SEMA3G antigen binding proteins and uses thereof

Abstract

The invention relates to the field of biological medicine, in particular to SEMA3G antigen binding protein and application thereof. The invention provides a separated antigen binding protein, the antigen binding protein is bound with SEMA3G protein, and the antigen binding protein comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises an HCDR1 as shown in any one of SEQ ID NO.3, 11 and 19, an HCDR2 as shown in any one of SEQ ID NO.4, 12 and 20, and an HCDR3 as shown in any one of SEQ ID NO.5, 13 and 21; the light chain variable region comprises an LCDR1 as shown in any one of SEQ ID NO.6, 14 and 22, an LCDR2 as shown in any one of SEQ ID NO.7, 15 and 23, and an LCDR3 as shown in any one of SEQ ID NO.8, 16 and 24; the antigen binding protein can bind to the target protein with high affinity and neutralize the inhibitory activity of the antigen binding protein to the T cell function, so that the effect of relieving the immune escape function of tumor cells is achieved, killing of immune cells to tumor cells in vivo and in vitro is promoted, and the antigen binding protein can be used for preparing drugs for adjusting immune response or having the anti-tumor effect.

Description

Technical Field

[0001] This application relates to the biomedical field, specifically to the SEMA3G antigen-binding protein and its uses. Background Technology

[0002] Immune checkpoint blockade (ICB) therapy enhances the body's immune response to tumors by targeting inhibitory immune regulatory molecules, opening new avenues for cancer treatment. Monoclonal antibody drugs, such as immune checkpoint inhibitors like CTLA-4, PD-1, PD-L1, and LAG-3, as well as chimeric antigen receptor T-cell (CAR-T) therapy, have demonstrated significant efficacy and durable responses in patients with various cancer types. Nevertheless, some patients receiving ICB treatment fail to respond, and some initially responsive patients eventually develop resistance and tumor progression. This suggests that some currently under-recognized immunosuppressive mechanisms may exist during tumor development, potentially promoting tumor immune escape. Therefore, there is an urgent need to further explore novel immune checkpoint molecules as potential targets for immunotherapy, aiming to improve the effectiveness and durability of treatment.

[0003] Semaphorins were initially discovered in studies of nervous system development, acting as signaling molecules to guide axonal growth. A series of studies have shown that semaphorins and their receptors are widely expressed in various cell types, including neurons, endothelial cells, and various tumor cells. These molecules play crucial roles in regulating physiological processes such as cell development, morphogenesis, and immune responses. Based on the similarity of their amino acid sequences and unique structural features, the semaphore family is classified into eight different subfamilies. Among them, members of the SEMA3 family are secreted proteins, including seven subtypes: A, B, C, D, E, F, and G. SEMA3 family proteins primarily exert their biological functions by binding to neuropilin and plexin receptor complexes. SEMA3G, as the most recently discovered member of the SEMA3 subfamily, still lacks in-depth research on its biological functions in the body, particularly its role in immune regulation and its mechanisms of action.

[0004] Neuropilins (NRPs) are a class of type I transmembrane glycoproteins with a molecular weight of approximately 120 to 130 kilodaltons (kDa). The molecular structure of NRPs consists of three main parts: an intracellular region, a transmembrane helical region, and an extracellular region. The intracellular region is responsible for signal transduction, the transmembrane helical region anchors the protein to the cell membrane, and the extracellular region participates in the specific binding of ligands. Recent studies have found that the expression of NRPs in various immune cell subsets plays an important role in the regulation of immune responses. NRPs not only participate in the migration, proliferation, and differentiation of immune cells, but may also affect tumor immune escape and the efficacy of immunotherapy by influencing the interaction between immune cells and tumor cells. These biological functions of NRPs suggest that they may play a key role in the tumor immune microenvironment. For example, NRPs may affect the distribution and function of immune cells in the tumor microenvironment by binding to specific members of the semaphorin family. Our research revealed that NRP1 is a high-affinity receptor for SEMA3G and plays a crucial role in SEMA3G-mediated tumor immunosuppression. Therefore, the development of novel SEMA3G modulators is of great significance. Currently, the development of highly specific and high-affinity SEMA3G antibodies remains to be achieved. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this application provides an isolated antigen-binding protein capable of binding to SEMA3G protein, including a SEMA3G antibody or its antigen-binding fragment. This antigen-binding protein exhibits high specificity and affinity for SEMA3G protein, enabling it to block the binding of SEMA3G protein to its receptor NRP protein, thereby blocking SEMA3G-induced T cell dysfunction. This protein can be used to prepare drugs with immunomodulatory or antitumor effects.

[0006] The first aspect of this application provides an isolated antigen-binding protein, the antigen-binding protein comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprising HCDR1 as shown in any one of SEQ ID NO. 3, 11, 19, HCDR2 as shown in any one of SEQ ID NO. 4, 12, 20, and HCDR3 as shown in any one of SEQ ID NO. 5, 13, 21; the light chain variable region comprising LCDR1 as shown in any one of SEQ ID NO. 6, 14, 22, LCDR2 as shown in any one of SEQ ID NO. 7, 15, 23, and LCDR3 as shown in any one of SEQ ID NO. 8, 16, 24.

[0007] A second aspect of this application provides a chimeric antigen receptor, said chimeric antigen receptor comprising the antigen-binding protein as described in the first aspect above.

[0008] A third aspect of this application provides a polynucleotide encoding an antigen-binding protein as described in the first aspect above, or a chimeric antigen receptor as described in the second aspect above.

[0009] A fourth aspect of this application provides a vector containing the polynucleotides described in the third aspect above.

[0010] The fifth aspect of this application provides a host cell containing an antigen-binding protein as described in the first aspect above, or a vector as described in the fourth aspect above, or a polynucleotide as described in the third aspect above integrated into its genome.

[0011] The sixth aspect of this application provides a method for preparing an antigen-binding protein or a chimeric antigen receptor, comprising culturing a host cell containing a vector of the fourth aspect described above or a host cell whose genome has integrated exogenous polynucleotides of the third aspect described above, under conditions that allow the expression of the antigen-binding protein or chimeric antigen receptor, and recovering the antigen-binding protein or chimeric antigen receptor from the cultured host cell culture.

[0012] The seventh aspect of this application provides a pharmaceutical composition comprising an antigen-binding protein as described in the first aspect above, or a chimeric antigen receptor as described in the second aspect above, or a polynucleotide as described in the third aspect above, or a carrier as described in the fourth aspect above, or a host cell as described in the fifth aspect above.

[0013] The eighth aspect of this application provides a kit containing an antigen-binding protein as described in the first aspect above, or a chimeric antigen receptor as described in the second aspect above, or a polynucleotide as described in the third aspect above, or a carrier as described in the fourth aspect above, or a host cell as described in the fifth aspect above.

[0014] The ninth aspect of this application provides a method for detecting the content of SEMA3G protein, comprising using an antigen-binding protein as described in the first aspect above, or a kit as described in the eighth aspect above, to detect the content of SEMA3G protein using immunoassay technology.

[0015] The tenth aspect of this application provides the use of the antigen-binding protein of the first aspect above, or the chimeric antigen receptor of the second aspect above, or the polynucleotide of the third aspect above, or the carrier of the fourth aspect above, or the host cell of the fifth aspect above, or the kit of the eighth aspect above in the preparation of an immunoassay product or a product for the preparation of a product for detecting SEMA3G protein content.

[0016] The eleventh aspect of this application provides the use of the antigen-binding protein of the first aspect above, or the chimeric antigen receptor of the second aspect above, or the polynucleotide of the third aspect above, or the carrier of the fourth aspect above, or the host cell of the fifth aspect above, or the pharmaceutical composition of the seventh aspect above in the preparation of a product that blocks the binding activity of SEMA3G protein to NRP1.

[0017] The twelfth aspect of this application provides the use of the antigen-binding protein of the first aspect above, or the chimeric antigen receptor of the second aspect above, or the polynucleotide of the third aspect above, or the carrier of the fourth aspect above, or the host cell of the fifth aspect above, or the pharmaceutical composition of the seventh aspect above, or the kit of the eighth aspect above in the preparation of a product, said product being used for any one or more of the following: modulating immune responses, diagnosing tumors; preventing tumors; treating tumors.

[0018] The beneficial effects of this application are as follows:

[0019] The NRP ligand SEMA3G disclosed in this application has the effect of binding to NRP1 to induce T cell dysfunction. This application creatively develops a SEMA3G-targeting antibody or antigen-binding fragment that can bind to the aforementioned target protein with high affinity and neutralize its inhibitory activity on T cell function, thereby relieving tumor cell immune escape and promoting the killing of tumor cells by immune cells in vitro and in vivo. This can be used to prepare drugs that regulate immune responses or have anti-tumor effects. Previously, no research has been found on SEMA3G inhibiting T cell function through NRP1, and the strong regulatory effect of the antibody or antigen-binding fragment on immune responses or anti-tumor activity is unexpected by those skilled in the art. This invention also creatively constructs a kit for the indirect quantitative detection of SEMA3G protein using a double-antibody sandwich method. Within the range of 0.1–100 ng / ml, the system exhibits good linearity (R² ≥ 0.99); simultaneously, the system has high sensitivity, with a detection limit of 0.1 ng / ml; and good repeatability and intermediate precision—characteristics that were unexpected by those skilled in the art. Attached Figure Description

[0020] Figure 1 For in vitro T cell proliferation experiments, SEMA3G significantly inhibited T cell proliferation under in vitro activation conditions.

