection, with a cell binding EC50 value between 0.1 and 1 nM; the cell binding activity is preferably determined according to the FACS assay of Example 6 or 10;

[0184] (c) It exhibits immune cross-reactivity with monkey B7-H3; and

[0185] (d) It has B7-H3-mediated endocytosis activity.

[0186] In some further preferred embodiments, the antibody according to the invention is a nanobody and has the high solubility and stability imparted by VHH, as well as excellent tissue penetration.

[0187] II. B7-H3 binding molecules

[0188] In a second aspect, the present invention provides a B7-H3 antigen-binding molecule based on an anti-B7-H3 antibody according to the first aspect of the present invention. As used herein, the term "antigen-binding molecule" refers to a molecule, such as a protein or polypeptide or a molecule derived therefrom, that contains an antigen-binding domain or antigen-binding site capable of binding to a target antigen. Thus, examples of antigen-binding molecules include, but are not limited to, antibodies and their antigen-binding fragments, as well as various fusions and conjugates constructed based on antibodies or antigen-binding fragments, such as immunoconjugates, immunofusions, antibody-drug conjugates (ADCs), multi / bispecific antibodies, chimeric antigen receptors (CARs), etc.

[0189] As will be apparent to those skilled in the art, the antigen-binding site of an antibody typically contains amino acid residues from a "complementarity-determining region" or "CDR". Accordingly, various antigen-binding molecules constructed by including the antigen-binding site of an anti-B7-H3 antibody or its antigen-binding fragment according to this disclosure (especially the B7-H3 VHH domain according to the invention) may be referred to herein as B3-H3 binding molecules and are considered in this invention. Similarly, the use of the anti-B3-H3 antibody or its antigen-binding fragment according to this disclosure (especially the B7-H3 VHH domain according to the invention) for the preparation of such antigen-binding molecules is also a part of this invention.

[0190] In some embodiments, the present invention provides immunofusions or immunoconjugates produced by fusing or conjugating the present invention's anti-B7-H3 antibody or its antigen-binding fragment to a heterologous molecule.

[0191] The term "heterogeneous" in the context of fusions or conjugates refers to a fusion or conjugate containing at least two sub-parts that do not coexist in the same molecule in nature. As an example, a polypeptide containing an anti-B7-H3 VHH domain can be directly or covalently linked to a heterologous polypeptide via a linker to form a fusion; or it can be directly or covalently linked to a heterologous small chemical molecule via a linker to form a conjugate.

[0192] In one embodiment, the present invention provides an immunofusion comprising the anti-B7-H3 antibody (or its antigen-binding fragment) of the present invention. In the immunofusion according to the invention, the antibody (or its antigen-binding fragment) may be linked directly to a heterologous peptide or polypeptide, or via an amino acid linker. Heterologous peptides or polypeptides that may be mentioned include, but are not limited to, proteins or polypeptides that impart another functional activity to the fusion, or tag peptides that facilitate the purification or detection of the immunofusion. Amino acid linkers that may be used for such linkages are typically short amino acid sequences consisting of amino acids, such as glycine (G) and / or serine (S) and / or threonine residues (T) used alone or in combination, or hinge regions derived from immunoglobulins. In the case of fused polypeptides, in some embodiments, the application of a linker will help ensure that the heterologous peptide / polypeptide moiety linked thereto continues to perform its intended function after fusion.

[0193] In some further embodiments, the immunofusion compound according to the invention may contain antigen-binding domains other than the B7-H3VHH domain according to the invention. Typically, the antibody forms an antigen-binding domain on the surface of the VH-VL dimer via three complementarity-determining regions (HCDR1-3) in its heavy chain variable region (VH) and three complementarity-determining regions (LCDR1-3) in its light chain variable region (VL), wherein the six CDRs confer specific binding of the antibody to the antigen. In the case of heavy chain antibodies, such as those derived from camelid heavy chain antibodies, the antibody confers specific binding to the antigen via three complementarity-determining regions (CDR1-3) in a single VH domain (i.e., the VHH domain). VH / VL pairs from conventional antibodies, as well as the VHH domain from heavy chain antibodies, can both serve as sources of the other antigen-binding domains in the B7-H3 binding molecule of the invention. Furthermore, the region where the receptor binds to its associated ligand can also serve as a source of the other antigen-binding domains in the B7-H3 binding molecule of the invention. In some embodiments, the immunofusion compound according to the invention is a multi(bi)specific antibody comprising one or more VHH domains according to the invention and one or more other antigen-binding domains.

[0194] In some further embodiments, the immune fusion product according to the invention is a chimeric antigen receptor (CAR) targeting B7-H3. In this regard, immune cells incorporating this chimeric antigen receptor of the invention, including but not limited to T cells, NK cells, and macrophages, are also within the scope of the invention.

[0195] In other embodiments, the present invention provides immunoconjugates comprising the anti-B7-H3 antibody of the present invention (or an antigen-binding fragment thereof). Examples of heterologous molecules that may be conjugated to the antibodies of the present invention include, but are not limited to, markers, pharmaceuticals, and cytotoxic agents, such as: radioisotopes; chemotherapeutic agents; growth inhibitors; enzymes and fragments thereof; fluorescent reporter proteins; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof; and various known antitumor or anticancer agents.

[0196] In one embodiment, the anti-B7-H3 antibody (or its antigen-binding fragment) of the present invention is conjugated with a therapeutic agent, diagnostic agent, or detectable agent.

[0197] In embodiments conjugated with diagnostic or detectable agents, such conjugates can be used as part of clinical testing methods (e.g., to determine the efficacy of a particular therapy) to monitor or predict the onset, formation, progression, and / or severity of B7-H3-related diseases or conditions (especially B7-H3-positive tumors). Such diagnostics and detections can be achieved by conjugating antibodies with detectable agents, including but not limited to a variety of enzymes such as horseradish peroxidase; prosthetic groups such as streptavidin / biotin and avidin / biotin; fluorescent substances; luminescent substances; radioactive substances; and positron-emitting metal and non-radioactive paramagnetic metal ions used in various positron emission tomography (PET) imaging techniques.

[0198] In embodiments conjugated with a therapeutic agent, a suitable therapeutic agent may be selected based on the therapeutic objective. In embodiments of conjugates for the treatment of B7-H3 positive tumors, the therapeutic agent may be selected from cytotoxic agents (e.g., cell growth inhibitors or cell killers), antitumor drugs, chemotherapeutic agents, or radioisotopes.

