Antigen binding protein specifically binding to SEMA4D and its application

Abstract

The present invention provides an antigen binding protein that specifically binds to SEMA4D and its application, and relates to the field of biomedicine technology. The antigen binding protein comprises the complementary determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region whose amino acid sequences are respectively shown as SEQ ID NO.1 to 3. The antigen binding protein can specifically bind to SEMA4D, and is expected to be used as a therapeutic drug for diseases related to the SEMA4D target, or play an auxiliary role in disease diagnosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an antigen binding protein specifically binding to SEMA4D and an application thereof. Background Art

[0002] SEMA4D (axon guidance protein 4D, also known as CD100) is a transmembrane homodimeric protein with a molecular weight of 150 kDa and is an important member of the semaphorin family [Kuklina, Elena, Irina Nekrasova, and Natalia Glebezdina. "Signaling from membrane semaphorin 4D in T lymphocytes." Molecular Immunology 129 (2021): 56-62]. SEMA4D is widely expressed in many human tumors, and its expression is associated with human invasive diseases. In recent years, a large number of studies have found that SEMA4D is highly expressed in many human tumor tissues and plays an important role in tumor angiogenesis and tumor invasion and metastasis [Evans, Elizabeth E., et al. "Antibody blockade of semaphorin 4D promotes immune infiltration into tumor and enhances response to other immunomodulatory therapies." Cancer immunology research 3.6 (2015): 689-701]. SEMA4D usually regulates the movement and differentiation of certain cells, including cells of the immune system and vascular system. In the tumor microenvironment, inflammatory cells and tumor cells express SEMA4D to regulate the infiltration, spatial distribution and activity of myeloid cells and lymphocytes. SEMA4D binds to the plexin receptors on myeloid cells in the tumor microenvironment. When the SEMA4D protein is blocked, the SEMA4D barrier can be eliminated. Once the barrier is breached, inflammatory dendritic cells and proinflammatory antigen-presenting cells migrate and infiltrate into the tumor. SEMA4D is also an effective angiogenesis-promoting molecule. Activation of Plexin-B1 by SEMA4D binding can transactivate c-Met in vitro and in vivo, promote tumor cell invasion, and promote angiogenesis. Immunohistochemical analysis of SEMA4D in larger tumor samples revealed that CD100 overexpression is a very common event in head and neck cancer, prostate cancer, colon cancer, breast cancer, and lung cancer.

[0003] SEMA4D / plexin B1 signaling can also induce endothelial cell migration and promote tumor cell migration (Conrotto et al., Blood 105:4321-4329 (2005); Giordano et al., Nature Cell Biology 4:720-724 (2002)). SEMA4D-induced endothelial cell migration can be prevented by SEMA4D-blocking antibodies and SEMA4D knockdown.

[0004] In addition to the demonstrated effects of SEMA4D on immune cells, SEMA4D has also been shown to play a direct role in the demyelination and axonal degeneration observed in neuroinflammatory diseases. The pathogenesis of inflammatory demyelinating diseases such as MS includes an inflammatory phase involving immune cells and a phase of selective demyelination and neurodegeneration. SEMA4D is expressed in oligodendrocytes of the central nervous system (CNS) and is an inhibitor of axonal degeneration. SEMA4D expression is upregulated in oligodendrocytes in the periphery of spinal cord injury (Moreau-Fauvarque et al., J. Neuroscience 23: 9229-9239 (2003)). Culturing long-term activated T cells expressing sSEMA4D with human multipotent neural precursors or primary oligodendrocytes from rat brain can induce apoptosis and process extension collapse (Giraudon et al., J. Immunol. 172: 1246-1255 (2004); Giraudon et al., NeuroMolecular Med. 7: 207-216 (2005)). Anti-SEMA4D antibodies can inhibit SEMA4D-induced apoptosis of neural precursors.

[0005] In summary, SEMA4D antibodies that specifically bind to SEMA4D are expected to play a role in targeted therapy in diseases such as tumors where SEMA4D is highly expressed.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide an antigen binding protein, which can specifically bind to SEMA4D. The present invention also aims to provide an application thereof for detecting SEMA4D or as a drug.

[0008] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0009] In a first aspect, an isolated antigen-binding protein is provided, wherein the antigen-binding protein specifically binds to SEMA4D, and the antigen-binding protein comprises complementary determining regions HCDR1, HCDR2 and HCDR3 of a heavy chain variable region;

[0010] The amino acid sequence of the HCDR1 is shown in SEQ ID NO.1; the amino acid sequence of the HCDR2 is shown in SEQ ID NO.2; and the amino acid sequence of the HCDR3 is shown in SEQ ID NO.3.

[0011] In an optional implementation, the heavy chain variable region contains a heavy chain framework region, and each heavy chain framework region is independently an antibody or a mutant thereof from the following species: camel, alpaca, mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey or human;

[0012] In an optional implementation, the heavy chain variable region contains heavy chain framework regions HFR1, HFR2, HFR3 and HFR4; the amino acid sequence of HFR1 is shown in SEQ ID NO.4; and / or, the amino acid sequence of HFR2 is shown in SEQ ID NO.5; and / or, the amino acid sequence of HFR3 is shown in SEQ ID NO.6; and / or, the amino acid sequence of HFR4 is shown in SEQ ID NO.7;

[0013] In an optional implementation, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.8.

[0014] In an optional implementation, the SEMA4D is human SEMA4D;

[0015] In an optional implementation, the isolated antigen-binding protein is a humanized antibody or an antigen-binding fragment thereof;

[0016] In an optional implementation, the separated antigen-binding protein is a nanobody;

[0017] In an optional implementation, the amino acid sequence of the Nanobody is as shown in SEQ ID NO.9.

[0018] In a second aspect, a fusion protein is provided, wherein the fusion protein comprises the antigen-binding protein in the first aspect.

[0019] In a third aspect, a biomaterial is provided, wherein the biomaterial is selected from any one of (I) to (III):

[0020] (I) a polynucleotide encoding the isolated antigen-binding protein of the first aspect, or the fusion protein of the second aspect;

[0021] (II) a vector, the vector carrying the polynucleotide;

[0022] (III) a cell, which is capable of expressing the isolated antigen-binding protein of the first aspect, or expressing the fusion protein of the second aspect, or carrying the polynucleotide, or containing the vector.

[0023] In a fourth aspect, a method for preparing the isolated antigen-binding protein of the first aspect is also provided, comprising expressing the antigen-binding protein in a cell.