[0021] Figure 2 To analyze the proportion of CD4 and CD8 cells in PBMCs by flow cytometry, the proportion of CD8 T cells was significantly reduced in the SEMA3G treatment group under in vitro activation conditions.

[0022] Figure 3 for Figure 2 The statistical results show that the proportion of CD8 T cells and the CD8 / CD4 ratio in the system decreased in a gradient manner with the increase of SEMA3G treatment concentration.

[0023] Figure 4 To detect the expression level of granzyme B in CD8 T cells under different concentrations of SEMA3G treatment by flow cytometry.

[0024] Figure 5 Enzyme-linked immunosorbent assay (ELISA) showed that SEMA3G has a much stronger affinity for NRP1 than for NRP2.

[0025] Figure 6 To detect the dose-response curve of SEMA3G binding to NRP1 in an enzyme-linked immunosorbent assay.

[0026] Figure 7 To detect the affinity between NRP1 protein and SEMA3G protein using biomembrane interferometry.

[0027] Figure 8 This study aims to statistically analyze the effects of SEMA3G protein and different concentrations of NRP1 protein on the proportion of CD8 T cells, CD8 T cell activation level, and cytotoxic function in the in vitro activated culture system under flow cytometry conditions.

[0028] Figure 9 To detect the blocking activity of purified antibodies from the supernatant of each hybridoma against SEMA3G-NRP1 binding in an enzyme-linked immunosorbent assay (ELISA).

[0029] Figure 10 The dose-response curve of the candidate antibody 2A6D8 binding to SEMA3G protein was obtained for enzyme-linked immunosorbent assay (ELISA).

[0030] Figure 11 To detect the affinity of antibody 2A6D8 for SEMA3G protein using biomembrane interferometry.

[0031] Figure 12 The dose-response curve of purified antibody 2A6D8 blocking SEMA3G-NRP1 binding was used to detect the effect of enzyme-linked immunosorbent assay.

[0032] Figure 13 Flow cytometry was used to demonstrate that the SEMA3G monoclonal antibody 2A6D8 can increase the activation and function of immune cells in a co-culture system, thereby promoting the killing effect of human peripheral blood mononuclear cells on various tumor cells.

[0033] Figure 14 The tumor growth curves and tumor weights of mice in the A375 mouse tumor model were obtained from the 2A6D8 antibody treatment group, the 18H1G2 antibody treatment group, and the control group.

[0034] Figure 15 This study analyzed the immune infiltration of the tumor microenvironment in the A375 mouse tumor model, specifically in the 2A6D8 antibody treatment group and the control group.

[0035] Figure 16The tumor growth curves and CD8 T cell infiltration levels in the tumor microenvironment of mice in the NCI-H520 and HCT-116 mouse tumor models were compared to those in the 2A6D8 antibody treatment group and the control group.

[0036] Figure 17 The dose-response curve of SEMA3G antibody 1C1A6,15H1F5 binding to SEMA3G was obtained for ELISA detection.

[0037] Figure 18 In the double-antibody sandwich ELISA detection method, a standard curve is plotted with the standard concentration as the x-axis (nanograms per milliliter) and the optical density OD450 value as the y-axis.

[0038] Figure 19 The dose-response curve for ELISA detection of the binding of humanized antibody 7K3Q to SEMA3G. Detailed Implementation

[0039] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0040] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0041] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0042] This application discovers that SEMA3G is a ligand for NRP and has the effect of binding NRP1 to induce T cell dysfunction. Therefore, this application has creatively developed a targeted antibody or antigen-binding fragment that can bind to SEMA3G, which can bind to the above-mentioned target protein with high affinity and neutralize its inhibitory activity on T cell function, thereby relieving the immune escape function of tumor cells and promoting the killing of tumor cells by immune cells in vivo and in vitro. It can be used to prepare drugs that regulate immune response or have anti-tumor effects.

[0043] This application first provides an isolated antigen-binding protein that binds to SEMA3G protein. The antigen-binding protein includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1 as shown in any one of SEQ ID NO. 3, 11, and 19, HCDR2 as shown in any one of SEQ ID NO. 4, 12, and 20, and HCDR3 as shown in any one of SEQ ID NO. 5, 13, and 21. The light chain variable region includes LCDR1 as shown in any one of SEQ ID NO. 6, 14, and 22, LCDR2 as shown in any one of SEQ ID NO. 7, 15, and 23, and LCDR3 as shown in any one of SEQ ID NO. 8, 16, and 24.

[0044] Antigen-binding proteins are a class of proteins that can specifically bind to specific antigen molecules. These proteins typically exhibit high specificity, recognizing and binding to specific epitopes of antigens.

[0045] The SEMA3G protein comprises human SEMA3G protein, which contains the amino acid sequence shown in SEQ SEQ ID NO:27.

[0046] SEQ ID NO:27:

[0047] MAPSAWAAICWLLGGLLLHGGSSGPSPGPSVPRLRLSYRDLLSANRSAIFLGPQGSLNLQAMYLDEYRDRLLFLGGLDALYSLRLDQAWPDPREVLWPPQPGQREECVRKGRDPLTECANFVRVLQPHNRTHLLACGTGAFQPTCALITVGHRGEHVLHLEPGSVESGRGRCPHEPSRPFASTFIDGELYTGLTADF LGREAMIFRSGGPRPALRSDSDQSLLHDPRFVMAARIPENSDQDNDKVYFFFSETVPSPDGGSNHVTVSRVGRVCVNDAGGQRVLVNKWSTFLKARLVCSVPGPGGAETHFDQLEDVFLLWPKAGKSLEVYALFSTVSAVFQGFAVCVYHMADIWEVFNGPFAHRDGPQHQWGPYGGKVPFPRPGVCPSKMTAQPG RPFGSTKDYPDEVLQFARAHPLMFWPVRPRHGRPVLVKTHLAQQLHQIVVDRVEAEDGTYDVIFLGTDSGSSVLKVIALQAGGSAEPEEVVLEELQVFKVPTPITEMEISVKRQMLYVGSRLGVAQLRLHQCETYGTACAECCLARDPYCAWDGASCTHYRPSLGKRRFRRQDIRHGNPALQCLGQSQEEEAVGLV AATMVYGTEHNSTFLECLPKSPQAAVRWLLQRPGDEGPDQVKTDERVLHTERGLLFRRLSRFDAGTYTCTTLEHGFSQTVVRLALVVIVASQLDNLFPPEPKPEEPPARGGLASTPPKAWYKDILQLIGFANLPRVDEYCERVWCRGTTECSGCFRSRSRGKQARGKSWAGLELGKKMKSRVHAEHNRTPREVEAT

[0048] In a specific embodiment of this application, the antigen-binding protein includes a SEMA3G antibody or its antigen-binding fragment.

[0049] The antibodies typically exist as one or more Y-shaped monomers, each consisting of four polypeptide chains, including two identical heavy chains and two identical light chains. The heavy and light chains are distinguished based on their molecular weight. Light chains are classified as κ or λ types based on differences in the structure of small polypeptide molecules, while heavy chains are classified as μ, δ, γ, α, or ε, defining the antibody class or type as IgM, IgD, IgG, IgA, and IgE, respectively.

[0050] The antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region. The variable region includes complementarity-determining regions (CDRs), which are key regions in the antibody molecule that specifically bind to antigens. CDR regions exhibit high sequence diversity, enabling the antibody to recognize and bind to a variety of different antigens. In the antibody's variable region, there are three CDR regions each in the light and heavy chains, denoted as CDR1, CDR2, and CDR3, respectively. The heavy chain variable regions can be denoted as HCDR1, HCDR2, and HCDR3, and the light chain variable regions can be denoted as LCDR1, LCDR2, and LCDR3. The CDR regions directly interact with specific epitopes of the antigen, and their sequence diversity determines the antibody's specificity and affinity. CDR regions are typically located at specific amino acid positions.

[0051] In one embodiment of this application, the antigen-binding protein includes HCDR1 as shown in SEQ ID NO.3, HCDR2 as shown in SEQ ID NO.4, HCDR3 as shown in SEQ ID NO.5, LCDR1 as shown in SEQ ID NO.6, LCDR2 as shown in SEQ ID NO.7, and LCDR3 as shown in SEQ ID NO.8.

[0052] HCDR1 as shown in SEQ ID NO.3: GYSFTKYW

[0053] HCDR2 as shown in SEQ ID NO.4: IDPSDSYI

[0054] HCDR3 as shown in SEQ ID NO.5: ARGPYYRFMDY

[0055] LCDR1 as shown in SEQ ID NO.6: HDINKN

[0056] LCDR2 as shown in SEQ ID NO.7: STS

[0057] LCDR3 as shown in SEQ ID NO.8: LHYDTLWT

[0058] In another embodiment of this application, the antigen-binding protein includes HCDR1 as shown in SEQ ID NO.11, HCDR2 as shown in SEQ ID NO.12, HCDR3 as shown in SEQ ID NO.13, LCDR1 as shown in SEQ ID NO.14, LCDR2 as shown in SEQ ID NO.15, and LCDR3 as shown in SEQ ID NO.16.