[0199] In some aspects, the anti-B7-H3 antibody of the present invention is particularly suitable as a component for preparing antibody-drug conjugates due to its specific high-level binding to a variety of B7-H3-positive tumor cells and its B7-H3-mediated endocytic activity. Therefore, in some embodiments, the present invention provides an immunoconjugate according to the invention, which is an antibody-drug conjugate (ADC) comprising the anti-B7-H3 antibody of the present invention.

[0200] The terms "antibody-drug conjugate" and "ADC" are used interchangeably in this disclosure, referring to an immunoconjugate formed by covalently conjugating an antibody or antibody fragment to a payload (i.e., a therapeutically active substance or active pharmaceutical ingredient), thereby enabling the therapeutically active substance or active pharmaceutical ingredient to target the antibody's binding target to exhibit its pharmacological function. In some embodiments of the ADC according to the invention, the covalent linking of the payload to the antibody or antibody fragment according to the invention can be performed in a non-site-specific manner or in a site-specific manner. In some embodiments of the ADC according to the invention, the payload is covalently linked to the antibody or antibody fragment according to the invention via a linker. A "linker," as a chemical module in the ADC that covalently links the antibody to the payload, typically has a bifunctional portion that links the drug to the antibody. Typically, such a linker has multiple chemical structural portions, including, for example, a linker group responsible for antibody conjugation; a degradable portion; a linker group responsible for drug conjugation; and optionally a hydrophilicity modulating module, such as a PEG segment. In some embodiments of the ADC according to the invention, the linker is preferably "degradable" or "cleavable," thereby enabling it to break and release the payload after the ADC is delivered to the target region (e.g., the target tumor tissue site). Such "cleavable linkers" available include, for example, acid-instable linkers, peptidase-sensitive linkers, photostable linkers, dimethyl linkers, or disulfide-containing linkers.

[0201] In this field, various cytotoxic agents with different mechanisms suitable as payloads have been reported, including, but not limited to,

[0202] (1) Microtubule inhibitors / disruptors: for example, but not limited to, auristatin class (e.g., MMAE or MMAF), maytansin derivatives (e.g., DM2, DM4), tubulosynins, cryptomycins, antimitotic EG5 inhibitors (e.g., spindle kinesin KSP inhibitors).

[0203] (2) DNA damaging agents: for example, but not limited to, pyrrolobenzodiazepines (e.g., pyrrolo[2,1-c][1,4]benzodiazepine (PBD)), ducarmycins, indolinobenzodiazepines; Duocarmycins; Calicheamicins;

[0204] (3) Topoisomerase inhibitors: for example, but not limited to, camptothecins (e.g., ixotecan and its derivative Dxd);

[0205] (4) Others: apoptosis inducers (Bcl-xL inhibitors), thailanstatin and its analogues, amatoxins, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, carbamycin.

[0206] In some embodiments of the ADC according to the present invention, the payload may be any compound selected from the above, such as microtubule inhibitors (e.g., monomethyl auristatin E (MMAE)) or topoisomerase I inhibitors (e.g., camptothecin compounds).

[0207] In some embodiments of the ADC according to the present invention, the ADC may have a DAR (drug:antibody ratio) of approximately 1-20. The term "drug:antibody ratio" or "DAR" refers to the ratio of the drug portion (D) coupled to the antibody portion to the antibody portion in the ADC conjugate. In this disclosure, DAR may refer to the ratio of the antibody portion to the drug portion in a single ADC molecule, and for example, in some embodiments of the ADC according to the present invention, the DAR may be 1 to 20, such as 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or any integer between 1 and 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or a range consisting of any two values ​​between 1 and 10. In this disclosure, DAR may also refer to the average DAR of the ADC molecular population in the product, i.e., the overall ratio of the drug moiety (D) coupled to the antibody moiety in the product to the antibody moiety, as determined by detection methods (e.g., conventional methods such as mass spectrometry, ELISA, electrophoresis, and / or HPLC). In some embodiments of the ADC according to the invention, the ADC has an average DAR value of 1 to 20, such as 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, 6-8, or 7-8, such as 1.0-8.0, 2.0-6.0, such as 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,

[0208] 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6. 8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, and ranges with two of these values ​​as endpoints.

[0209] In some embodiments, this disclosure provides immunoconjugates, particularly antibody-drug conjugates, wherein the B7H3 antibody or its antigen-binding fragment according to the invention is conjugated to a radioactive isotope, an antitumor drug, a chemotherapeutic agent, or a cytotoxin via a linker. In some embodiments, the B7H3 antibody or its antigen-binding fragment according to the invention is conjugated to a cytotoxin via a linker. In some embodiments, the cytotoxin is a topoisomerase I inhibitor. In some preferred embodiments, the cytotoxin is a camptothecin compound, particularly eczemab, Dxd, or a derivative thereof. In some embodiments, the linker is an enzymatically cleavable linker. In some preferred embodiments, the linker comprises a GGFG moiety (i.e., Gly-Gly-Phe-Gly).

[0210] In some preferred embodiments, this disclosure provides antibody-drug conjugates comprising a camptothecin compound conjugated to a B7H3 antibody or an antigen-binding fragment thereof according to the invention. In some embodiments, the antibody-drug conjugate (ADC) comprises a GGFG-DXD moiety conjugated to the antibody or an antigen-binding fragment thereof, and preferably the ADC has an average DAR of about 1-20, for example about 1-8, 1-6, 2-5, or 3-4, or about 3.8. In some preferred embodiments, the GGFG-DXD moiety has the following structure:

[0211] Where A represents the antibody linked to the GGFG-DXD portion.

[0212] III. Polynucleotide, Vector, Host, and Antibody Production Methods

[0213] In a third aspect, this disclosure provides a nucleic acid encoding an antibody or a fragment thereof according to the first aspect of the present invention, a vector containing the nucleic acid, a host cell containing the nucleic acid or the vector, and a method for preparing the same.

[0214] In some embodiments, this disclosure provides one or more polynucleotides encoding an anti-B7-H3 antibody or a fragment thereof according to the invention. As those skilled in the art will appreciate, due to codon degeneracy, the amino acid sequence of each antibody or polypeptide chain can be encoded by multiple nucleic acid sequences. In some embodiments, this disclosure provides one or more vectors comprising the one or more polynucleotides. In some embodiments, the one or more polynucleotides are present in a single vector; in other embodiments, the one or more polynucleotides are present in multiple separate vectors. In some embodiments, the invention also provides host cells comprising the one or more polynucleotides or the one or more vectors. Depending on the circumstances, a single or multiple vectors comprising the one or more polynucleotides may be introduced into the same host cell to express the desired product in one host cell; or a single or multiple vectors comprising the one or more polynucleotides may be introduced separately into different host cells to express, respectively, intermediates (e.g., haptens) comprising different chains or combinations of different chains of antibody in different host cells, and, under conditions suitable for assembling a complete antibody, to produce an antibody according to the invention by mixing the intermediates.