[0024] In a fifth aspect, an immunoconjugate is provided, which contains the isolated antigen-binding protein of the first aspect and at least one therapeutic agent and / or at least one diagnostic agent coupled to the antigen-binding protein.

[0025] In a sixth aspect, there is also provided the use of the isolated antigen-binding protein of the first aspect, or the fusion protein of the second aspect, or the biomaterial of the third aspect, or the preparation method of the fourth aspect, or the immunoconjugate of the fifth aspect in any of the following:

[0026] (i) preparing a blocking agent for blocking PlexinB1 and / or PlexinB2 molecules;

[0027] (ii) blocking PlexinB1 and / or PlexinB2;

[0028] (iii) preparing a medicament for treating, preventing or alleviating a disease and / or condition associated with SEMA4D signaling;

[0029] (iv) preparing a kit for detecting SEMA4D;

[0030] (v) Detection of SEMA4D.

[0031] In the seventh aspect, a pharmaceutical composition is also provided, which comprises the isolated antigen-binding protein of the first aspect, or the fusion protein of the second aspect, or the biomaterial of the third aspect, or the immunoconjugate of the fifth aspect.

[0032] In the eighth aspect, a kit for detecting Sema4D is also provided, the kit comprising the isolated antigen-binding protein of the first aspect, or the fusion protein of the second aspect, or the biological material of the third aspect, or the immunoconjugate of the fifth aspect.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention provides an antigen binding protein that can specifically bind to SEMA4D. The antigen binding protein has high affinity activity and specificity, has good application prospects, and is expected to be used as a therapeutic drug for diseases related to the SEMA4D target. It can also be used as a main component of a kit for detecting the expression abundance of patient-related indicators, and plays an auxiliary role in disease diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 This is a graph showing the experimental results of the binding of the humanized nanobody of Example 2 to the positive overexpressing cell 293T-Sema4D overexpressing SEMA4D;

[0037] Figure 2 This is a diagram showing the experimental results of Example 3 in which humanized antibodies blocked the binding of Sema4D to 293T-PlexinB1#7 cells;

[0038] Figure 3 This is a graph showing the experimental results of Example 4 in which the humanized antibody blocked the binding of Sema4D to 293T-PlexinB2#7 cells. DETAILED DESCRIPTION

[0039] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] Definition of terms:

[0041] Herein, the term "isolated antigen-binding protein" generally refers to a protein with antigen-binding ability obtained by artificial means from a natural state. The "isolated antigen-binding protein" may include a portion that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that promotes the antigen-binding portion to bind to the antigen. The antigen-binding protein may include, for example, an antibody-derived protein scaffold or an alternative protein scaffold or an artificial scaffold having a transplanted CDR or CDR derivative. Such scaffolds include, but are not limited to, antibody-derived scaffolds that include mutations introduced, for example, to stabilize the three-dimensional structure of the antigen-binding protein and fully synthetic scaffolds that include, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53 (1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20: 639-654 (2004). In addition, peptide antibody mimics ("PAMs") and scaffolds based on antibody mimics using fibronectin components can be used as scaffolds. In this application, the term "antigen binding protein" generally refers to an immunoglobulin or antibody polypeptide fragment that competes with an intact antibody (i.e., the intact antibody from which they are derived) for binding (i.e., specific binding) to an antigen. The antigen binding protein may include, but is not limited to, nanobodies, Fab, Fab', F(ab'), 2 and Fv fragments, linear antibodies, single-chain antibodies, diabodies, and multispecific antibodies formed from antibody fragments.

[0042] In the context of the present invention, the term "antibody" (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of any of them that has the ability to specifically bind to an antigen under typical physiological conditions, wherein the half-life is a significant period of time, such as at least about 30 min, at least about 45 min, at least about one hour, at least about two hours, at least about four hours, at least about eight hours, at least about 12 hours, about 24 hours or longer, about 48 hours or longer, about three, four, five, six, seven days or longer, etc., or any other relevant functionally defined period of time (such as sufficient to induce, promote, enhance, and / or regulate the physiological response associated with the antibody binding to the antigen and / or sufficient to recruit effector activity of the antibody). The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with the antigen. The constant region of the antibody (Ab) can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system (such as C1q, the first component in the classical pathway of complement activation). The antibody may also be a bispecific antibody, a diabody, a multispecific antibody or a similar molecule.

[0043] The term "variable region" or "variable domain" as used herein refers to the domain of the amino terminus of the heavy chain or light chain of an antibody that recognizes and binds to an antigen. The composition and arrangement of the amino acids in this segment determine the specificity of the antibody in recognizing an antigen. The heavy chain variable domain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL", which contains an antigen binding site. The variable regions of the heavy and light chains are each composed of three complementarity-determining regions (CDRs) (also known as hypervariable regions) connected by four framework regions (FRs).

[0044] "CDR" sequences are highly variable and / or form structure-defining loops. There are three CDRs (HCDR1, HCDR2, HCDR3) in VH and three CDRs (LCDR1, LCDR2, LCDR3) in VL. In natural antibodies, HCDR3 and LCDR3 show most of the diversity of the six CDRs, and HCDR3 in particular is considered to play a unique role in conferring fine specificity to antibodies. Nanobodies include three CDRs, HCDR1, HCDR2 and HCDR3. See, e.g., Xu et al, Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003).

[0045] The extent of the framework region and CDR has been precisely defined, for example, in Kabat (see Sequences of Proteins of Immunological Interest, E. Kabat et al.) and Chothia. Any CDR determination method well known in the art, including a combination of methods, can identify the CDRs of the variable domain. The CDRs in each chain are held together closely by FRs to form the variable region. Generally, the variable regions VL / VH of the heavy and light chains can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0046] The term "constant region" or "constant domain" as used herein refers to the constant region of the antibody light chain alone or in combination or the constant region of the antibody heavy chain. The heavy chain of an antibody has a variable domain (VH), which generally contains one or more of a hinge, CH1, CH2, CH3, and CH4, the CH1 domain being adjacent to the VH domain and at the amino terminus of the hinge region of the antibody heavy chain, and not forming part of the Fc region of the antibody; the hinge region includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain; the N-terminus of CH2 is generally the CH3 domain, which generally forms the C-terminal portion of the antibody, and in some antibody types, such as IgM and IgE, the constant region also includes the CH4 domain. The constant region of an antibody can be derived from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, as well as the constant regions of their subclasses and mutant forms. The constant domain is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions, such as the involvement of the antibody in antibody-dependent cellular toxicity.