[0059] HCDR1 as shown in SEQ ID NO.11: GFSLPNYG

[0060] HCDR2 as shown in SEQ ID NO.12: IWRSGNT

[0061] HCDR3 as shown in SEQ ID NO.13: AKNDGRGYFDY

[0062] LCDR1 as shown in SEQ ID NO.14: GNIHNY

[0063] LCDR2: NAE as shown in SEQ ID NO.15

[0064] LCDR3 as shown in SEQ ID NO.16: QHFWSTPFT

[0065] In another embodiment of this application, the antigen-binding protein includes HCDR1 as shown in SEQ ID NO.19, HCDR2 as shown in SEQ ID NO.20, HCDR3 as shown in SEQ ID NO.21, LCDR1 as shown in SEQ ID NO.22, LCDR2 as shown in SEQ ID NO.23, and LCDR3 as shown in SEQ ID NO.24.

[0066] HCDR1 as shown in SEQ ID NO.19: GYTFTTYT

[0067] HCDR2 as shown in SEQ ID NO.20: INPSGYT

[0068] HCDR3 as shown in SEQ ID NO.21: ARCYYTDYEAMDF

[0069] LCDR1 as shown in SEQ ID NO.22: ENVASY

[0070] LCDR2 as shown in SEQ ID NO.23: GAS

[0071] LCDR3 as shown in SEQ ID NO.24: GQSYSYPFT

[0072] In specific embodiments of this application, the heavy chain variable region of the antigen-binding protein includes an amino acid sequence as shown in any one of SEQ ID NO. 1, 9, and 17, and the light chain variable region of the antigen-binding protein includes an amino acid sequence as shown in any one of SEQ ID NO. 2, 10, and 18.

[0073] In one embodiment of this application, the antigen-binding protein includes a heavy chain variable region as shown in SEQ ID NO.1 and a light chain variable region as shown in SEQ ID NO.2.

[0074] The heavy chain variable region as shown in SEQ ID NO.1:

[0075] QVQLQQPGAEFVKPGASVKLSCKASGYSFTKYWMHWVKLRPGQGLEWIGEIDPSDSYITDNQKFKDKATLSVDKSSSTAFMQLSSLTSDDSAVYYCARGPYYRFMDYWGQGTSVTVSS

[0076] The light chain variable region as shown in SEQ ID NO.2:

[0077] DIQMTQSPSSSLSASLGGKVTITCKASHDINKNLAWYQHKPGKGPRLLIHSTSTLQPGIPS RFSGSGSGRDYSFSISDLEPEDIATYYCLHYDTLWTFGGGTKLEIT

[0078] In another embodiment of this application, the antigen-binding protein includes a heavy chain variable region as shown in SEQ ID NO.9 and a light chain variable region as shown in SEQ ID NO.10.

[0079] Heavy chain variable region as shown in SEQ ID NO.9:

[0080] QVQLKQSGPGLVQPSQSLSITCTVSGFSLPNYGVHWIRQSPGKGLEWLGVIWRSGNTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYYCAKNDGRGYFDYWGQGTTLTVSS as shown in SEQ ID NO.10, light chain variable region:

[0081] DIQMTQSPASSLSASVGETVTITCRASGNIHNYLAWYQQKQGYSPHLLVYNAETLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPFTFGSGTKLEIK

[0082] In another embodiment of this application, the antigen-binding protein includes a heavy chain variable region as shown in SEQ ID NO.17 and a light chain variable region as shown in SEQ ID NO.18.

[0083] Heavy chain variable region as shown in SEQ ID NO.17:

[0084] QVQLQQSAAELARPGASVKMSCKASGYTFTTYTLHWVKQRPGQGLEWIGYINPNSGYTYYTQKFKDKTTLTADKSSSTTYMQLTSLTSEDSAVFYCARCYYTDYEAMDFWGPGTSLTVSS

[0085] The light chain variable region as shown in SEQ ID NO.18:

[0086] NIVMTQSPKSMSVSVGERVTLSCKASENVASYVSWYQQKPEQSPKLLIYGASSRYTGVPDRFTGSGSATDFTLTITSVQAEDLADYHCGQSYSYPFTFGSGSKLEIK

[0087] In another embodiment of this application, the antigen-binding protein includes a heavy chain variable region as shown in SEQ ID NO.25 and a light chain variable region as shown in SEQ ID NO.26, specifically, a humanized antibody derived from a murine antibody after humanization design.

[0088] Heavy chain variable region as shown in SEQ ID NO.25:

[0089] QVQLVQSGPEVKKPGTSVRVSCKASGYSFTKYWMHWVRLARGQRLEWIGEIDPSDSYITDNQKFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARGPYYRFMDYWGQGTMVTVSS

[0090] The light chain variable region as shown in SEQ ID NO.26:

[0091] DIVMTQTPGTLSLSPGERATLSCKASHDINKNLAWYQHKPGQAPRLLIHSTSTLQPGIPDRFSGSGSGRDYTLTISRLEPEDFAVYYCLHYDTLWTFGQGTKVEIKRTV

[0092] In specific embodiments of this application, the SEMA3G antibody may be a polyclonal antibody, a monoclonal antibody, a single-chain antibody, an antigen-binding domain, a bispecific antibody, a multispecific antibody, or an antigen-binding portion of a chimeric antigen receptor.

[0093] SEMA3G antibodies can be either murine or humanized, with the latter being a chimeric antibody. Specifically, murine antibodies are those whose encoding genes are entirely derived from mice. Humanized antibodies are those whose constant region is partially or entirely encoded by human antibody genes. Humanized antibodies can significantly reduce the immune side effects caused by heterologous antibodies in the human body. Humanized antibodies include chimeric antibodies, modified antibodies, and fully humanized antibodies. Chimeric antibodies are monoclonal antibodies produced by inserting the variable regions of the light and heavy chains of murine monoclonal antibodies into a vector containing the constant region of human antibodies using DNA recombination technology, followed by transformation into mammalian cells for expression. Chimeric antibodies can achieve a humanization rate of up to 70%, fully retaining the variable region and parental activity of the murine monoclonal antibody, while the introduction of the human antibody constant region reduces immunogenicity.

[0094] Those skilled in the art will recognize that antibodies in non-complete tetrameric forms, including but not limited to Fab, Fab', F(ab') or F(ab')2, nanobodies (VHH), single-chain antibodies (scFv), BsFv, dsFv, (dsFv)2, or Fv, can also exert the effect of specifically binding antigens. Therefore, this application also includes the antigen-binding fragment of the SEMA3G antibody.

[0095] In the general case of this application, the antibody further includes a heavy chain constant region and a light chain constant region, which may be derived from human or mouse sources. The heavy chain constant region is selected from IgG, IgA, IgM, IgD, or IgE types; and / or, the light chain constant region is selected from κ or λ types. IgG is a globulin required for normal immune function maintenance in the body. Human IgG has four subtypes: IgG1, IgG2, IgG3, and IgG4.

[0096] The antigen-binding protein of this application can also be modified into a chimeric antigen receptor, i.e., a CAR. Therefore, this application also provides a chimeric antigen receptor, which includes the antigen-binding protein described above. Specifically, the chimeric antigen receptor includes an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain. The antigen-binding domain contains the antigen-binding protein as described above.

[0097] CARs also include a transmembrane domain, which is a key region connecting the extracellular antigen recognition portion and the intracellular signal transduction portion of the CAR. The transmembrane domain typically consists of a hydrophobic amino acid sequence that allows the CAR to anchor to the cell membrane of T cells or other immune cells. In specific embodiments of this application, the transmembrane domain can be the transmembrane region of CD8α, CD28, CD3ε, CD3ζ, CD45, CD4, CD5, CD9, CD16, CD20, CD22, CD27, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD152, CD154, DAP10, DAP12, PD-1, or the α, β, or ζ chain of the T cell receptor, etc.

[0098] CARs also include intracellular signal transduction domains, which are responsible for transmitting activation signals to T cells after the CAR binds to tumor cell surface antigens, thereby activating T cells and triggering an immune response. The intracellular signal transduction domains include primary signal transduction domains and co-stimulatory signal transduction domains. The primary signal transduction domains contain immune receptor tyrosine activation motifs (ITAMs), which, upon receptor activation, trigger downstream signal transduction pathways, thereby activating T cells. The co-stimulatory signal transduction domains provide necessary second signals to enhance T cell activation, proliferation, and survival.

[0099] In a specific embodiment of this application, the intracellular signal transduction domain sequentially comprises at least one co-stimulatory signal transduction domain and a primary signal transduction domain from the N-terminus to the C-terminus. In a specific embodiment of this application, the primary signal transduction domain may be an intracellular signal transduction domain of CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD22, CD79a, DAP10, CD79b, or CD66d, etc. The co-stimulatory signal transduction domain may be an intracellular signal transduction domain of CD137(4-1BB), CD27, CD28, ICOS, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, etc., or a combination thereof.

[0100] The CAR also includes a hinge region located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. This hinge region is a structural domain situated between the antigen recognition domain and the transmembrane domain, and is typically derived from the hinge region of an antibody. The main function of the hinge region is to provide the necessary flexibility and adaptability, allowing the CAR to move freely in space, thereby effectively recognizing and binding to antigens on the surface of tumor cells. In specific embodiments of this application, the hinge region can be a hinge region of CD8α, CD28, IgG1, or IgG4.

[0101] CAR also includes a signal peptide located at the N-terminus of the chimeric antigen receptor polypeptide, which is a short peptide chain that can enhance the expression level of CAR on the surface of T cells, affecting the surface stability and antigen recognition efficiency of CAR-T cells, and influencing the immune activation and effector function of CAR-T cells. In specific embodiments of this application, the signal peptide may be derived from HLA-A, CD8α, CD33, Igκ, IL-2, or GM-CSFRα, etc.