[0215] In this disclosure, there are no particular limitations on the vectors that can be used, including cloning vectors and expression vectors. An example of a suitable expression vector is a eukaryotic expression vector, including but not limited to viruses, plasmids, granules, λ phages, or yeast artificial chromosomes (YAC). In this disclosure, there are no particular limitations on the host cells that can be used. Suitable host cells include prokaryotic microorganisms such as *Escherichia coli*, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as mammalian host cells, insect cells, etc. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7), human embryonic kidney (HEK293 or 293F cells), young hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), CHO cells, NSO cells, and myeloma cell lines such as YO, NSO, P3X63, and Sp2 / 0. In some embodiments, mammalian cell lines suitable for suspension culture may be used. In some embodiments, the host cell is CHO or HEK293 cells.

[0216] In some embodiments, the present invention provides a method for producing the anti-B7-H3 antibody of the present invention or a fragment thereof. To produce the antibody of the present invention, the polypeptide chain constituting the antibody of the present invention can be obtained, for example, by solid-state peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production, and assembled under suitable conditions. In the case of recombinant production, the assembly can occur in a host cell for expressing the polypeptide chain; or, if necessary, the assembly of the antibody of the present invention can be performed in vitro after harvesting the expressed intermediate polypeptide chain from the host cell.

[0217] The antibodies prepared by the method described herein can be purified using known existing techniques such as high-performance liquid chromatography, ion-exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. After purification, the purity of the antibody protein of this invention can be determined by any of a variety of well-known analytical methods, such as SEC-HPLC.

[0218] The physical / chemical properties and / or biological activity of the antibody molecules provided herein can be identified, screened, or characterized using a variety of assays known in the art.

[0219] IV. Pharmaceutical compositions and pharmaceutical preparations

[0220] In a fourth aspect, this disclosure provides compositions comprising an antibody according to the first aspect of the invention or a B7-H3 binding molecule (e.g., an immunoconjugate / fusion) according to the second aspect. In some embodiments, the compositions are pharmaceutical compositions.

[0221] The term "pharmaceutical composition" refers to a composition that is present in a form that allows for the effective biological activity of the active ingredient contained therein, and that does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition. Pharmaceutical compositions may optionally contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art. For a review of pharmaceutical excipients, see "Handbook of Pharmaceutical Excipients," 5th edition, R.C. Rowe, P.J. Seskey, and S.O. Wen, Pharmaceutical Press, London, Chicago.

[0222] In some embodiments, this disclosure relates to preparing a pharmaceutical formulation comprising the invention by mixing an antibody, immunoconjugate, or immunofusion having the desired purity of the present invention with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)).

[0223] In the pharmaceutical compositions and formulations of the present invention, the antibody or its antigen-binding fragment of the first aspect of the present invention, or the antigen-binding molecule of the second aspect of the present invention (e.g., an immunoconjugate or immunofusion compound), may be the sole active agent or may be combined with other active agents. Other active agents that may be combined with the antibodies of the present invention include, but are not limited to, therapeutic agents (e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, antitumor drugs, etc.) that have beneficial therapeutic effects on the disease and / or condition to be treated. The antibodies of the present invention and the other active agents may be combined in the pharmaceutical compositions and formulations in an amount effective for the intended use and in a manner suitable for their respective effects.

[0224] In some embodiments, this disclosure also provides a combination product comprising an antibody or antigen-binding fragment thereof from the first aspect of the invention, or an antigen-binding molecule (e.g., an immunoconjugate or immunofusion compound) from the second aspect of the invention, and one or more other active agents (e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, antitumor drugs, etc.). The components constituting the combination product, such as the antibody of the present invention and other active agents, can be formulated separately in different formulations and preferably contained in different containers. Depending on the disease to be treated and individual conditions, those skilled in the art can determine the administration method and order of administration of the components of the combination product. The combination product of the present invention can be used in treatment methods according to the present invention. In some embodiments, the combination product is used for the prevention or treatment of B7-H3 positive tumors.

[0225] IX. Methods and Uses

[0226] In five aspects, this disclosure provides methods and uses for applying antibodies or antigen-binding fragments thereof from the first aspect of the invention, or antigen-binding molecules (e.g., immunoconjugates or immunofusions) from the second aspect of the invention.

[0227] In some embodiments, the methods and uses according to the invention relate to the treatment of a disease in an individual subject. In other embodiments, the methods and uses according to the invention relate to the detection of the presence of B3-H7 in a sample, for example, from a subject. In still other embodiments, the methods and uses according to the invention relate to use in the preparation of products (e.g., pharmaceutical compositions or pharmaceutical products or combination products or diagnostic products) for said treatment or diagnosis.

[0228] In some embodiments, this disclosure provides a method for treating or preventing cancer, comprising administering to an individual in need an effective amount of an antibody of the first aspect of the invention or an antigen-binding fragment thereof, or an antigen-binding molecule of the second aspect of the invention (e.g., an immunoconjugate or immunofusion).

[0229] The term "treatment" refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of existing symptoms, conditions, ailments, or diseases. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of this invention are used to delay the development of B3-H7 positive tumors.

[0230] In this document, the term "prevention" refers to a medical intervention implemented before the onset of at least one symptom of a disease to suppress, delay, or prevent the occurrence or development of the disease or specific disease symptoms. Therefore, in some embodiments, the prevention method according to the invention includes administering a drug to a subject before the onset of the disease or symptoms.

[0231] The term "effective amount" refers to the quantity or dose of the antibody, immunofusion, or immunoconjugate of the present invention, which, when administered to a patient in a single or multiple doses, produces the intended effect in a patient requiring treatment or prevention. The effective amount can be readily determined by a physician skilled in the art by considering a variety of factors, such as: the species of the mammal; weight, age, and general health status; the specific disease involved; the degree or severity of the disease; the individual patient's response; the specific antibody administered; the administration pattern; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapies. In some cases, the "effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%, and still more preferably at least about 80% or 90%, relative to an untreated subject. The ability of a compound to inhibit measurable parameters (e.g., cancer) can be evaluated in animal model systems that predict efficacy in human tumors.

[0232] In this document, the terms "cancer" and "tumor" are used interchangeably to refer to or describe a physiological disorder in mammals that is typically characterized by unregulated cell growth. Examples of cancers suitable for treatment according to the methods of the present invention include, but are not limited to, carcinomas, solid tumors, and liquid tumors.