[0047] In this article, the term "nanobody" is an antibody that naturally lacks light chains. The antibody only contains a heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but it is not as easy to stick to each other or even aggregate into clumps as the artificially modified single-chain antibody fragment (scFv). More importantly, the VHH structure cloned and expressed separately has the same structural stability and antigen binding activity as the original heavy chain antibody, and is the smallest unit known to bind to the target antigen.

[0048] As used herein, the term "SEMA4D protein" generally refers to axon guidance protein 4D (SEMA4D). As used herein, the term encompasses complete SEMA4D and functionally active fragments, variants, homologues, analogs and derivatives thereof.

[0049] In the text, the term "specific binding" or "specific" generally refers to a measurable and reproducible interaction, such as the binding between a target and an antibody, which can be determined in the presence of a heterogeneous population of molecules (including biomolecules) to determine the presence of the target. For example, an antibody that specifically binds to a target (which can be an epitope) is an antibody that binds to the target with greater affinity, avidity, more readily, and / or with greater duration than it binds to other targets. In one embodiment, the degree to which the antibody binds to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, specific binding can include but is not required to bind exclusively.

[0050] As used herein, the term "humanized antibody" generally refers to an antibody that is derived from a non-human species but whose protein sequence has been modified to increase its similarity to naturally occurring antibodies in humans.

[0051] In this article, the term "fully humanized antibody" generally refers to fully humanized antibodies, i.e., the constant region and variable region of the antibody are both derived from humans. The fully humanized antibody can be achieved by phage antibody library technology, transgenic mice to prepare humanized antibodies, ribosome display technology, EBV-transformed B cell cloning technology, single B cell cloning and other technologies.

[0052] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably and generally refer to polymers of amino acid residues. The term may be used to refer to amino acid polymers in which one or more amino acid residues are synthetic chemical mimetics of their corresponding natural amino acids, and may also be used to refer to natural amino acid polymers, those containing modified residues, and non-natural amino acid polymers.

[0053] In this article, the term "fusion protein" generally refers to a protein consisting of two or more polypeptides, which, although usually not combined in the native state, are combined together through their respective amino and carboxyl termini via peptide bonds to form a continuous polypeptide. It should be understood that the two or more polypeptide components can be directly combined or indirectly combined through a peptide linker / spacer.

[0054] In this article, the term "nucleic acid molecule" generally refers to any length polynucleotide, "polynucleotide" or "nucleic acid molecule" refers to a polymer form of nucleotides of any length, and polynucleotides include ribonucleotides and / or deoxyribonucleotides. Examples of polynucleotides include but are not limited to single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. The target protein encoded by the polynucleotide, the encoding is optionally for encoding the sense strand or the antisense strand. The polynucleotide can be naturally occurring, synthetic, recombinant or any combination thereof. The terms "polynucleotide", "nucleic acid" and "nucleic acid molecule" are used interchangeably herein.

[0055] As used herein, the term "purified" or "isolated" in connection with a polypeptide (protein) or polynucleotide means that the polypeptide or polynucleotide is not in its natural medium or in its natural form. Thus, the term "isolated" includes a polypeptide or polynucleotide removed from its original environment, such as if it is naturally present, from its natural environment. For example, an isolated polypeptide is generally free of at least some proteins or other cellular components that are normally associated with it or are normally mixed with it or in solution. Isolated polypeptides include naturally produced polypeptides contained in cell lysates, polypeptides in purified or partially purified form, recombinant polypeptides, polypeptides expressed or secreted by cells, and polypeptides in heterologous host cells or cultures. In connection with a polynucleotide, the term isolated or purified indicates that, for example, the nucleic acid is not in its natural genomic context (e.g., in a vector, as an expression cassette, connected to a promoter, or artificially introduced into a heterologous host cell).

[0056] In this article, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. The vector can be transformed, transduced or transfected into the host cell so that the genetic material elements it carries are expressed in the host cell. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. The types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain a variety of elements that control expression, including promoter sequences, transcription start sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site. A vector may also include components that assist it in entering cells, such as viral particles, liposomes, or protein coats, but is not limited to these substances.

[0057] In this article, the term "cell" generally refers to a single cell, cell line or cell culture that can be or has been a recipient of a subject polynucleotide or vector, including a nucleic acid molecule of the present invention or a vector of the present invention. A cell may include the offspring of a single cell. Due to natural, accidental or intentional mutations, the offspring may not necessarily be identical to the original parent cell (in the form of the total DNA complement or in the genome). A cell may include a cell transfected in vitro with the vector of the present invention. The cell can be a bacterial cell (e.g., E. coli), a yeast cell or other eukaryotic cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a CHO-K1 cell, a LNCAP cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NS0 cell, a human non-small cell lung cancer A549 cell, a human skin squamous cell carcinoma A431 cell, a renal clear cell adenocarcinoma 786-O cell, a human pancreatic cancer MIA PaCa-2 cell, an erythroleukemia K562 cell, an acute T-cell leukemia Jurkat cell, a human breast cancer MCF-7 cell, a human breast cancer MDA-MB-231 cell, a human breast cancer MDA-MB-468 cell, a human breast cancer SKBR3 cell, a human ovarian cancer SKOV3 cell, a lymphoma U-937 cell, a lymphoma Raji cell, a human myeloma U266 cell, or a human multiple myeloma RPMI8226 cell. In certain embodiments, the cell is a mammalian cell.

[0058] In this article, the term "pharmaceutical composition" generally refers to a composition suitable for application to a patient, preferably a human patient. For example, the pharmaceutical composition may include the separated antigen-binding proteins of the present invention, the immunoconjugates of the present invention, the nucleic acid molecules of the present application, the carriers of the present application and / or the cells of the present application, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may also include one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. The acceptable ingredients of the composition are preferably nontoxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present invention includes, but is not limited to, liquid, frozen and lyophilized compositions.

[0059] As used herein, the term "pharmaceutically acceptable carrier" generally refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration and are generally safe, non-toxic, and neither biologically nor otherwise undesirable.

[0060] As used herein, the term "treatment" refers to any type of intervention or treatment performed on a subject, or the administration of an active agent to a subject, with the purpose of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of symptoms, complications, conditions or biochemical indicators associated with a disease. In some embodiments, the disease is a malignant blood disease. In other embodiments, the disease is a solid tumor.

[0061] As used herein, "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, monkeys, sheep, and guinea pigs. In some embodiments, the subject is a human. The terms "subject" and "patient" and "individual" are used interchangeably herein.