[0102] In a specific embodiment of this application, the chimeric antigen receptor CAR comprises, from its N-terminus to its C-terminus, the signal peptide, the antigen-binding domain, the hinge region, the transmembrane domain, the co-stimulatory signal transduction domain, and the primary signal transduction domain.

[0103] This application also provides a polynucleotide encoding the antigen-binding protein described above, or the chimeric antigen receptor described above.

[0104] The preparation method of the polynucleotide is an existing technology, and conventional methods can be selected according to specific circumstances. For example, it can be prepared by automated DNA synthesis, or it can be prepared by recombinant DNA technology, or it can be isolated from a suitable natural source.

[0105] Polynucleotides are polymers of nucleotides typically linked from one deoxyribose or ribose to another. There are no size limitations for polynucleotides in this application, and they may include polynucleotides containing modifications, particularly modified nucleotides. In some embodiments, polynucleotides may be RNA, DNA, or cDNA, etc.

[0106] In one embodiment of this application, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:28, 29.

[0107] In another embodiment of this application, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:30, 31.

[0108] In another embodiment of this application, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:32, 33.

[0109] In another embodiment of this application, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:34, 35.

[0110] Based on the antibody-based recombinant expression method, this application also provides a vector containing the aforementioned polynucleotides.

[0111] In this application, "vector" refers to a polynucleotide capable of carrying at least one polynucleotide fragment. The vector can exist in a circular or linear (linearized) form and includes vector fragments, or it can be an artificial chromosome or similar polynucleotide containing a transferable exogenous nucleic acid fragment. The vector may contain at least one expression cassette containing a regulatory sequence for the proper expression of the polynucleotide incorporated therein. The polynucleotide to be introduced into the cell (e.g., a polynucleotide encoding a target product or a selectable marker) can be inserted into the expression cassette of the vector for expression therefrom. The vector can also integrate exogenous DNA sequences into the host cell's genome, thereby achieving stable expression of the exogenous gene. It typically contains homologous arms or specific DNA sequences that can undergo homologous recombination with specific locations in the host cell's genome. The vector can be selected from DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR / Cas9 vectors, or viral vectors, etc. For example, in a specific embodiment of this application, the vector is pcDNA3.1.

[0112] Based on the above-mentioned polynucleotides and vectors, this application may also provide a host cell containing the above-mentioned antigen-binding protein, or containing the above-mentioned vector, or having the above-mentioned polynucleotides integrated into its genome.

[0113] The host cell can be selected from bacterial cells, fungal cells, insect cells, plant cells, mammalian cells, etc. Specifically, it can be selected from Escherichia coli, Streptomyces, Salmonella typhimurium, yeast, filamentous fungi, Drosophila S2 or Sf9 cells, CHO cells, COS cells, HEK293F cells, Bowes melanoma cells, NSO cells, BHK cells, PER.C6 cells, etc. For example, in a specific embodiment of this application, the host cell is a mammalian 293 cell. Further, the host cell is a 293 cell infused with a vector containing the aforementioned polynucleotides. The method for incorporating the vector into the host cell can be conventional, depending on the actual situation. For example, methods such as microinjection, gene gun, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, liposome transfection, and PEI can be selected. The host cell can also be an immune cell, such as a T cell.

[0114] This application also provides a method for preparing an antigen-binding protein or a chimeric antigen receptor, comprising culturing a host cell containing the aforementioned vector or a host cell whose genome has integrated exogenous polynucleotides, under conditions that allow the expression of the antigen-binding protein or chimeric antigen receptor, and recovering the antigen-binding protein or chimeric antigen receptor from the cultured host cell culture.

[0115] Specifically, the conditions that allow the expression of the anti-antigen binding protein mentioned above may refer to effective transfection or transduction methods, favorable promoter conditions, stable gene copies, suitable culture conditions, optimized post-translational modifications, etc.

[0116] Specifically, the effective transfection or transduction methods mentioned above can include microinjection, gene gun, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, liposome transfection, PEI, and other methods.

[0117] Specifically, the recovered antibody or its antigen-binding fragment can be selected according to the actual situation using conventional methods. For example, the antibody described in this application can be purified by Protein A affinity chromatography and the antibody protein can be eluted with glycine.

[0118] This application also provides a pharmaceutical composition comprising, as described above, an antigen-binding protein, as described above, a chimeric antigen receptor, as described above, a polynucleotide, as described above, a carrier, or a host cell, as described above.

[0119] The effective amount refers to the amount of a pharmaceutical compound or composition that causes a measurable clinical, biological, or pharmaceutical change or response in a biomarker, cell, tissue, system, or patient.

[0120] The form of the pharmaceutical composition is not limited and can be in various forms such as solid, liquid, gel, semi-liquid, or aerosol.

[0121] In specific embodiments of this application, the pharmaceutical composition may further include pharmaceutically acceptable excipients or carriers. Pharmaceutically acceptable refers to non-toxic materials that do not interfere with the bioactivity and effectiveness of the active ingredient. Specific examples of pharmaceutically acceptable excipients or carriers include sterile water or physiological saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphate, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.), binders, etc. Furthermore, it may also contain other low-molecular-weight peptides; proteins such as serum albumin, gelatin, or immunoglobulins; amino acids such as glycine, glutamine, asparagine, arginine, and lysine; sugars or carbohydrates such as polysaccharides and monosaccharides; and sugar alcohols such as mannitol or sorbitol. When preparing aqueous solutions for injection, such as physiological saline, isotonic solutions containing glucose or other excipients like D-sorbitol, D-mannose, D-mannitol, or sodium chloride, appropriate solubilizers such as alcohols (ethanol, etc.), polyols (propylene glycol, PEG, etc.), and nonionic surfactants (Tween 80, HCO-50) can be used. The excipients or carriers can be adjusted according to the desired dosage form, and the ratio can be adjusted based on actual needs.

[0122] In specific embodiments of this application, the antigen-binding protein or chimeric antigen receptor in the pharmaceutical composition can be a single active ingredient, or it can be combined with one or more other active ingredients that have therapeutic effects on regulating immune responses or on tumors to form a combined formulation, wherein the efficacy and safety of the components do not conflict. The content of each component in the combined formulation is usually a safe and effective amount, which can be adjusted based on actual usage (e.g., patient weight, type of application, disease condition, severity).

[0123] The drug composition is preferably administered to mammals, such as, but not limited to, humans, primates, livestock (e.g., sheep, cattle, horses, donkeys, pigs), pets (e.g., dogs, cats), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs, hamsters) or captured wild animals (e.g., foxes, deer); preferably, the subject is a primate; more preferably, the subject is a human.

[0124] This application also provides a kit containing the antigen-binding protein described above, or the chimeric antigen receptor described above, or the polynucleotide described above, or the carrier described above, or the host cell described above.

[0125] In a specific embodiment of this application, the kit may be an ELISA kit, wherein the antigen-binding protein serves as a capture antibody and / or a detection antibody, and is used in a double-antibody sandwich ELISA to detect the SEMA3G protein content; the kit may also contain other reagents necessary for ELISA detection, such as a solid-phase carrier, negative control, positive control, sample diluent, blocking buffer, washing buffer, chromogenic solution, and reaction termination solution.

[0126] In a specific embodiment of this application, the antigen-binding protein that serves as the detection antibody is labeled with a marker. The marker can be linked to the detection antibody by coupling and is used to catalyze the substrate to generate a detectable signal, thereby achieving quantitative detection. The marker includes, but is not limited to, enzymes, biotin, chemiluminescent groups, or isotopic groups.

[0127] In one embodiment of this application, for example, the kit includes SEMA3G antibody 1C1A6 and SEMA3G antibody 15H1F5, wherein 1C1A6 is used as a capture antibody and 15H1F5 is used as a detection antibody and is biotinylated.

[0128] This application also provides a method for detecting the content of SEMA3G protein, including using the antigen-binding protein described above or the kit described above on the test subject, and employing immunoassay technology to detect the content of SEMA3G protein.

[0129] The SEMA3G protein content refers to the amount of SEMA3G protein in a sample (such as a vaccine, blood, tissue fluid, etc.). This amount can be expressed as mass concentration (e.g., micrograms per milliliter, ug / mL), molar concentration (e.g., moles per liter, M) or other appropriate concentration units.

[0130] In a specific embodiment of this application, the immune detection technology is a double-antibody sandwich ELISA detection.

[0131] In a specific embodiment of this application, the antigen-binding protein in the method serves as a capture antibody and / or a detection antibody.

[0132] In a specific embodiment of this application, the method uses SEMA3G antibody 1C1A6 and SEMA3G antibody 15H1F5, wherein 1C1A6 is used as a capture antibody and 15H1F5 is used as a detection antibody and is biotinylated. The content of SEMA3G protein is detected by double antibody sandwich ELISA.

[0133] In a sandwich ELISA using two antibodies, the capture antibody is a specific antibody that is first linked to the solid-phase support. Its role is to immobilize the antigen to be tested onto the solid-phase support, forming a solid-phase antigen-antibody complex. The detection antibody, also known as a secondary antibody or enzyme-labeled antibody, is a specific antibody used to detect and quantify the antigen that has been immobilized on the solid-phase support by the capture antibody.

[0134] The double-antibody sandwich ELISA can employ conventional procedures found in existing technologies. For example, capture antibodies are typically first coated into the microwells of an ELISA plate, then a sample is added. Samples containing the target antigen bind to the capture antibody, and unbound components are removed by washing. Next, a labeled detection antibody is added, which binds to the antigen on the solid-phase immune complex, forming a sandwich complex. Finally, a labeled substrate is added, producing a colored product. The intensity of the product color indicates the concentration of the antigen to be tested. In experiments, capture and detection antibodies typically target different epitopes of the same target molecule, ensuring they can bind simultaneously to the target molecule without interference. The primary function of the capture antibody is to immobilize the target molecule, while the function of the detection antibody is to reflect the presence and / or quantity of the antigen through the signal generated by its labeling.