[0233] In some embodiments, cancers suitable for treatment by the antibodies or immune conjugates or immune fusions of the present invention include B7-H3 positive tumors / cancers, including their primary and metastatic forms.

[0234] In some embodiments, the cancer is a B7-H3 positive solid tumor or a hematologic malignancy. In some embodiments, the cancer is selected from: non-small cell lung cancer, colorectal cancer, breast cancer, pancreatic cancer, gastric cancer, ovarian cancer, and glioma.

[0235] In another aspect, the present invention provides methods and kits for detecting B7-H3 in a sample, wherein the method comprises: (a) contacting the sample with an antibody of the present invention or an antigen-binding fragment or immunoconjugate thereof; and (b) detecting the formation of a complex between the antibody or an antigen-binding fragment or immunoconjugate thereof and the B7-H3 protein. In some embodiments, the sample is derived from a cancer patient. The detection may be in vitro or in vivo. In some embodiments, the detection is a quantitative or qualitative detection. Exemplary methods for the detection may include immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads with antibody molecules, ELISA assays, PCR techniques (e.g., RT-PCR), etc. In some embodiments, the biological sample is blood, serum, or other liquid samples of biological origin. In some embodiments, the biological sample comprises cells or tissues. In some embodiments, the biological sample is derived from hyperplastic or cancerous lesions.

[0236] Any or all of the features described above and throughout this application may be combined in various embodiments of the invention. The following examples further illustrate the invention; however, it should be understood that the examples are for illustrative purposes only and should not be construed as constituting any limitation. Example

[0237] Example 1: Construction of a camel nanobody phage library

[0238] Camels were immunized with B7H3 recombinant antigen (SEQ ID NO.:10), peripheral blood mononuclear cells (PBMCs) were isolated and total RNA was extracted for reverse transcription. The reverse transcription product was used as a template to amplify the variable domain of the heavy-chain of heavy chain antibody (VHH) and ligated into a phage display vector. The VHH was then electroporated into E. coli TG1 competent cells to construct a camel immune library.

[0239] Specifically, camels were immunized every two weeks for a total of four times. Each injection consisted of 0.8 mg of recombinant B7H3 extracellular protein (NCBI: Q5ZPR3-1), supplemented with Freund's complete / incomplete adjuvant (Sigma, F5881, F5506), administered via subcutaneous multi-site injection. Two weeks after each immunization, 1 mL of blood was collected to separate serum. Using the immunogen as the assay antigen, the titers of total antibodies (IgG) and heavy chain antibodies (HcAb) in the serum were measured by ELISA. Once the serum titers met the requirements for library construction, 100 mL of camel peripheral blood was collected, and PBMCs were isolated using a separation kit (Tianjin Haoyang, Cat: TBD2011CM). Total RNA was extracted from the PBMCs, and cDNA was obtained by reverse inversion, serving as a template for subsequent amplification of the VHH fragment. Based on the camel-derived VHH antibody genes retrieved from relevant literature and databases, primers for VHH antibody library construction were designed and synthesized, and the variable region gene sequence of the antibody was amplified by PCR. Subsequently, the vector and amplified antibody fragment were digested with endonucleases. The ligation product was constructed using T4 ligase and then transferred into *E. coli* TG1 strain using electroporation. A strain with a diversity of 1.8 × 10⁻⁶ was ultimately constructed. 8 A camel anti-human B7H3 VHH antibody immunoassay library was developed for screening specific anti-human B7H3 nanobodies. To assess the library's accuracy, 50 clones were randomly selected for colony PCR, and the results showed an insertion rate of 90%.

[0240] The constructed camel immune library was screened using a solid-phase screening method to obtain specific phage-display nanobodies. Through original library presentation, screening, and identification, phage-display nanobodies A9 binding to recombinant human B7H3 protein were obtained.

[0241] Example 2: Preparation of anti-human B7H3 antibody and control antibody

[0242] The variable region gene of the target control antibody DS7300 (sequence source: CN103687945B) was synthesized, and its light and heavy chain variable region sequences are shown in SEQ ID NO.1 and SEQ ID NO.2. The light and heavy chain sequences were cloned into eukaryotic transient expression vectors containing the constant regions of the human lambda / IgG1 light and heavy chains, respectively, to obtain light and heavy chain expression plasmids of the control antibody. These plasmids were transformed into *E. coli* for amplification, and a large number of plasmids containing the light and heavy chains of the control antibody were isolated. The plasmids were extracted and precipitated with ethanol. Following the instructions of the transfection reagent 293fectin (Cat:12347019, Gibco), the light and heavy chain plasmids of the control antibody were transformed into HEK293 cells for recombinant expression. Five to six days after cell transfection, the culture supernatant was collected and purified using a protein A affinity chromatography column to obtain the control antibody.

[0243] Based on the sequencing results of the nanobody displayed by bacteriophage, primers were designed to clone nanobody A9 into a eukaryotic transient expression vector containing the encoding gene of human IgG1 Fc(hFc) (SEQ ID NO:9) via PCR, and recombinantly expressed in HEK293 cells. Five to six days after cell transfection, the culture supernatant was collected and purified using a protein A affinity chromatography column to obtain the recombinant chA9 protein. The variable region sequence is shown in SEQ ID NO.3.

[0244] Example 3: Detection of chimeric antibody affinity

[0245] Using the Fortebio Octet QKe system, antibody affinity was determined by employing an anti-human antibody Fc fragment capture antibody (AHC) bioprobe to capture the antibody Fc fragment. During the assay, the chA9 chimeric antibody and the control antibody DS7300 were diluted to 4 μg / ml with 1×HBS-EP buffer (Cat: BR-1006-69, Cytiva) and flowed through the AHC probe (Cat: 18-5060, Sartorius) for 120 s. The B7H3 recombinant protein (ECD, His Tag) (purchased from Yiqiao, Cat: 11188-H08H) at a concentration of 60 nM was used as the mobile phase, with a binding time of 300 s and a dissociation time of 300 s. After the experiment, the blank control response value was subtracted, and the kinetic constants of antigen-antibody binding were calculated using a 1:1 Langmuir binding model fitting.

[0246] The results are shown in Table 1 below. The results indicate that chA9 can bind to the B7H3 recombinant protein, but its binding activity is slightly weaker than that of DS7300.