[0062] In this application, the terms "sample" and "specimen" are used interchangeably herein and refer to a composition obtained or derived from a subject (e.g., an individual of interest) that contains cells and / or other molecular entities to be characterized and / or identified based on, for example, physical, biochemical, chemical and / or physiological characteristics. For example, the phrase "disease sample" or a variant thereof refers to any sample obtained from a subject of interest that is expected or known to contain cells and / or molecular entities to be characterized. Samples include, but are not limited to, tissue samples (e.g., tumor tissue samples), primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymphatic fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates, and tissue culture fluid, tissue extracts such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof.

[0063] In a first aspect, an isolated antigen-binding protein that specifically binds to SEMA4D is provided, comprising complementary determining regions HCDR1, HCDR2 and HCDR3 of a heavy chain variable region;

[0064] The amino acid sequence of the HCDR1 is shown in SEQ ID NO.1;

[0065] The amino acid sequence of the HCDR2 is shown in SEQ ID NO.2;

[0066] The amino acid sequence of HCDR3 is shown in SEQ ID NO.3.

[0067] In an optional embodiment, the heavy chain variable region of the isolated antigen-binding protein contains a heavy chain framework region, wherein each heavy chain framework region is independently an antibody or a mutant thereof from the following species: camel, alpaca, mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey or human.

[0068] In an optional embodiment, the heavy chain framework regions of the isolated antigen-binding proteins are all derived from the heavy chain framework regions of human antibodies or mutants thereof.

[0069] In an alternative embodiment, the heavy chain variable region contains heavy chain framework regions HFR1, HFR2, HFR3 and HFR4.

[0070] In an optional embodiment, the amino acid sequence of HFR1 is shown as SEQ ID NO.4.

[0071] In an optional embodiment, the amino acid sequence of HFR2 is shown as SEQ ID NO.5.

[0072] In an optional embodiment, the amino acid sequence of HFR3 is shown as SEQ ID NO.6.

[0073] In an optional embodiment, the amino acid sequence of HFR4 is shown as SEQ ID NO.7.

[0074] In an optional embodiment, the amino acid sequence of HFR1 is shown as SEQ ID NO.4, the amino acid sequence of HFR2 is shown as SEQ ID NO.5, the amino acid sequence of HFR3 is shown as SEQ ID NO.6, and the amino acid sequence of HFR4 is shown as SEQ ID NO.7.

[0075] In an optional embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.8.

[0076] In an alternative embodiment, the isolated antigen binding protein is an antibody or an antigen binding fragment thereof.

[0077] In an optional embodiment, the isolated antigen-binding protein is selected from a nanobody, a full-length antibody, a F(ab') 2 , Fab', Fab, Fv, scFv, dsFv, diabodies or multispecific antibodies.

[0078] In an alternative embodiment, the isolated antigen-binding protein further comprises a constant region or a portion of a constant region of an antibody.

[0079] In an optional embodiment, the isolated antigen-binding protein contains a constant region or a portion of a constant region whose entire or partial sequence originates from species including one or more of camels, alpacas, mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, cows, pigs, horses, monkeys and humans.

[0080] In an alternative embodiment, the constant region contained in the isolated antigen-binding protein can be selected from the sequence or partial sequence of any one of the constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD, as well as their subclasses and mutant forms.

[0081] In an alternative embodiment, the isolated antigen binding protein is a humanized antibody or an antigen binding fragment thereof.

[0082] In an alternative embodiment, the isolated antigen binding protein is a fully human antibody or an antigen binding fragment thereof.

[0083] In an alternative embodiment, the isolated antigen binding protein is a Nanobody.

[0084] In an optional embodiment, the CH2 and CH3 regions of the nanobody are derived from the constant regions of human antibodies.

[0085] In an optional embodiment, the amino acid sequence of the constant region of the Nanobody is as shown in SEQ ID NO.10.

[0086] In an optional embodiment, the amino acid sequence of the nanobody is as shown in SEQ ID NO.9.

[0087] In the second aspect, a fusion protein is also provided, wherein the fusion protein contains the antigen-binding protein in the first aspect.

[0088] Exemplary fusion proteins are, for example, but not limited to, antigen chimeric receptors, chimeric antigen receptors (CAR) are recombinant transmembrane molecules mainly composed of an extracellular domain, a transmembrane domain, and an intracellular signal transduction domain, and are artificially constructed receptors that can recognize specific antigens. After the tumor cell antigen binds to the antigen binding fragment, the signal is transmitted to the cell through the hinge transmembrane region and the intracellular signal transduction domain to be converted into an activation signal and activate effector cells, so that immune cells produce cytokines or perforins that kill tumor cells, and proliferate at the same time to further expand the killing effect. The separated antigen binding protein of the first aspect can be combined with other parts of CAR, for example, as a heavy chain variable region of a single-chain antibody in CAR to bind to tumor cell antigens.

[0089] In a third aspect, a biomaterial is also provided, wherein the biomaterial is selected from any one of (I) to (III):

[0090] (I) A polynucleotide encoding the isolated antigen-binding protein of the first aspect, or the fusion protein of the second aspect.

[0091] In an optional embodiment, the polynucleotide also encodes a signal peptide fused to the mature heavy chain. The coding sequence on the polynucleotide can be operably linked to a regulatory sequence to ensure expression of the coding sequence, such as a promoter, enhancer, ribosome binding site, transcription termination signal, etc.

[0092] In an optional embodiment, when the isolated antigen-binding protein also contains a light chain or a light chain variable region, the nucleic acids encoding the heavy and light chains may be present in a separated form, or may be cloned into one or more vectors. The polynucleotides encoding the heavy and light chains may be, for example, connected as a continuous polynucleotide in an expression vector, or may be separate, for example, each cloned into its own expression vector.

[0093] In alternative embodiments, the polynucleotides may be synthesized by, for example, solid-state synthesis of overlapping oligonucleotides or PCR.

[0094] In an alternative embodiment, the polynucleotide is an isolated polynucleotide.

[0095] (II) A vector carrying the polynucleotide described in (I).

[0096] In an optional embodiment, the vector is an expression vector, a vector for cloning the polynucleotide, or a vector for integrating the polynucleotide into the genome of a target cell.

[0097] In an optional embodiment, the vector is an expression vector, which refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. In addition to the polynucleotide described above, the expression vector also includes an expression control sequence operably linked to the nucleic acid sequence. The vector can be transformed, transduced or transfected into a host cell so that the genetic material elements it carries can be expressed in the host cell. In addition to the origin of replication, the expression vector may also contain marker genes and other translation control elements.