[0135] The method for detecting SEMA3G protein content in this application exhibits good linearity in the range of 0.1-100 (unit: nanograms / mL), R 2 It is 0.9997.

[0136] In specific embodiments of this application, the detection method is a detection method for non-disease diagnosis and treatment purposes. The non-disease diagnosis and treatment purposes can be to detect the content of SEMA3G protein in the following scenarios: cell biology and developmental biology research, SEMA3G as a drug target or biomarker in drug development, biomedical marker research, identifying health differences and disease tendencies in different populations in large-scale epidemiological studies, and assessing an individual's physiological functions and intercellular communication status in the field of functional medicine.

[0137] This application also provides the use of the antigen-binding protein described above, or the chimeric antigen receptor described above, or the polynucleotide described above, or the carrier described above, or the host cell described above, or the kit described above in the preparation of immunoassay products.

[0138] Specifically, the immunoassay product can be a double-antibody sandwich ELISA assay for detecting SEMA3G protein, wherein the antigen-binding protein can serve as a capture antibody and / or a detection antibody.

[0139] This application also provides the use of the antigen-binding protein described above, or the chimeric antigen receptor described above, or the polynucleotide described above, or the carrier described above, or the host cell described above, or the kit described above in the preparation of products for detecting SEMA3G protein content.

[0140] Specifically, the product for detecting SEMA3G protein content uses immunoassay technology to quantitatively determine the SEMA3G protein content in biological samples. The immunoassay technology can be a double-antibody sandwich ELISA.

[0141] This application also provides the use of the antigen-binding protein described above, or the chimeric antigen receptor described above, or the polynucleotide described above, or the carrier described above, or the host cell described above, or the pharmaceutical composition described above in the preparation of a product that blocks the binding activity of SEMA3G protein to NRP1.

[0142] This application found that NRP1 can bind to SEMA3G protein in a concentration-dependent manner, and the binding of SEMA3G to NRP1 is much stronger than that of SEMA3G to NRP2 under the same concentration conditions. After using SEMA3G antibody, the binding of SEMA3G protein to the extracellular region of NRP1 protein can be blocked.

[0143] This application also provides the use of the above-mentioned antigen-binding protein, or the above-mentioned chimeric antigen receptor, or the above-mentioned polynucleotide, or the above-mentioned carrier, or the above-mentioned host cell, or the above-mentioned pharmaceutical composition, or the above-mentioned kit in the preparation of products, said products being used for any one or more of the following: regulating immune responses; tumor diagnosis; tumor prevention; and tumor treatment.

[0144] The diagnosis refers to the process by which a physician or other medical professional assesses a patient's health status based on the patient's symptoms, signs, medical history, laboratory test results, imaging results, and other relevant information, and determines whether the patient has a disease and the nature and extent of the disease.

[0145] Prevention refers to taking measures before a disease occurs to prevent its occurrence; or reducing the development and impact of a disease through early detection, diagnosis, and treatment; or reducing the adverse consequences of a disease that has already occurred, and improving the patient's quality of life and functional status through rehabilitation and disease management.

[0146] The treatment refers to a series of methods, techniques and procedures designed to alleviate disease symptoms, treat the disease itself, improve the patient's health or enhance their quality of life.

[0147] The immune response includes, but is not limited to, autoimmune diseases, immunodeficiency diseases, allergic reactions, tumor immune escape, neurodegenerative diseases, cardiovascular diseases, diabetes, and infectious diseases; among which, the autoimmune diseases include systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), etc.; immunodeficiency diseases include congenital or acquired immunodeficiency syndrome (AIDS); allergic reactions include allergic rhinitis, asthma, etc.; and neurodegenerative diseases include Alzheimer's disease (AD) and Parkinson's disease, etc.

[0148] The tumors mentioned include, but are not limited to, squamous cell carcinoma of the lung, colorectal cancer, melanoma, lung cancer, breast cancer, prostate cancer, stomach cancer, liver cancer, pancreatic cancer, ovarian cancer, cervical cancer, thyroid cancer, skin cancer, brain cancer, leukemia, lymphoma, multiple myeloma, kidney cancer, bladder cancer, head and neck cancer, testicular cancer, bone cancer, soft tissue sarcoma, neuroendocrine tumors, gallbladder cancer, and esophageal cancer.

[0149] The products may be drugs, combinations of drugs, drug compositions, reagents, or kits.

[0150] This application also provides a method for treating tumors, comprising administering to a subject an effective amount of the above-described antigen-binding protein, or the above-described chimeric antigen receptor, or the above-described pharmaceutical composition.

[0151] This method can be used in combination with other methods for treating tumors, either simultaneously or sequentially. Other methods for treating tumors include, but are not limited to, surgery, radiotherapy, chemotherapy, targeted therapy, immunotherapy, hormone therapy, and stem cell transplantation.

[0152] The tumors mentioned include, but are not limited to, squamous cell carcinoma of the lung, colorectal cancer, melanoma, lung cancer, breast cancer, prostate cancer, stomach cancer, liver cancer, pancreatic cancer, ovarian cancer, cervical cancer, thyroid cancer, skin cancer, brain cancer, leukemia, lymphoma, multiple myeloma, kidney cancer, bladder cancer, head and neck cancer, testicular cancer, bone cancer, soft tissue sarcoma, neuroendocrine tumors, gallbladder cancer, and esophageal cancer.

[0153] This application also provides a method for regulating an immune response, comprising administering to a subject an effective amount of the antigen-binding protein described above, or the chimeric antigen receptor described above, or the pharmaceutical composition described above.

[0154] The immune response includes, but is not limited to, autoimmune diseases, immunodeficiency diseases, allergic reactions, tumor immune escape, neurodegenerative diseases, cardiovascular diseases, diabetes, and infectious diseases; among which, the autoimmune diseases include systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), etc.; immunodeficiency diseases include congenital or acquired immunodeficiency syndrome (AIDS); allergic reactions include allergic rhinitis, asthma, etc.; and neurodegenerative diseases include Alzheimer's disease (AD) and Parkinson's disease, etc.

[0155] The method described can be used in combination with other methods for modulating immune responses, either simultaneously or sequentially. Other methods for modulating immune responses include the administration of immunomodulators, such as drugs of microbial origin (e.g., BCG), human or animal immune system products (e.g., monoclonal antibodies, thymosin, transfer factor, interferon, interleukin, etc.), chemically synthesized drugs (e.g., levamisole, polyinosinic-polycytidylic acid), and traditional Chinese medicine and others (e.g., ginseng, astragalus, etc.).

[0156] The effective amount in the above methods refers to the amount of a drug compound or composition that causes measurable clinical, biological, or pharmaceutical changes or responses in biomarkers, cells, tissues, systems, or patients. This can be adjusted according to individual circumstances.

[0157] The preferred subjects for applying the above method are mammals, such as, but not limited to, humans, primates, livestock (e.g., sheep, cattle, horses, donkeys, pigs), pets (e.g., dogs, cats), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), or captured wild animals (e.g., foxes, deer). Preferably, the subject is a primate. More preferably, the subject is a human.

[0158] The present application will be further illustrated by the following examples, but these examples do not limit the scope of the application. Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques have been well described in existing literature.

[0159] Example 1: SEMA3G protein inhibits cytotoxic T cell activation, proliferation, and function.

[0160] Human PBMCs (Heyousheng, Shanghai, China) were revived and cultured in AIM-V medium (Gibco, CA, USA) overnight at 37°C with 0.5% CO2. Cells were accurately counted using an automated cell counter, and 5 × 10^6 cells were harvested. Cells were then washed twice with PBS to remove residual culture medium. After centrifugation and discarding the supernatant, the cells were resuspended in 0.5 mL of PBS to prepare a single-cell suspension. Cell Proliferation Dye eFluor TM Thermo Fisher Scientific (MA, USA) dye 670 was dissolved in anhydrous DMSO to prepare a stock solution concentration of 5 mM. This stock solution was further diluted in PBS to prepare a 10 μM working solution, which was then preheated to room temperature. 10 μM eFluor... TMThe 670 dye working solution was mixed with an equal volume of the previously prepared single-cell suspension, and then incubated in the dark at 37°C for 10 minutes to allow the dye to fully penetrate and label the cells. After incubation, 4-5 volumes of pre-chilled complete culture medium were quickly added, and the cells were incubated on ice for 5 minutes to terminate the staining process. The cells were washed three times with complete culture medium to remove all unbound dye. The cells were resuspended in AIM-V medium and the cell density was adjusted to 2 x 10^6 cells per milliliter. Medium concentrations of 1 μg / mL anti-CD3 antibody (16-0037-81, Invitrogen) and anti-CD28 antibody (Thermo Fisher Scientific, MA, USA) and 50 ng / mL recombinant human interleukin-2 (Thermo Fisher Scientific, MA, USA) were added and thoroughly mixed. 200 μL of cell suspension was added to each well of a clear 96-well plate (Corning, NY, USA). Each group was given SEMA3G protein at final concentrations of 0, 1, 2, 4, 8, 10, 15, and 20 μg / mL, respectively, and incubated at 37°C with 0.5% CO2. Cell samples were collected after 96 hours. Cells were washed twice with cell staining buffer, then incubated at 4°C for 30 minutes in the dark with a mixture of CD4 and CD8 fluorescent antibodies diluted in cell staining buffer. After washing with cell staining buffer again, 500 μL of 1× permeabilization working solution was added to each tube, and the cells were incubated at 4°C for 45 minutes to permeabilize. Cells were washed twice with 1× Permeabilization Buffer, and the supernatant was discarded. Cells were resuspended in 100 μL of cell staining buffer, and fluorescently labeled granzyme B antibody was added. Cells were incubated at room temperature for 45 minutes, washed twice with 1× Permeabilization Buffer, and the supernatant was discarded. Cells were resuspended in cell staining buffer and analyzed.