[0247] Table 1. Affinity assay results between chimeric antibody and B7H3 recombinant protein

[0248] Example 4: Species-specificity of anti-B7H3 chimeric antibody detected by ELISA

[0249] Human B7H3 recombinant protein (ECD, His Tag) (purchased from Yiqiao, Cat:11188-H08H), cynomolgus monkey B7H3 recombinant protein (ECD, His Tag) (purchased from Yiqiao, Cat:90806-C08H), and mouse B7H3 recombinant protein (ECD, His Tag) (purchased from Yiqiao, Cat:50973-M08H) were used to coat ELISA plates overnight at 4°C with a coating concentration of 1 μg / mL. After washing the plates three times with PBS, 5% BSA PBS was added, and the plates were blocked at 37°C for 60 min. The plates were then washed three times with PBST. ChA9 and control antibody DS7300 at different dilutions (starting at 0.4 μg / mL, with 4 concentrations diluted 5-fold) were added, and the plates were incubated at 37°C for 60 min. The plates were then washed four times with PBST. Finally, HRP-anti-human diluted 1:5000 was added. Fc (Cat: 109-035-098, Jackson Immuno Research), incubated at 37°C for 45 min, washed 4 times with PBST; added TMB substrate for color development, incubated at 37°C for 10 min, and then added 2M HCl to terminate the reaction; using 630 nm as the reference wavelength, the absorbance A450nm-630nm of the well plate at a wavelength of 450 nm was read and recorded.

[0250] Experimental results showed that both chA9 and the control antibody DS7300 could specifically bind to human and cynomolgus monkey B7H3 recombinant protein (as shown in Figure 1), and chA9 had no cross-reactivity with mouse B7H3.

[0251] Example 5: ELISA detection of binding epitopes of anti-B7H3 chimeric antibody

[0252] Different truncated segments of recombinant human 4Ig B7H3, namely B7H3-ECD, B7H3-IgV1, B7H3-IgV2, B7H3-IgC1, B7H3-IgC2, and B7H3-IgV1-C1 (all KN-expressed and His-tagged), were coated onto ELISA plates overnight at 4°C with a coating concentration of 1 μg / mL. After washing the plates three times with PBS, 5% BSA PBS was added, and the plates were blocked at 37°C for 60 min, followed by three washes with PBST. Then, 0.4 μg / mL of chA9 and control antibody DS7300 were added, and the plates were incubated at 37°C for 60 min, followed by four washes with PBST. Finally, 1:5000 dilution of HRP-anti-human Fc (Cat: 109-035-098, Jackson Immuno) was added. (Research), incubate at 37℃ for 45 min, wash the plate 4 times with PBST; add TMB substrate for color development, incubate at 37℃ for 10 min, then add 2M HCl to terminate the reaction; use 630nm as the reference wavelength, read and record the absorbance A450nm-630nm of the well plate at a wavelength of 450nm.

[0253] The experimental results are shown in Figure 2. Both chA9 and the control antibody DS7300 can specifically and strongly bind to the extracellular domain (ECD) and IgV1-C1 region of human B7H3, and bind to some C1 and C2 regions, but basically do not bind to V1 and V2 regions.

[0254] Example 6: FACS detection of the binding activity of anti-B7H3 chimeric antibody on different tumor cells

[0255] Two E5 MDA-MB-231 (Cat:CBP60382, Kebai), LS1034 (Cat:CBP60013, Kebai), and C2BBe1 (Cat:CBP60007, Kebai) cells were collected, and their binding to the anti-B7H3 chimeric antibody was detected. chA9, control antibody DS7300, and isotype control (NC) were prepared into 66 nM solutions, then serially diluted 3-fold to obtain 8 concentrations, which were added to the cells. After incubation at 4°C in the dark for 60 min, and thorough washing with PBS, 1:200 diluted FITC-labeled goat anti-human IgG antibody (Sigma, F9512) was added. After incubation at 4°C in the dark for 30 min, the cells were washed thoroughly with PBS, resuspended in 200 μL of PBS, and analyzed by flow cytometry.

[0256] The results are shown in Figures 3, 4, and 5. chA9 specifically bound to different tumor cells, exhibiting a binding ability superior to the control antibody DS7300. EC50 values ​​are shown in Table 2.

[0257] Table 2. EC50 of anti-B7H3 antibody on different tumor cells as detected by FACS

[0258] Example 7: FACS detection of internalization activity of anti-B7H3 chimeric antibody on different tumor cells

[0259] use Fabfluor-pH antibody labeling reagent (Cat.4722, Sartorius) was used to detect the internalization of anti-B7H3 chimeric antibodies on different tumor cells. 1E5 MDA-MB-231, LS1034, and C2BBe1 cells were seeded in 96-well plates. Four concentrations of chA9, control antibody DS7300, and isotype control were prepared: 6.6 nM, 2.2 nM, 0.73 nM, and 0.24 nM. Antibody Internalization Human Reagent (Cat.4722, Sartorius) was added to each concentration according to the manufacturer's instructions, and the cells were incubated for a period to allow labeling of the test antibody. The mixture was then added to the cells. The cells were incubated at 37°C in the dark for 120 min, and the immunofluorescence signal of the internalized cells was detected by flow cytometry.

[0260] The results are shown in Figures 6, 7, and 8. chA9 underwent strong internalization in different tumor cells, with internalization activity comparable to DS7300.

[0261] Example 8: Humanization and Recombinant Expression Analysis of Anti-B7H3 Chimeric Antibody

[0262] First, a comprehensive analysis of the camel-derived antibody VHH sequence was performed to determine the antigen complementarity determinant (CDR) region for antibody-antigen binding and the framework region supporting the conserved three-dimensional conformation of the antibody. Then, based on homology alignment results, the most similar human antibody germline sequence was selected as the template. Combined with full-sequence BLAST results, CDR transplantation was performed to humanize the chA9 variable region (VHH) within the framework region. The amino acid sequences of the obtained humanized antibodies hzA9-1 and hzA9-2 are shown in SEQ ID NO:4 and SEQ ID NO:5, respectively.

[0263] The humanized variable region sequences of hzA9-1 and hzA9-2 were fully synthesized and cloned into a eukaryotic expression vector containing the human Fc(hFc) (SEQ ID NO:9) encoding gene. After obtaining the expression plasmid with the correct sequence, it was transformed into HEK293 cells for recombinant expression. Five to six days after cell transfection, the culture supernatant was collected and purified using a protein A affinity chromatography column to obtain the humanized antibody recombinant protein.