[0098] (III) a cell, wherein the cell is capable of expressing the isolated antigen-binding protein of the first aspect, or expressing the fusion protein of the second aspect, or carrying the polynucleotide of (I), or containing the vector of (II). The cell includes a prokaryotic cell and a eukaryotic cell. The prokaryotic cell includes a bacterial cell. The eukaryotic cell includes a protist cell, an animal cell, a plant cell, and a fungal cell. The animal cell includes a mammalian cell, an avian cell, and an insect cell.

[0099] In an optional embodiment, the cell is a host cell for expressing the separated antigen-binding protein of the first aspect, or expressing the fusion protein of the second aspect. Usually, after expressing the antigen-binding protein or fusion protein, the host cell also includes separating and enriching the antigen-binding protein or fusion protein to obtain purified protein.

[0100] In an optional embodiment, the cell is an immune cell modified by a chimeric antigen receptor. "Chimeric antigen receptor-modified immune cells (CAR-modified immune cells)" are well known in the art. They use genetic modification technology to express immune cells with antigen-specific chimeric receptors (such as tumor antigens) and can target and kill. In an optional embodiment, the immune cell is selected from T cells (e.g., α / β T cells and γ / δ T cells), natural killer cells (NK cells), monocytes, macrophages, NKT cells (Natural killer T cells), dendritic cells, granulocytes, B cells, lymphocytes, leukocytes and / or peripheral blood mononuclear cells.

[0101] In a fourth aspect, a method for preparing the isolated antigen-binding protein of the first aspect is also provided, comprising expressing the antigen-binding protein in a cell.

[0102] In an optional embodiment, the cell comprises the cell in any embodiment of the biomaterial of the second aspect.

[0103] In an optional embodiment, the preparation method comprises culturing the cell of the second aspect under conditions suitable for expressing the antigen binding protein and recovering the antigen binding protein.

[0104] In an optional embodiment, the polynucleotide encoding the antigen-binding protein or the vector containing the same can be transformed or transfected into cells using conventional techniques well known to those skilled in the art. Exemplary transformation or transfection methods include but are not limited to: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation or liposome packaging, etc.

[0105] In a fifth aspect, an immunoconjugate is also provided, wherein the immunoconjugate contains the antigen-binding protein of the first aspect and at least one therapeutic agent and / or at least one diagnostic agent coupled thereto.

[0106] Exemplary therapeutic agents include, but are not limited to, one or more of a cytotoxic agent, a compound drug, a radionuclide, a boron atom, an immunomodulator, an anti-apoptotic agent, a photosensitizing therapeutic agent, and a polynucleotide.

[0107] Exemplary compound drugs include, but are not limited to, one or more of methotrexate, fluorouracil, mercaptopurine, hydroxyurea, cytarabine, nitrogen mustard, cyclophosphamide, thiotepa, cisplatin, mitomycin, bleomycin, camptothecin, podophyllotoxin, actinomycin D, doxorubicin, daunorubicin, vinblastine, paclitaxel, cephalotaxel alkaloids, and L-asparaginase.

[0108] Exemplary radionuclides include, but are not limited to 110 In, 111 In, 177 Lu, 18 F. 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y. 90 Y. 89 Zr, 94 mTc, 94 Tc, 99 mTc, 120 I. 123 I. 124 I. 125 I. 131 I. 154-158 Gd, 32 P. 11 C. 13 N. 15 O. 186 Re, 188 Re, 51 Mn, 52 mMn, 55 Co. 72 As, 75 Br, 76 Br, 82 mRb and 83 One or more of Sr.

[0109] Exemplary immunomodulators include, but are not limited to, one or more of cytokines, chemokines, stem cell growth factors, lymphotoxins, hematopoietic factors, colony stimulating factors (CSFs), interferons, erythropoietins, thrombopoietins, tumor necrosis factors (TNFs), interleukins (ILs), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), and stem cell growth factors.

[0110] Exemplary polynucleotides include, but are not limited to, one or more of shRNA, miRNA, and siRNA.

[0111] Exemplary diagnostic agents include, but are not limited to, one or more of radiocontrast agents, paramagnetic ions, metals, luminescent materials, enzymes, prosthetic groups, ultrasound contrast agents, photosensitizers, biotin, streptavidin, etc.

[0112] Exemplary radionuclides include, but are not limited to 110 In, 111 In, 177 Lu, 18 F. 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y. 90 Y. 89 Zr, 94 mTc, 94 Tc, 99 mTc, 120 I. 123 I. 124 I. 125 I. 131 I. 154-158 Gd, 32 P. 11 C. 13 N. 15 O. 186 Re, 188 Re, 51 Mn, 52 mMn, 55 Co. 72 As, 75 Br, 76 Br, 82 mRb and 83 One or more of Sr.

[0113] Exemplary paramagnetic ions include, but are not limited to, one or more of chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and erbium (III).

[0114] Exemplary fluorescent labels include, but are not limited to, Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1,3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresol fast violet, cresol blue violet, brilliant cresol blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinylfluorescein, rare earth metal cryptates, trisbipyridyl diamine europium, europium cryptates or chelates, diamines, bicyanines, La Jolla blue dye, allophycocyanin, allococyanin B, one or more of phycocyanin C, phycocyanin R, thiamine, phycoerythrin, phycoerythrin R, REG, rhodamine green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine and Texas Red.

[0115] Exemplary radionuclides include, but are not limited to 111 In, 111 At 177 Lu, 211 Bi, 212 Bi, 213 Bi, 211 At 62 Cu, 67 Cu, 90 Y. 125 I. 131 I. 133 I. 32 P. 33 P. 47 Sc, 111 Ag, 67 Ga, 153 Sm, 161 Tb, 152 Dy, 166Dy, 161 Ho, 166 Ho, 186 Re, 188 Re, 189 Re, 211 Pb, 212 Pb, 223 Ra, 225 Ac, 77 As, 89 Sr, 99 Mo, 105 Rh, 149 Pm, 169 Er, 194 Ir, 58 Co, 80 mBr, 99 mTc, 103 mRh, 109 Pt, 119 Sb, 189 mOs, 192 Ir, 219 Rn, 215 Po, 221 Fr, 255 Fm, 11 C, 13 N, 15 O, 75 Br, 198 Au, 199 Au, 224 Ac, 77 Br, 113 mIn, 95 Ru, 97 Ru, 103 Ru, 105 Ru, 107 Hg, 203 Hg, 121 mTe, 122 mTe, 125 mTe, 165 Tm, 167 Tm, 168 Tm, 197 Pt, 109 Pd, 142 Pr, 143 Pr, 161 Tb, 57 Co, 58 Co, 51 Cr, 59 Fe, 75 Se, 201 Tl, 76 Br and 169One or more of Yb.