[0161] The results are as follows Figure 1 As shown, under the conditions of 1 μg / mL anti-CD3 antibody and anti-CD28 antibody (16-0289-81, Invitrogen) and 50 ng / mL recombinant human interleukin-2 activation, with the increase of the added SEMA3G protein concentration, eFluor in T cells increased. TM The fluorescence intensity of 670 gradually decreased with dilution. Figures 2 to 3 As shown, the proportion of CD8-positive cells and the CD8 / CD4 ratio decreased in a gradient with increasing concentration of added SEMA3G protein. Meanwhile, as... Figure 4 As shown, the expression level of granzyme B, an important factor mediating T cell killing function in CD8 cells, was significantly decreased after SEMA3G treatment. These results indicate that SEMA3G protein treatment significantly inhibited antibody-induced T cell activation, proliferation, and functional activity in a SEMA3G concentration-dependent manner.

[0162] Example 2: NRP1 as a high-affinity acceptor for SEMA3G

[0163] 1. Enzyme-linked immunosorbent assay (ELISA) showed that SEMA3G has a much stronger affinity for NRP1 than for NRP2.

[0164] SEMA3G protein at a concentration of 2 μg / mL was coated onto an ELISA plate (Costar, ME, USA) and incubated overnight at 4°C, discarding the supernatant. After washing five times with 300 μL of PBST per well, each well was blocked with 200 μL of 3% bovine serum albumin (Sangon Biotech, Shanghai, China) at 37°C for 90 minutes. After washing five times with PBST, 100 μL of NRP1-hFc or NRP2-hFc recombinant protein at concentrations of 4, 2, 1, and 0 μg / mL were added to each well, and incubated at 37°C for 1 hour. After washing five times with 300 μL of PBST per well, each well was incubated with working solution of horseradish peroxidase-labeled specific anti-human Fc fragment antibody diluted in PBS (Jackson, PA, USA) at 37°C for 30 minutes. After washing with PBST 5 times, prepare TMB chromogenic solution (Sangon Biotech, Shanghai, China), 100 μL per well, and incubate in an incubator for 10 minutes. Then add 50 μL of stop solution (Sangon Biotech, Shanghai, China) to stop the reaction. Detect the optical density at a wavelength of 450 nm using an ELISA reader (Thermo Fisher Scientific, MA, USA).

[0165] The results are as follows Figure 5 As shown, both NRP1 and NRP2 exhibit concentration-dependent binding to SEMA3G protein. However, within the detected concentration range, the binding of SEMA3G to NRP1 is much stronger than that of SEMA3G to NRP2 at the same concentration.

[0166] 2. Enzyme-linked immunosorbent assay (ELISA) to verify the binding of SEMA3G to NRP1.

[0167] Using ELISA plates (Costar, ME, USA), SEMA3G protein was coated at concentrations of 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.01563, and 0 (µg / mL). The plates were incubated overnight at 4°C, and the supernatant was discarded. After washing five times with 300 µL of PBST per well, each well was blocked with 200 µL of 3% bovine serum albumin (Sangon Biotech, Shanghai, China) at 37°C for 90 min. After washing five times with PBST, 100 µL of NRP1-hFc recombinant protein (2 µg / mL) was added, and the plates were incubated at 37°C for 1 h. After washing five times with 300 µL of PBST per well, each well was incubated with working solution of horseradish peroxidase-labeled specific anti-human Fc fragment antibody diluted in PBS (Jackson, PA, USA) at 37°C for 30 min. After washing with PBST 5 times, prepare the chromogenic solution (Sangon Biotech, Shanghai, China), 100 μL per well, and incubate in an incubator for 10 minutes. Then add 50 μL of stop solution (Sangon Biotech, Shanghai, China) and measure the optical density under 450 nm light using a microplate reader (Thermo Fisher Scientific, MA, USA).

[0168] The results are as follows Figure 6 As shown, different concentrations of SEMA3G bind to NRP1 in a dose-dependent manner, and the nonlinear fitting curve results show that the EC50 of the combination of the two in ELISA is 2.51 (unit: micrograms / mL).

[0169] 3. Detection of the affinity between NRP1 protein and SEMA3G protein using biomembrane interferometry.

[0170] Further verification of the affinity between SEMA3G and NRP1 proteins was conducted using Gator (GatorBio, CA, USA). First, the equilibrated Protein A probe was immersed in buffer-diluted NRP1-Fc recombinant protein (5 μg / mL) to immobilize the NRP1-Fc protein on the sensor surface. The sensor, immobilized with a known concentration of NRP1-Fc recombinant protein, was then immersed in buffer. After baseline stabilization, it was immersed in sample solutions containing different concentrations of the target SEMA3G protein. When the immobilized molecules interacted with molecules in the solution, the biofilm thickness increased, and the interference spectrum shifted towards increasing wavelength. The stabilized sensor was then immersed in buffer for dissociation. The phase shift of the light wave was detected in real time by the Gator detection system workstation, and the kinetic constant of the SEMA3G-NRP1 binding was quantitatively determined by analyzing the phase shift.

[0171] The results are as follows Figure 7 As shown, the affinity of the SEMA3G protein for the receptor NRP1, measured by biomembrane interferometry, is KD = 3.67 × 10^-7 (M).

[0172] Example 3: SEMA3G inhibits cytotoxic T cell activation and function by binding to NRP1 on the surface of T cells.

[0173] Human PBMCs (Heyousheng, Shanghai, China) were resuscitated and cultured overnight in AIM-V medium (Gibco, CA, USA) at 37°C with 0.5% CO2. The cells were resuspended in AIM-V medium and the cell density was adjusted to 2 × 10⁶ cells / mL. Anti-CD3 antibody (Thermo Fisher Scientific, MA, USA), anti-CD28 antibody (Thermo Fisher Scientific, MA, USA), and 50 ng / mL recombinant human interleukin-2 were added to a final concentration of 1 μg / mL and mixed thoroughly. 300 μL of cell suspension was added to each well of a clear 48-well plate (Corning, NY, USA). Except for the control group, SEMA3G protein was added to a final concentration of 10 μg / mL. NRP1 protein was also added to each group at the concentrations shown in the figure. The plates were then returned to a 37°C, 0.5% CO2 incubator for further culture. Cell samples were collected after 72 hours. After washing the cells twice with cell staining buffer (Invitrogen, CA, USA), a mixture of fluorescent antibodies diluted with cell staining buffer was added and incubated at 4°C in the dark for 30 minutes to stain and analyze the expression levels of CD4, CD8, CD25, CD69, HLADR, CD38 on the surface of T cells and granzyme B in the cells.

[0174] like Figure 8 Flow cytometry results showed that, under in vitro activation conditions, SEMA3G treatment significantly reduced the CD8 / CD4 ratio, granzyme B expression level, and the proportion of activated CD8 T cells. Simultaneously, treatment with different concentrations of recombinant NRP1 extracellular protein gradually inhibited the inhibitory effect of SEMA3G until it was completely neutralized with increasing NRP1 recombinant protein concentration. These results indicate that SEMA3G inhibits T cell activation, proliferation, and cytotoxic function by binding to NRP1 on the T cell surface.

[0175] Example 4: Screening of mouse monoclonal antibodies against human SEMA3G

[0176] This embodiment provides a method for preparing mouse anti-human SEMA3G monoclonal antibody, which specifically includes the following steps performed in sequence:

[0177] 1. Preparation of SEMA3G mouse hybridoma antibodies

[0178] Mice were immunized with SEMA3G protein emulsified with CFA (complete Freund's adjuvant) as an immunogen, and multiple immunizations were administered to enhance the effect: For the primary immunization, 50 micrograms of antigen were injected subcutaneously at multiple sites, 3 weeks apart; for the secondary immunization, the dosage and route were the same, but with Freund's incomplete adjuvant, 3 weeks apart; for the third immunization, the dosage was the same, but without adjuvant, injected intraperitoneally, 3 weeks apart; for the fourth booster immunization, the dosage was the same, injected intraperitoneally. Blood was collected 3 days after the final injection to determine the titer. After confirming the desired immunization effect, lymphocytes from the hind leg inguinal lymph nodes and spleen cells of the immunized mice were fused with myeloma cells, and hybridoma cell lines containing anti-SEMA3G protein monoclonal antibodies were obtained through culture, screening, and multiple cloning processes.

[0179] 2. Enzyme-linked immunosorbent assay (ELISA) was used to determine the blocking effect of various hybridoma antibodies on the binding of SEMA3G-NRP1.