[0264] Example 9: Detection of affinity for humanized B7H3 antibody

[0265] Using the Fortebio Octet QKe system, antibody affinity was determined by employing an anti-human antibody Fc fragment capture antibody (AHC) bioprobe to capture the antibody Fc fragment. During the assay, chA9, hzA9, and the control antibody DS7300 were diluted to 4 μg / ml with 1×HBS-EP buffer and flowed through the AHC probe (Cat:18-0015, PALL) for 120 s. B7H3 recombinant protein (ECD, His Tag) (purchased from Yiqiao, Cat:11188-H08H) at a concentration of 60 nM was used as the mobile phase, with a binding time of 300 s and a dissociation time of 300 s. After the experiment, the blank control response value was subtracted, and the kinetic constants of antigen-antibody binding were calculated using a 1:1 Langmuir binding model fitting.

[0266] The results are shown in Table 3 below. The results indicate that the humanized antibody and the chimeric antibody have basically the same affinity, and the affinity of different humanized molecules is basically the same.

[0267] Table 3. Affinity assay results between humanized antibody and recombinant B7H3 protein

[0268] Example 10: FACS detection of the binding activity of anti-B7H3 humanized antibody on different tumor cells

[0269] Two E5 MDA-MB-231 (Cat:CBP60382, Kebai), LS1034 (Cat:CBP60013, Kebai), and NCI-H1975 (Cat:CBP60121, Kebai) cells were collected, and their binding to the anti-B7H3 humanized antibody was detected. chA9, hzA9-1, hzA9-2, and control antibodies DS7300 and isotype control (NC) were prepared into 66 nM solutions, then serially diluted 3-fold to obtain 8 concentrations, which were added to the cells. After incubation at 4°C in the dark for 60 min, and thorough washing with PBS, 1:200 diluted FITC-labeled goat anti-human IgG antibody (Sigma, F9512) was added. After incubation at 4°C in the dark for 30 min, the cells were washed thoroughly with PBS, resuspended in 200 μL of PBS, and analyzed by flow cytometry.

[0270] The results are shown in Figures 9, 10, and 11. hzA9-1 and hzA9-2 can specifically bind to different tumor cells, and their binding ability is comparable to that of chA9, which is superior to that of the control antibody DS7300. EC50 is shown in Table 4.

[0271] Table 4. EC50 of anti-B7H3 humanized antibody on different tumor cells as detected by FACS

[0272] Example 11: FACS detection of internalization activity of anti-B7H3 humanized antibody on different tumor cells

[0273] use Fabfluor-pH antibody labeling reagent (Cat.4722, Sartorius) was used to detect the internalization of anti-B7H3 humanized antibodies on different tumor cells. 1E5 MDA-MB-231, LS1034, and NCI-H1975 cells were seeded in 96-well plates. For LS1034 and NCI-H1975 cells, four concentrations of chA9, hzA9-1, hzA9-2, and control antibodies DS7300 and isotype control (NC) were set at 6.6 nM, 2.2 nM, 0.73 nM, and 0.24 nM; for MDA-MB-231 cells, three concentrations were set at 6.6 nM, 2.2 nM, and 0.73 nM. Following the manufacturer's instructions, the antibody internalization human reagent (Cat.4722, Sartorius) was added to the above-mentioned test antibodies, and after incubation for a period of time, the mixture was added to the cells. Incubate at 37°C in the dark for 120 min, and then detect the immunofluorescence signal of the cells internalized by flow cytometry.

[0274] The results are shown in Figures 12, 13, and 14. Both hzA9-1 and hzA9-2 underwent strong internalization on different tumor cells, and their internalization activity was comparable to that of the chimeric antibody chA9 and the control antibody DS7300.

[0275] Example 12: Modification and Recombinant Expression Analysis of Anti-B7H3 Humanized Antibody

[0276] To further optimize the properties of the humanized antibody, the hzA9-1 sequence was further optimized, and the mutation sites are shown in Table 5 below (underlined).

[0277] Table 5. Humanized Sequence Design of hzA9-1

[0278] The mutant sequence was fully synthesized and cloned into a eukaryotic expression vector containing the human Fc(hFc) (SEQ ID NO:9) encoding gene. After obtaining the expression plasmid with the correct sequence, it was transfected into HEK293 cells for recombinant expression. Five to six days after cell transfection, the culture supernatant was collected and purified using a protein A affinity chromatography column to obtain the humanized mutant antibody recombinant protein.

[0279] Example 13: Affinity Detection of Humanized B7H3 Modified Antibody

[0280] Using the Fortebio Octet QKe system, antibody affinity was determined by employing an anti-human antibody Fc fragment capture antibody (AHC) bioprobe to capture the antibody Fc fragment. During the assay, chA9, hzA9, and the control antibody DS7300 were diluted to 4 μg / ml with 1×HBS-EP buffer and flowed through the AHC probe (Cat:18-0015, PALL) for 120 s. B7H3 recombinant protein (ECD, His Tag) (purchased from Yiqiao, Cat:11188-H08H) at a concentration of 60 nM was used as the mobile phase, with a binding time of 300 s and a dissociation time of 300 s. After the experiment, the blank control response value was subtracted, and the kinetic constants of antigen-antibody binding were calculated using a 1:1 Langmuir binding model fitting.

[0281] The results are shown in Table 6 below. The results indicate that there are certain differences in affinity among the modified antibodies, with hzA9-1mut5, hzA9-1mut6, hzA9-1mut7, hzA9-1mut8, and hzA9-1mut11 showing the best affinity.

[0282] Table 6. Affinity assay results between humanized antibody and recombinant B7H3 protein

[0283] Example 14: FACS detection of the binding activity of anti-B7H3 humanized antibody on different tumor cells

[0284] Two E5 DLD-1 (Cat:CL-0074, Pronosai) and MDA-MB-231 (Cat:CBP60382, Kebai) cells were collected, and their binding to the anti-B7H3 humanized antibody was detected. hzA9-1 and its mutant were prepared at 66 nM, then serially diluted 3-fold to obtain three concentrations, which were added to the cells. After incubation at 4°C in the dark for 60 min, and thorough washing with PBS, 1:200 diluted FITC-labeled goat anti-human IgG antibody (Sigma, F9512) was added. After incubation at 4°C in the dark for 30 min, the cells were washed thoroughly with PBS, resuspended in 200 μL of PBS, and analyzed by flow cytometry.

[0285] The results are shown in Figures 15 and 16. The binding activities of different mutants vary to some extent, with hzA9-1mut8 and hzA9-1mut11 showing relatively better binding activities.