[0116] Exemplary enzymes include, but are not limited to, alkaline phosphatase and horseradish peroxidase.

[0117] In a sixth aspect, there is also provided the use of the isolated antigen-binding protein of the first aspect, or the fusion protein of the second aspect, or the biomaterial of the third aspect, or the preparation method of the fourth aspect, or the immunoconjugate of the fifth aspect in any of the following:

[0118] (i) preparing a blocking agent for blocking PlexinB1 and / or PlexinB2 molecules;

[0119] (ii) blocking PlexinB1 and / or PlexinB2;

[0120] (iii) preparing a medicament for treating, preventing or alleviating a disease or condition associated with SEMA4D signaling;

[0121] (iv) Preparation of a kit for detecting SEMA4D.

[0122] (v) Detection of SEMA4D.

[0123] In an optional embodiment, the blocking agent in (i) further contains a carrier acceptable in the art, including but not limited to a buffer and / or a culture medium.

[0124] In an alternative embodiment, the blocking of PlexinB1 and / or PlexinB2 in (ii) is for non-diagnostic and non-therapeutic purposes.

[0125] In an optional embodiment, the blocking of PlexinB1 and / or PlexinB2 comprises allowing the cells to which the binding of SEMA4D to PlexinB1 and / or PlexinB2 is to be blocked to be fully contacted with the antigen binding protein of the first aspect or the immunoconjugate of the fifth aspect.

[0126] In an alternative embodiment, the detection of SEMA4D in (v) is for non-diagnostic and non-therapeutic purposes.

[0127] In an alternative embodiment, the detecting of SEMA4D comprises administering the antigen binding protein of the first aspect or the immunoconjugate of the fifth aspect to the subject.

[0128] In an optional embodiment, the detection of SEMA4D is an in vitro and / or ex vivo method.

[0129] In an alternative embodiment, the detecting of SEMA4D comprises contacting the sample with the antigen binding protein of the first aspect or the immunoconjugate of the fifth aspect.

[0130] In alternative embodiments, the labeling for detection and / or analysis and / or diagnostic purposes depends on the specific detection / analysis / diagnosis technique and / or method used, such as immunohistochemical staining of (tissue) samples, flow cytometry, laser scanning cytometry detection, fluorescent immunoassay, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), bioassay (e.g., phagocytosis assay), Western blot application, etc. Suitable labels for detection / analysis / diagnosis techniques and / or methods known in the art are well known to those skilled in the art.

[0131] In an optional embodiment, for the kit for detecting SEMA4D prepared in (iv), a person skilled in the art can select the reagent composition in the kit according to the detection and / or analysis means of the kit, for example, a SEMA4D detection kit based on immunohistochemistry staining, a SEMA4D detection kit based on flow cytometry, a SEMA4D detection kit based on laser scanning cytometry, a SEMA4D detection kit based on ELISA detection or a SEMA4D detection kit based on protein blotting is prepared according to the above-mentioned detection and / or analysis means. Other components in the exemplary kit include, but are not limited to, one or more of a buffer, a washing solution, an eluent, a solid phase carrier (e.g., a magnetic particle, an ELISA plate or an NC membrane), an enzyme for reaction, a reaction substrate, a secondary antibody, a positive control, a negative control and a blank control.

[0132] In the seventh aspect, a pharmaceutical composition is also provided, comprising the isolated antigen-binding protein of the first aspect, or the fusion protein of the second aspect, or the biomaterial of the third aspect, or the immunoconjugate of the fifth aspect.

[0133] In an optional embodiment, the separated antigen binding protein of the first aspect, or the fusion protein of the second aspect, or the biomaterial of the third aspect, or the immunoconjugate of the fifth aspect can be used in combination with existing therapeutic drugs, as long as it can produce a better therapeutic effect on the targeted indication. The combined administration includes simultaneous administration or non-simultaneous administration. Exemplary existing therapeutic drugs include, but are not limited to, protein molecules, including but not limited to peptides, polypeptides, proteins, including post-translationally modified proteins, fusion proteins, antibodies, etc.; small molecules, including inorganic or organic compounds; nucleic acid molecules, including but not limited to double-stranded or single-stranded DNA, or double-stranded or single-stranded RNA (such as antisense (molecules), RNAi, etc.), intron sequences, triple-helix nucleic acid molecules and aptamers; or vaccines. Or any one or more of the therapeutic agents in the aforementioned fourth aspect.

[0134] In an optional embodiment, the pharmaceutical composition further includes other therapeutic agents for treating tumors, autoimmune diseases or inflammation, preferably one or more selected from tumor-associated antigen monoclonal antibodies and their related ADCs (antibody drug conjugates), chemotherapeutic drugs and radiotherapeutic drugs.

[0135] In an optional embodiment, the pharmaceutical composition further includes a pharmaceutically acceptable carrier, including but not limited to any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the Food and Drug Administration for use in humans or animals, and various forms of carriers that have no side effects on the composition of the pharmaceutical composition.

[0136] In an optional embodiment, the pharmaceutical composition is used to treat, prevent or alleviate diseases and / or conditions associated with SEMA4D signaling.

[0137] In the eighth aspect, a kit for detecting Sema4D is also provided, the kit comprising the isolated antigen binding protein of the first aspect, or the fusion protein of the second aspect, or the biomaterial of the third aspect, or the immunoconjugate of the fifth aspect. The detection of Sema4D includes detecting the isolated Sema4D protein in the sample or subject, or a complex containing it, or a cell expressing it, for example, detecting the abundance of SEMA4D expression in a clinical sample, which can be used in clinical diagnosis and prognosis. The detection of Sema4D includes qualitative and / or quantitative detection.

[0138] In an optional embodiment, a kit for detecting Sema4D comprises the isolated antigen-binding protein of the first aspect, and the detection kit is used to detect the presence and / or content of the SEMA4D protein in a sample or a subject.

[0139] In optional embodiments, the diseases and / or conditions associated with SEMA4D signaling in any of the above embodiments include, but are not limited to, autoimmune diseases mediated by cells expressing SEMA4D, inflammatory diseases (including central nervous system (CNS) and peripheral nervous system (PNS) inflammatory diseases), cancer, and invasive angiogenesis.