[0180] Dilute SEMA3G protein to 2 μg / mL using ELISA coating buffer (Solarbio, Beijing, China). Add 100 μL of coating buffer to each well of a 96-well polystyrene microplate (Costar, ME, USA), and add 100 μL of coating buffer without SEMA3G recombinant protein to each negative control well. Coat overnight at 4°C. Discard the supernatant, wash each well three times with 300 μL of PBST, and then add 200 μL of 3% bovine serum albumin (Sangon Biotech, Shanghai, China) prepared with phosphate buffer to each well for blocking. Incubate at 37°C for 90 min, and wash three times with PBST. Mix the purified monoclonal antibodies and NRP1-Fc and add to each well to achieve a final concentration of 4 μg / mL for the monoclonal antibodies and 2 μg / mL for the NRP1-Fc. Incubate at 37°C for 1.5 h, and wash five times with PBST. Add horseradish peroxidase-labeled anti-human Fc antibody (Jackson, PA, USA), incubate at 37°C for 30 min, and wash 5 times with PBST. Add 100 μL of chromogenic buffer (Invitrogen, CA, USA) to each well and incubate at 37°C for 15 min. Add 50 μL of stop solution (Sangon Biotech, Shanghai, China) to terminate the reaction, and measure the optical density at 450 nm using a microplate reader (Thermo Fisher Scientific, MA, USA).

[0181] The results are as follows Figure 9 As shown, various hybridoma antibodies can block the binding of SEMA3G and NRP1 to varying degrees, with the 2A6D8 antibody exhibiting the best blocking effect. Furthermore, this invention was compared with existing patent technology (CN202210206150.2), and the results are as follows... Figure 9The results show that the 2A6D8 antibody has a significantly better blocking effect on the binding of SEMA3G and NRP1 than the antibody 18H1G2 described in the existing patent technology (CN202210206150.2).

[0182] 3. Enzyme-linked immunosorbent assay (ELISA) was used to verify the affinity of antibody 2A6D8 for SEMA3G.

[0183] Dilute SEMA3G protein to 0.5 μg / mL using ELISA coating buffer (Solarbio, Beijing, China). Add 100 μL of coating buffer to each well of a 96-well polystyrene microplate (Costar, ME, USA). Add 100 μL of coating buffer without SEMA3G recombinant protein to each negative control well. Coat overnight at 4°C. Discard the supernatant. Wash each well three times with 300 μL of PBST. Block each well with 200 μL of 3% bovine serum albumin (Sangon Biotech, Shanghai, China) prepared with phosphate buffer. Incubate at 37°C for 90 min. Wash three times with PBST. Add 100 μL of the solution to each well as... Figure 10 The serially diluted 2A6D8 antibody was incubated at 37°C for 1 hour. After washing five times with 300 μL of PBST per well, each well was incubated with working solution of horseradish peroxidase-labeled specific anti-mouse Fc fragment antibody diluted in PBS (Jackson, PA, USA) at 37°C for 30 minutes. After washing five times with PBST, 100 μL of chromogenic solution (Sangon Biotech, Shanghai, China) was prepared per well and incubated for 10 minutes. Then, 50 μL of stop solution (Sangon Biotech, Shanghai, China) was added, and the absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, MA, USA).

[0184] The results are as follows Figure 10 As shown, different concentrations of 2A6D8 antibody bind to SEMA3G in a dose-dependent manner. The nonlinear fitting curve results show that the EC50 of antibody 2A6D8 binding to SEMA3G antigen is 21.1 (unit: nanograms / mL).

[0185] 4. Gator assay for the affinity between hybridoma antibody and SEMA3G protein.

[0186] Further verification of the affinity between the 2A6D8 antibody and the SEMA3G antigen was conducted using Gator (GatorBio, CA, USA). First, the equilibrated Ni-NTA probe was immersed in buffer-diluted SEMA3G-His recombinant protein (5 μg / mL) to immobilize the SEMA3G-His recombinant protein on the sensor surface. The sensor, immobilized with a known concentration of NRP1-Fc recombinant protein, was then immersed in buffer. After baseline stabilization, it was immersed in sample solutions containing different concentrations of the 2A6D8 antibody to be tested. When the immobilized antigen interacted with the antibody molecules in the solution, the biofilm thickness increased, and the interference spectrum shifted towards increasing wavelength. The stabilized sensor was then immersed in buffer for dissociation. The phase shift of the light wave was detected in real time by the Gator detection system workstation, and the kinetic constant of the binding between the antibody 2A6D8 and the SEMA3G antigen protein was obtained by analyzing and quantifying the phase shift.

[0187] The results are as follows Figure 11 As shown, the affinity KD for the binding of antibody 2A6D8 to the SEMA3G antigen protein was measured by biomembrane interference technique to be 2.28 × 10^-10 (M).

[0188] 5. Blocking activity of candidate SEMA3G antibody 2A6D8 at the protein level

[0189] First, dilute the NRP1 extracellular region protein to 2 μg / mL with ELISA coating buffer (Solarbio, Beijing, China). Add 100 μl to each well of a 96-well flat-bottom polystyrene microplate (Costar, ME, USA) and coat overnight at 4°C. Discard the supernatant, wash twice with 300 μL PBST per well, and then block each well with 200 μL of 3% bovine serum albumin prepared with phosphate buffer (Sangon Biotech, Shanghai, China). Incubate at 37°C for 90 min. Then, use 2 μg / mL biotin-labeled SEMA3G recombinant protein and... Figure 12 Different concentrations of purified 2A6D8 antibody were pre-incubated at 37°C for 30 min. After blocking, the ELISA plate was washed twice with PBST, and then a mixture of antibody and biotin-labeled SEMA3G recombinant protein was added to each well, and incubated at 37°C for 60 min. After washing five times with PBST, 100 μL of horseradish peroxidase-labeled avidin (GenScript, Nanjing, China) detection working solution was added to the reaction wells, and the plate was sealed and incubated at room temperature for 30 min. After washing five times with PBST, 100 μL of chromogenic substrate TMB was added to the reaction wells, and the plate was sealed and incubated at room temperature in the dark for 10 min. The reaction was terminated by adding 50 μL of stop solution, and the optical density was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, MA, USA).

[0190] like Figure 12 As shown, this experiment demonstrates that the binding of SEMA3G protein to the extracellular region of NRP1 can be blocked by antibody 2A6D8, and the blocking effect is antibody concentration-dependent. The nonlinear fitting curve results show that the IC50 of antibody 2A6D8 blocking the binding of SEMA3G protein to the extracellular region of NRP1 is 0.94 (unit: micrograms / mL).

[0191] Example 5: Determination of the promoting effect of SEMA3G monoclonal antibody 2A6D8 on the killing of tumor cells by PBMC cells.

[0192] Resuscitated human PBMCs (Heyousheng, Shanghai, China) were resuspended in AIM-V medium and the cell density was adjusted to 3 x 10⁻⁶ cells / mL. 6Add 1 μg / mL of anti-CD3 antibody (16-0037-81, Invitrogen) and anti-CD28 antibody (16-0289-81, Invitrogen) to each well of cells and mix thoroughly. Incubate at 37°C and 0.5% CO2 for 5 days to activate cells. Use HCT-116 colon cancer cells, LOVO colon cancer cells, and MCF-7 breast cancer cells. Count and plate the cells one day in advance. On the second day, add activated PBMC cells at a 1:1 cell ratio, along with monoclonal antibody or control serum. Incubate for 12 hours. Collect cells from each well into EP tubes (Axygen, CA, USA), centrifuge at 400 rcf for 5 minutes, and discard the supernatant. After resuspending and washing the co-cultured cells twice with cell staining buffer (Invitrogen, CA, USA), a portion of the cells were incubated at 4°C for 30 min in a mixture of diluted fluorescent antibody and cell staining buffer. Different staining groups were set up to analyze the expression levels of CD45, CD4, CD8, CD25, CD69, HLADR, CD38, and GITR on the surface of T cells. After washing with cell staining buffer, 500 μL of 1× permeabilization working solution was added to each tube, and the membrane was permeabilized at 4°C for 45 min. The cells were washed twice with 1× Permeabilization Buffer, and the supernatant was discarded. The cells were resuspended in 100 μL of cell staining buffer, and fluorescently labeled granzyme B antibody was added. The cells were incubated at room temperature for 45 min, washed twice with 1× Permeabilization Buffer, and the supernatant was discarded. The cells were resuspended in cell staining buffer and analyzed. Another portion of the samples was resuspended and washed with 500 μL of cell staining buffer (Invitrogen, CA, USA), and the centrifugation and washing process was repeated. After centrifugation at 400 rcf for 5 minutes, the supernatant was discarded, and the cells were resuspended and washed with 1 mL of binding buffer (Beyotime, Shanghai, China). Centrifugation and washing were repeated. Depending on the conditions, 200 μL of binding buffer, 5 μL of Annexin V-FITC (Beyotime, Shanghai, China) and 10 μL of PI (Beyotime, Shanghai, China) were added, and the cells were incubated on ice for 15 minutes. The cells were then transferred to flow cytometry tubes (Falcon, NY, USA) for analysis.

[0193] The results are as follows Figure 13As shown, this result indicates that, compared with the control, the monoclonal antibody 2A6D8, which blocks the binding of SEMA3G and NRP1, significantly increased the killing effect of PBMCs on tumor cells. Flow cytometry analysis of CD45-positive immune cells showed that in the antibody 2A6D8 treatment group, the expression level of granzyme B in the co-culture system was significantly increased, and the proportions of activated CD8 T cells with CD25+CD69+ double positivity, CD38+HLA-DR+ double positivity, and GITR positive labels were all significantly increased. These results indicate that antibody 2A6D8 treatment increased the activation and function of immune cells in the co-culture system, thereby promoting the killing effect of immune cells on tumor cells.