[0286] 2E5 cells were collected from DLD-1 (Cat:CL-0074, Pronosai), Calu-6 (Cat:CL-0327, Pronosai), human lung cancer A549 (Cat:GCL-0016, Pronosai), human large cell lung cancer NCI-H460 (Cat:CBP60138, Nanjing Kebai), human breast cancer MDA-MB-468 (Cat:CBP60387, Nanjing Kebai), and human epidermal carcinoma A431 (…). The binding of human pancreatic cancer PANC-1 (Cat:C5138, Baidi), human cervical cancer HeLa (Cat:YC-A012, UBIGENE), human colorectal cancer RKO (Cat:CBP60006, Nanjing Kebai), and human lung squamous cell carcinoma EBC-1 (Cat:CBP60091, Nanjing Kebai) cells to the anti-B7H3 humanized antibody hzA9-1 was detected. hzA9-1, the control antibody DS7300, and the isotype control (NC) were prepared at 66 nM, then serially diluted 3-fold to a total of 7 concentrations, and added to the cells. Incubate at 4°C in the dark for 60 min, wash thoroughly with PBS, add FITC-labeled goat anti-human IgG antibody (sigma, F9512) diluted 1:200, incubate at 4°C in the dark for 30 min, wash thoroughly with PBS, resuspend in 200 μL of PBS, and detect by flow cytometry.

[0287] The results are shown in Figures 17 to 26. hzA9-1 could specifically bind to different tumor cells, and its binding ability was superior to that of the control antibody DS7300. EC50 is shown in Table 7.

[0288] Table 7. EC50 of anti-B7H3 humanized antibody on different tumor cells as detected by FACS

[0289] Example 15: FACS detection of internalization activity of anti-B7H3 humanized antibody on different tumor cells

[0290] use Fabfluor-pH antibody labeling reagent (Cat.4722, Sartorius) was used to detect the internalization of anti-B7H3 humanized antibodies on DLD-1 tumor cells. Two concentrations were set: 6.6 nM and 0.66 nM for hzA9-1 and its mutant. Following the manufacturer's instructions, the antibody internalization human reagent (Cat.4722, Sartorius) was added to the above-mentioned test antibodies, and after incubation for a period of time, the mixture was added to the cells. The cells were incubated at 37°C in the dark for 120 min, and the immunofluorescence signal of the internalized cells was detected by flow cytometry.

[0291] As shown in Figure 27, each mutant underwent strong internalization on tumor cells.

[0292] Furthermore, 1E5 DLD-1, Calu-6, human lung cancer A549 (Cat:GCL-0016, Pronosai), human large cell lung cancer NCI-H460 (Cat:CBP60138, Nanjing Kebai), human breast cancer MDA-MB-468 (Cat:CBP60387, Nanjing Kebai), human epidermal carcinoma A431 (Cat:CBP60330, Nanjing Kebai), human pancreatic cancer PANC-1 (Cat:C5138, Baidi), human cervical cancer HeLa (Cat:YC-A012, Ubigene), human colorectal cancer RKO (Cat:CBP60006, Nanjing Kebai), and human lung squamous cell carcinoma EBC-1 (Cat:CBP60091, Nanjing Kebai) cells were seeded into 96-well plates. hzA9-1 and control antibody DS7300 and isotype control (NC) were prepared at four concentrations: 6.6 nM, 2.2 nM, 0.73 nM, and 0.24 nM. Following the manufacturer's instructions, the antibody internalization human reagent (Cat.4722, Sartorius) was added to the above-mentioned test antibodies. After incubation for a period of time, the mixture was added to the cells. The cells were incubated at 37°C in the dark for 120 min, and the immunofluorescence signal of the internalized cells was detected by flow cytometry.

[0293] As shown in Figures 28-37, hzA9-1 underwent strong internalization in different tumor cells, with internalization activity comparable to or slightly better than the control antibody DS7300, especially at low concentrations (0.73, 0.24 nM), where its internalization was superior to that of the control antibody DS7300.

[0294] Further, 1E5 cells each of lung cancer HCC827 (Cat:GCL-0094, Pronosai), colorectal cancer DLD-1 (Cat:CBP60037, Nanjing Kebai), and human pancreatic cancer Aspc-1 (Cat:GCL-0027, Pronosai) were seeded into 96-well plates. hzA9-1, control antibody DS7300, and isotype control (NC) were added at a concentration of 0.66 nM. Following the manufacturer's instructions, the antibody internalization human reagent (Cat.4722, Sartorius) was added to the above-mentioned test antibodies, and after incubation for a period of time, the mixture was added to the cells. The cells were incubated at 37°C in the dark for 1, 2, 3, and 5 hours, and the immunofluorescence signal of the internalized cells was detected by flow cytometry.

[0295] As shown in Figures 38-40, hzA9-1 underwent strong internalization on different tumor cells, and the internalization intensity increased significantly with the extension of internalization time, showing better internalization ability than the control antibody DS7300.

[0296] Example 16: Preparation of antibody-drug conjugates targeting B7H3

[0297] B7H3 antibody hzA9-1 and DS7300 were conjugated with the drug-containing linker GGFG-DXD via cysteine ​​coupling. The conjugation method for DS7300 is described in patents PCT / JP2021 / 041496 and WO 2014 / 057687. The GGFG-DXD drug-containing linker is shown below. The resulting ADC was then prepared. The ADC was characterized, and the results are shown in Table 8.

[0298] Table 8. Characterization results of antibody-drug conjugates

[0299] Example 17: Activity evaluation of antibody-drug conjugates targeting B7H3

[0300] Human lung cancer Calu-6 cells were seeded into 96-well plates at a density of 3000 cells / well. Antibody-drug conjugates diluted to working concentrations (starting at 100 μg / mL, with 8 sequential 5-fold dilutions) were added and thoroughly mixed with the cells. After incubation at 37°C for 7 days, the cells were analyzed using a CTG assay kit. (Luminescent Cell Viability Assay, DD1101-02, Vazyme), to detect the killing effect of ADC samples on target cells.

[0301] The test results are shown in Figure 41. The results indicate that the killing activity of hzA9-1-DXD is superior to that of the control antibody-drug conjugate DS7300-DXD.

[0302] Example 18: Pharmacodynamic evaluation of antibody-drug conjugates targeting B7H3 in the Nude mouse Calu-6 model of human lung cancer

[0303] Calu-6 human lung cancer cells were subcutaneously instilled into the right anterior flank of female Nude mice. The day of instillation was designated as day 0. Tumors were counted when they reached 200 mm. 3 The animals were divided into groups of 6 each, and the dosage was 5 mg / kg, administered intraperitoneally, once.

[0304] The tumor volume was measured twice a week using calipers, and the mice were weighed using an electronic balance. The major and minor diameters of the tumor were measured, and the volume was calculated using the formula: Volume (TV) = 0.5 × Major diameter × Minor diameter 2 The T / C value is calculated based on tumor volume. The tumor volume ratio (T / C%) of the treatment group / control group is calculated as follows: T / C (%) = RTV of the treatment group / RTV of the control group × 100%. The average RTV of the tumor volume in the treatment group (T) / control group (C) is the ratio of tumor volume after administration to tumor volume before administration. The tumor growth inhibition rate (TGITV) (%) is calculated as follows: TGITV (%) = (1 - T / C) × 100%.