[0140] Exemplary cancers include, but are not limited to, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, colon cancer, breast cancer, lymphoma, leukemia, and lung cancer. Exemplary autoimmune diseases include, but are not limited to, arthritis and multiple sclerosis, arthritis being an inflammatory disease of the joints; exemplary inflammatory diseases include, but are not limited to, inflammatory diseases of the central nervous system (CNS) and peripheral nervous system (PNS).

[0141] In an alternative embodiment, the disease or disorder associated with SEMA4D signaling comprises an autoimmune disease or an inflammatory disease.

[0142] In an optional embodiment, the antigen binding protein of the first aspect specifically binding to SEMA4D or a pharmaceutical composition containing it is used to treat multiple sclerosis (MS). Multiple sclerosis, also known as diffuse sclerosis or diffuse encephalomyelitis, is an autoimmune disease in which the immune system attacks the central nervous system, leading to demyelination. The name multiple sclerosis refers to scars (sclerosis, also known as plaques or lesions) formed in the nervous system. MS lesions generally include white matter areas close to the cerebellar ventricles, brainstem, basal ganglia and spinal cord and optic nerves. MS causes the destruction of oligodendrocytes, which are responsible for the production and maintenance of myelin. MS causes myelin to decrease or disappear completely, and as the disease progresses, it also causes neuronal transection. Neurological symptoms can change with MS, and the disease often develops to physical and cognitive disability. MS has a variety of forms, with new symptoms independently attacking (relapsing form) or slowly accumulating over time (progressive form). Between attacks, symptoms may disappear completely, but permanent nerve damage is often produced, especially when the disease progresses deeper.

[0143] Antigen binding proteins that specifically bind to SEMA4D or pharmaceutical compositions containing the same can reduce the severity of MS through several different mechanisms, for example, the immune maturation and activation of SEMA4D can be blocked to reduce the relapse rate by reducing the secondary immune response to CNS antigens, the role of soluble SEMA4D in mediating oligodendrocyte apoptosis in the CNS can be blocked, and the severity of the disease can be alleviated by reducing demyelination.

[0144] In an optional embodiment, the disease or condition associated with SEMA4D signaling comprises arthritis, which is an inflammatory disease of the joints that may be caused by an autoimmune condition in which the immune system attacks the joints. In certain embodiments, the arthritis is selected from osteoarthritis, gouty arthritis, ankylosing spondylitis, psoriatic arthritis, active arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, infectious arthritis, inflammatory arthritis, septic arthritis, degenerative arthritis, destructive arthritis (arthritis mutilans) and Lyme arthritis. In one embodiment, the arthritis is rheumatoid arthritis (RA).

[0145] In an optional embodiment, the present invention also provides a method for diagnosing a disease or condition associated with the expression of SEMA4D protein in a subject, the method comprising: contacting a sample derived from the subject with the isolated antigen-binding protein of the first aspect or the immunoconjugate of the fifth aspect, and determining the presence and / or content of a substance capable of specifically binding to the antigen-binding protein in the sample.

[0146] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0147] Example 1 Screening of anti-SEMA4D nanobodies

[0148] Camels immunized with human SEMA4D were used to screen and identify nanobodies. Human-monkey crosses were screened, and clones with good binding activity and blocking activity were selected. After analyzing the target sequence, they were humanized to obtain humanized nanobodies MY8457, MY8458, and MY8459. The CDR and HFR sequences of nanobody MY8457 are shown in Table 1.

[0149] Table 1 Sequence of humanized SEMA4D antibody MY8457

[0150] name sequence Serial Number HFR1 EVQLVESGGGLVQPGGSLKLSCVASGYGYC SEQ ID NO.4 HCDR1 RYNMN SEQ ID NO.1 HFR2 WYRQAPGKGLEFVS SEQ ID NO.5 HCDR2 AIDSDGSTSYADSVKG SEQ ID NO.2 HFR3 RFTISRDNAYNVVTLQMNSLKPEDAAMYYCKT SEQ ID NO.6 HCDR3 DSSDMICRDYGMDS SEQ ID NO.3 HFR4 WGKGTLVTVSS SEQ ID NO.7

[0151] The amino acid sequence of the variable region of the nanobody is SEQ ID NO.8, the constant region sequence is SEQ ID NO.10, and the full-length sequence is SEQ ID NO.9:

[0152] SEQ ID NO.8:

[0153] EVQLVESGGGLVQPGGSLKLSCVASGYGYCRYNMNWYRQAPGKGLEFVSAIDSDGSTSYADSVKGRFTISRDNAYNVVTLQMNSLKPEDAAMYYCKTDSSDMICRDYGMDSWGKGTLVTVSS.

[0154] SEQ ID NO.9:

[0155] EVQLVESGGGLVQPGGSLKLSCVASGYGYCRYNMNWYRQAPGKGLEFVSAIDSDGSTSYADSVKGRFTISRDNAYNVVTLQMNSLKPEDAAMYYCKTDSSDMICRDYGMDSWGKGTLVTVSSDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG.

[0156] SEQ ID NO.10:

[0157] DHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG.

[0158] Example 2 Detection of the binding of anti-SEMA4D humanized antibody to SEMA4D overexpressing positive overexpressing cells 293T-Sema4D

[0159] Human embryonic kidney cells 293T-SEMA4D overexpressing human SEMA4D were collected and 5 × 10 5cells were added to a 96-well deep-well plate; different concentrations of anti-SEMA4D humanized antibodies MY8457, MY8458 and MY8459 and control drug L553 (sequence information refers to patent application disclosure WO2010 / 129917) were added and incubated on ice for 30 minutes; after washing with FACS washing solution, PE fluorescent-labeled goat anti-human IgG Fc secondary antibody (Goat Anti-Human IgG Fc Secondary Antibody, Invitrogen) was added and incubated on ice in the dark for 30 minutes; FACS washing solution was used for two times; 400 μL of 1% paraformaldehyde fixative (Solarbio) was added to each tube to fix the cells, and after mixing, the percentage of PE fluorescent positive cells was detected on the machine to analyze the binding ability of the three anti-SEMA4D humanized antibodies to the positive overexpressing cells 293T-Sema4D overexpressing SEMA4D.

[0160] The results are as follows Figure 1 shown. Figure 1 The data show that the affinity of the three humanized Anti-Sema4D antibodies MY8457, MY8458 and MY8459 for binding to 293T-human Sema4D cells is close to the binding activity of the control drug L553.