[0305] The tumor growth inhibition of D47 is shown in Table 9, Figure 42, and Figure 43. The results indicate that, compared with the isotype control (NC) group, each ADC administration group had a clear antitumor effect, and the efficacy (TGI) of hzA9-1-DXD was significantly higher. TV (100%) significantly superior to the control antibody-drug conjugate DS7300-DXD (TGI) TV (57%).

[0306] Table 9. Tumor growth inhibition status in each group

[0307] Example 19: Pharmacodynamic evaluation of antibody-drug conjugates targeting B7H3 in a Nude mouse model of human colorectal cancer.

[0308] Human colorectal cancer cells DLD-1 were subcutaneously injected into the right anterior flank of female Nude mice. The day of injection was designated as day D0. Tumors were cultured until they reached 200 mm in size. 3 The animals were divided into groups of 6 each, and the dosage was 5 mg / kg, administered once a week for a total of 2 weeks.

[0309] The tumor growth inhibition is shown in Table 10 and Figure 44. The results indicate that hzA9-1-DXD has significant antitumor efficacy compared with the isotype control (NC) group.

[0310] Table 10. Tumor growth inhibition status in each group

[0311] Sequence List Overview

Claims

1. An antibody that specifically binds to B7-H3 or an antigen-binding fragment thereof, wherein the antibody comprises a VHH domain, the VHH domain comprising three CDRs contained in the heavy chain variable region selected from SEQ ID NOs:3-5 and 11-21, optionally wherein the CDRs are defined according to Kabat, AbM, IMGT, Chothia or a combination thereof.

2. The antibody or its antigen-binding fragment according to claim 1, wherein the VHH domain comprises CDR1, CDR2, and CDR3, wherein: (1) CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:6; CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:7; and CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:8; (2) CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:22; CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:7; and CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:8; (3) CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:23; CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:7; and CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:8; (4) CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:24; CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:7; and CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:8; (5) CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:25; CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:7; and CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:8; (6) CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:26; CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:7; and CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:8; (7) CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:6; CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:7; and CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:27; (8) CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:6; CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:7; and CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:28; (9) CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:6; CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:7; and CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:29; (10) CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:6; CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:7; and CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:30; (11) CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:6; CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:7; and CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:31; (12) CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:6; CDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:7; and CDR3 contains or consists of the amino acid sequence shown in SEQ ID NO:

32.

3. The antibody or antigen-binding fragment thereof according to any one of claims 1-2, wherein the VHH domain comprises or is composed of the following amino acid sequence: (i) an amino acid sequence selected from SEQ ID NOs:3-5 and 11-21; or (ii) an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the same amino acid sequence as (i); or (iii) An amino acid sequence having at least 1-30 (e.g., 1-20, 1-15, 1-10, or 1-5) amino acid additions, deletions, and / or substitutions relative to the amino acid sequence of (i).

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the VHH domain comprises, or is composed of, an amino acid sequence selected from, SEQ ID NOs:4-5, 11, 15-18 and 20-21.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the antibody is a chimeric, murine, or humanized antibody.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the antibody is a nanobody or a single-domain antibody.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the antibody further comprises an immunoglobulin constant region, such as the immunoglobulin Fc region.

8. The antibody or antigen-binding fragment thereof according to any one of claims 7, wherein the antibody is a heavy chain antibody.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1-7, wherein the antibody is a monospecific, bispecific, or multispecific antibody.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1-9, wherein the antibody has one or more properties selected from the following: (a) Binds to human B7-H3, optionally, with affinity K D Value less than 1x10 -9 M, preferably about 0.1-10x10 -10 M; (b) Binding to tumor cells expressing human B7-H3, preferably as detected by FACS, with a cell binding EC50 value between 0.1 and 1 nM; (c) It exhibits immune cross-reactivity with monkey B7-H3; and (d) It has B7-H3-mediated endocytosis activity.

11. A polynucleotide encoding an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-10.

12. A vector, preferably an expression vector, comprising the polynucleotide of claim 11.

13. A host cell comprising the polynucleotide of claim 11 or the vector of claim 12, wherein the host cell is optionally a mammalian cell.

14. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1-10, the method comprising: Host cells containing the polypeptide chain encoding the antibody or its antigen-binding fragment are cultured under conditions suitable for expressing the antibody or its antigen-binding fragment.

15. An immunoconjugate or immunofusion comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-10.

16. The immunoconjugate of claim 15, wherein the antibody or its antigen-binding fragment is conjugated to a radioactive isotope, an antitumor drug, a chemotherapeutic agent, or a cytotoxin via a linker.

17. The immunoconjugate of claim 16, wherein the linker is an enzymatically cleavable linker, preferably comprising a GGFG moiety.

18. The immunoconjugate according to claim 16 or 17, wherein the cytotoxin is a topoisomerase I inhibitor, for example, a camptothecin compound, preferably eczemab, Dxd or a derivative thereof.

19. The immunoconjugate according to any one of claims 15-18, wherein the immunoconjugate is an antibody-drug conjugate (ADC).

20. The immunoconjugate of claim 19, wherein the antibody-drug conjugate (ADC) comprises a GGFG-DXD portion conjugated to the antibody or its antigen-binding fragment, preferably the ADC having an average DAR of about 1-20, for example about 1-8, 1-6, 2-5 or 3-4.

21. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-10, or an immunoconjugate or immunofusion compound as described in any one of claims 15-20, and a pharmaceutically acceptable carrier.

22. Use of the antibody or antigen-binding fragment of any one of claims 1-10 or the immunoconjugate or immunofusion of any one of claims 15-20 as a medicament or the pharmaceutical composition of claim 21 for the preparation of a medicament, optionally for the treatment or prevention of cancer.

23. A method of treating or preventing cancer, comprising administering to an individual in need an effective amount of an antibody or antigen-binding fragment thereof as claimed in any one of claims 1-10, an immunoconjugate or immunofusion compound as claimed in any one of claims 15-20, or a pharmaceutical composition as claimed in claim 21.

24. The method of claim 23, wherein the cancer is a B7-H3 positive solid tumor or hematologic malignancy, selected from, for example, lung cancer, small cell lung cancer, colorectal cancer, breast cancer, pancreatic cancer, gastric cancer, ovarian cancer, and glioma.

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