[0161] Example 3 Evaluation of the Binding Activity of Three Humanized Antibodies to Block Sema4D Binding to 293T-PlexinB1#7 Cells

[0162] Different concentrations of anti-SEMA4D humanized antibodies MY8457, MY8458 and MY8459 and control drug L553 were mixed with Sema4D-Biotin diluted to 1 μg / mL in equal proportions and incubated in a 37°C incubator for 1 h. Human embryonic kidney cells 293T-PlexinB1 overexpressing human PlexinB1 were collected and 5×10 5 cells were added to a 96-well deep-well plate; 100 μL of the incubated antibody and Sema4D-biotin mixture was added to each well and incubated on ice for 30 min; after washing with FACS washing solution, PE fluorescent-labeled secondary antibody (PE-Streptavidin, Cat: 554061, BD) was added and incubated on ice in the dark for 30 min; FACS washing solution was used for two times; 400 μL of 1% paraformaldehyde fixative (Solarbio) was added to each tube to fix the cells, and after mixing, the percentage of PE fluorescent-positive cells was detected on the machine, and the ability of the three anti-SEMA4D humanized antibodies to block the binding of Sema4D-biotin to the positive cells 293T-PlexinB1 overexpressing Human PlexinB1 was analyzed.

[0163] The results are as follows Figure 2 shown. Figure 2 The results show that the activities of three humanized Anti-Sema4D antibodies MY8457, MY8458 and MY8459 in blocking the binding of Sema4D-biotin to 293T-PlexinB1 cells are comparable to those of the positive control drug L553, and MY8457 has the best blocking activity among the three humanized antibodies.

[0164] Example 4 Evaluation of humanized antibody binding activity to block Sema4D binding to 293T-PlexinB2#7 cells

[0165] Different concentrations of anti-SEMA4D humanized antibodies MY8457, MY8458 and MY8459 and control drug L553 were mixed with Sema4D-Biotin diluted to 1 μg / mL in equal proportions and incubated in a 37°C incubator for 1 h. Human embryonic kidney cells 293T-PlexinB2 overexpressing human PlexinB2 were collected and 5×10 5 cells were added to a 96-well deep-well plate; 100 μL of the incubated antibody and Sema4D-biotin mixture was added to each well and incubated on ice for 30 min; after washing with FACS washing solution, PE fluorescently labeled secondary antibody (PE-Streptavidin, Cat: 554061, BD) was added and incubated on ice in the dark for 30 min; FACS washing solution was used for two times; 400 μL of 1% paraformaldehyde fixative (Solarbio) was added to each tube to fix the cells, and the average fluorescence intensity of PE fluorescence was detected on a machine after mixing, and the ability of the three anti-SEMA4D humanized antibodies to block the binding of Sema4D-biotin to the positive cells 293T-PlexinB2 overexpressing Human PlexinB2 was analyzed.

[0166] The results are as follows Figure 3 shown. Figure 3 The results show that among the three humanized antibodies of Anti-Sema4D, MY8458 and MY8459 have similar activities in blocking the binding of Sema4D-biotin to 293T-PlexinB2 cells, both of which are stronger than the control drug L553; MY8457 has similar blocking activities as L553. MY8458 and MY8459 have better activities in blocking the binding of Sema4D to the receptor PlexinB2.

[0167] Sema4D promotes angiogenesis and leakage by binding to the PlexinB1 receptor, and mediates pro-inflammatory responses by binding to PlexinB2, indirectly promoting angiogenesis and leakage. Based on the interaction results between Sema4D and these two receptors, the antibody MY8457, which binds better to PlexinB1 and has higher PlexinB1 blocking activity, is preferred to achieve the purpose of treating SEMA4D-related diseases described in the present invention, especially diseases related to angiogenesis.

[0168] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nanobody that specifically binds to human SEMA4D, characterized in that: The nanobody comprises the complementarity determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region; The amino acid sequence of the HCDR1 is shown in SEQ ID NO.1; the amino acid sequence of the HCDR2 is shown in SEQ ID NO.2; and the amino acid sequence of the HCDR3 is shown in SEQ ID NO.

3.

2. The Nanobody according to claim 1, characterized in that The heavy chain variable region contains a heavy chain framework region, and each heavy chain framework region is independently an antibody or a mutant thereof from the following species: camel, alpaca, mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey or human.

3. The Nanobody according to claim 2, characterized in that The heavy chain variable region contains heavy chain framework regions HFR1, HFR2, HFR3 and HFR4; the amino acid sequence of HFR1 is shown as SEQ ID NO.4; and / or, the amino acid sequence of HFR2 is shown as SEQ ID NO.5; and / or, the amino acid sequence of HFR3 is shown as SEQ ID NO.6; and / or, the amino acid sequence of HFR4 is shown as SEQ ID NO.

7.

4. The Nanobody according to claim 3, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

8.

5. The Nanobody according to any one of claims 1 to 4, characterized in that The amino acid sequence of the nanobody is shown in SEQ ID NO.

9.

6. Biomaterial, characterized in that Select any one of (I) to (III): (I) a polynucleotide encoding the Nanobody according to any one of claims 1 to 5; (II) a vector carrying the polynucleotide described in (I); (III) A cell capable of expressing the Nanobody according to any one of claims 1 to 5, or carrying the polynucleotide according to (I), or containing the vector according to (II).

7. The method for preparing a nanobody according to any one of claims 1 to 5, characterized in that: Comprising expressing said Nanobody in a cell.

8. An immunoconjugate, characterized in that Composed of the Nanobody according to any one of claims 1 to 5 and at least one diagnostic agent coupled to the Nanobody; The diagnostic agent includes one or more of a radioactive contrast agent, a paramagnetic ion, a metal, a luminescent material, an enzyme, a prosthetic group, an ultrasound contrast agent, a photosensitizer, biotin, and streptavidin.

9. Use of the Nanobody according to any one of claims 1 to 5, or the biomaterial according to claim 6, or the immunoconjugate according to claim 8 in any of the following: (i) preparing a kit for detecting SEMA4D; (ii) Detection of SEMA4D for non-diagnostic and therapeutic purposes.

10. Use of the nanobody according to any one of claims 1 to 5, or the biomaterial according to claim 6, in the preparation of a drug for treating, preventing or alleviating diseases and / or conditions associated with SEMA4D signaling; The diseases and / or conditions associated with SEMA4D signaling are head and neck cancer and multiple sclerosis.

11. A pharmaceutical composition, characterized in that Comprising the nanobody according to any one of claims 1 to 5, or the biomaterial according to claim 6.

12. A kit for detecting SEMA4D, characterized in that: Comprising the nanobody according to any one of claims 1 to 5, or the biomaterial according to claim 6.

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