[0194] Example 6: In vivo experimental analysis of the antitumor effect of monoclonal antibody 2A6D8

[0195] 1. Evaluation of the antitumor effect of SEMA3G antibody in a subcutaneous melanoma cell xenograft model of A375 melanoma cells.

[0196] Five-week-old NCG mice (Jicui Pharmaceutical, Jiangsu, China) were selected and an A375 human melanoma mouse model was established through a mixed inoculation with PBMCs. The tumor volume of the mice reached approximately 50-100 mm. 3Mice were randomly and evenly divided into groups of seven mice each, based on tumor size. The groups included a solvent control group and a SEMA3G antibody 2A6D8 treatment group. This invention was also compared with existing patent technology (CN202210206150.2), which used the SEMA3G antibody 18H1G2 to set up a control antibody treatment group. The antibody treatment group received intraperitoneal injections of 10 mg of antibody 18H1G2 or 2A6D8 per kilogram of body weight per mouse, administered every two days. The solvent control group received the same administration formula, with the corresponding volume of solvent. Tumor growth curves were monitored. After treatment, tumor tissue samples were collected and weighed. A portion of the tumor samples was fixed with tissue fixative and analyzed for immune cell infiltration in the tumor microenvironment using immunohistochemical staining. Another portion of the samples was ground and filtered to obtain a single-cell suspension. Immune cells were isolated and collected, and their immune infiltration in the tumor microenvironment was studied using flow cytometry. After resuspending and washing cells twice with cell staining buffer (Invitrogen, CA, USA), a mixture of fluorescent antibodies diluted with cell staining buffer was added and incubated at 4°C in the dark for 30 min. Different groups were set up to stain and analyze the expression levels of CD45, CD4, CD8, CD25, CD69, HLADR, CD38, PD1, and TIM3 on the surface of T cells. For the detection of intracellular granzyme B and Foxp3 expression, after staining the cell surface, the cells were washed with staining buffer, and 500 μL of 1× permeabilization working solution was added to each tube. The cells were incubated at 4°C for 45 min to permeabilize the membrane. The cells were washed twice with 1× Permeabilization Buffer, and the supernatant was discarded. The cells were resuspended in 100 μL of cell staining buffer, and fluorescently labeled granzyme B antibody and Foxp3 antibody were added. The cells were incubated at room temperature for 45 min, washed twice with 1× Permeabilization Buffer, and the supernatant was discarded. The cells were resuspended in cell staining buffer and analyzed.

[0197] The results are as follows Figure 14 As shown, compared with the solvent control group, the SEMA3G antibody 2A6D8, which blocks SEMA3G-NRP1 binding activity, significantly inhibited tumor growth and had a therapeutic effect on melanoma. This invention was compared with existing patent technology (CN202210206150.2). The SEMA3G antibody 2A6D8 in this invention, which blocks SEMA3G-NRP1 binding activity, exhibits significant anti-tumor activity; while the SEMA3G antibody 18H1G2 described therein did not show effective activity in inhibiting A375 tumor growth.

[0198] like Figure 15Immunohistochemical staining results showed that the proportion of CD8 T cells infiltrating the tumor microenvironment of mice treated with SEMA3G antibody was significantly increased. Flow cytometry analysis of tumor-infiltrating immune cells showed that the expression level of granzyme B in tumor-infiltrating immune cells was significantly increased in the antibody treatment group, and the proportion of activated CD8 T cells labeled with CD25+CD69+ double positive or CD38+HLA-DR+ double positive were significantly increased. Conversely, the proportion of Foxp3-labeled Treg cells and PD1+TIM3+ double positive exhausted T cells in the tumor microenvironment of mice treated with SEMA3G antibody 2A6D8 was significantly reduced. These results indicate that antibody 2A6D8 treatment increased the activation and function of immune cells in the tumor microenvironment, thereby promoting the anti-tumor immune response.

[0199] 2. The SEMA3G monoclonal antibody 2A6D8 significantly inhibited tumor growth in NCI-H520 and HCT116 subcutaneous xenograft models.

[0200] Five-week-old NCG mice (Jicui Pharmaceutical, Jiangsu, China) were selected and NCI-H520 lung squamous cell carcinoma models and HCT-116 colorectal cancer models were established through PBMC co-inoculation. The tumors in the mice reached approximately 50-100 mm in size. 3 Mice were randomly and evenly divided into a solvent control group and a SEMA3G antibody 2A6D8 treatment group based on tumor size. The antibody treatment group received intraperitoneal injections of 10 mg of antibody A6D8 per kilogram of body weight per mouse, administered every two days. The solvent control group received the same administration formula, but with the corresponding volume of solvent. Tumor growth curves were monitored. After treatment, tumor tissue samples were collected, fixed, and immunohistochemically stained to study immune infiltration in the tumor microenvironment.

[0201] The results are as follows Figure 16 As shown, compared with the solvent control group, the SEMA3G antibody 2A6D8, which blocks the binding activity of SEMA3G-NRP1, significantly inhibited the growth of NCI-H520 lung squamous cell carcinoma and HCT-116 colorectal cancer tumors in mice. Furthermore, immunohistochemical staining results indicated a significantly increased proportion of CD8 T cells infiltrating the tumor microenvironment in the SEMA3G antibody treatment group, suggesting that the SEMA3G antibody enhances the activation and function of immune cells in the tumor microenvironment and has a therapeutic effect on tumors such as lung squamous cell carcinoma and colorectal cancer.

[0202] Example 7: Establishment of a method for detecting SEMA3G levels in samples using a double-antibody sandwich ELISA and detection of SEMA3G secretion levels in cell culture supernatant.

[0203] 1. Antibody affinity assay

[0204] Dilute SEMA3G protein to 0.5 μg / mL using ELISA coating buffer; add 100 μL to each well of a 96-well polystyrene microplate (Costar, ME, USA) and incubate overnight at 4°C. Discard the supernatant, wash twice with 300 μL PBST per well, then add 200 μL of 3% bovine serum albumin (Sangon Biotech, Shanghai, China) prepared with phosphate buffer per well for blocking, and incubate at 37°C for 90 min. Wash three times with PBST, then add 100 μL of serially diluted SEMA3G antibody 1C1A6 or 15H1F5 as shown in the figure, and incubate at 37°C for 1 h. Wash five times with 300 μL PBST per well, then add phosphate buffer diluted specific anti-human Fc fragment antibody (Jackson, PA, USA), and incubate at 37°C for 30 min for binding. After washing five times with PBST, prepare the chromogenic solution (Sangon Biotech, Shanghai, China). Add 100 μL of the chromogenic solution to each well and incubate at room temperature for 5 minutes. Stop the reaction by adding 50 μL of stop solution (Sangon Biotech, Shanghai, China) to each well. Measure the optical density at 450 nm using a microplate reader (Thermo Fisher Scientific, MA, USA). Results are as follows: Figure 17 As shown, the two antibodies bound to the SEMA3G protein under different concentration conditions, and the binding was significantly dose-dependent. The nonlinear fitting curve results showed that the EC50 of the two antibodies binding to SEMA3G was 19.5 and 27 (unit: micrograms / mL), respectively.

[0205] 2. Establishment of a double-antibody sandwich ELISA detection method

[0206] Add 1 mg of the antibody 15H1F5 to be labeled to a 0.5 mL ultrafiltration tube, and make up the total volume to 0.5 mL with labeling buffer. Centrifuge at 12000×g for 10 min, discard the liquid flowing through the collection tube, and recover and adjust the concentrated protein concentration to approximately 2 mg / mL. Immediately add 26.6 μL of 10 mM NHS-Biotin to the protein solution, gently pipette to mix, seal the tube, and incubate at 37°C in the dark for 30 min to perform the labeling reaction. Add 10 μL of 1 M Tris (pH 8.7) per 100 μg of protein, mix well, and incubate at room temperature for 10 min to terminate the blocking reaction. Add an appropriate amount of 1×PBS to the above reaction solution, repeat the centrifugation and ultrafiltration operation 2-3 times, collect the biotin-labeled antibody, adjust the concentration, add an equal volume of glycerol, and store at -20°C for later use.

[0207] Using a 96-well flat-bottom polystyrene ELISA plate as the solid-phase carrier, the monoclonal antibody 1C1A6 purified by Protein G affinity chromatography was diluted to 2 μg / mL with antibody coating buffer. 100 μL of the diluted antibody was added to each well of the ELISA plate, and the plate was sealed with sealing film and incubated overnight at 4°C. The supernatant was discarded, and each well was washed twice with 300 μL of PBST. Then, 200 μL of 3% bovine serum albumin (Sangon Biotech, Shanghai, China) prepared in phosphate buffer was added to each well for blocking, and the plate was incubated at 37°C for 90 min. After washing three times with PBST, 100 μL of serially diluted SEMA 3G protein standard was added to each well, and a blank control was set up. The plate was then sealed and incubated at room temperature for 2 h. After washing five times with PBST, 100 μL of biotin-labeled antibody 15H1F5-Biotin working solution was added to each well, and the plate was sealed and incubated at room temperature for 1 h. Wash five times with PBST, add 100 μL of Streptavidin-HRP (GenScript, Nanjing, China) detection working solution to the reaction wells, seal the plate, and incubate at room temperature for 45 minutes. Wash five times with PBST, add 100 μL of chromogenic substrate TMB to the reaction wells, seal the plate, and incubate at room temperature in the dark for 15 minutes. Stop the reaction by adding 50 μL of stop solution, and measure the optical density at 450 nm using a microplate reader (Thermo Fisher Scientific, MA, USA). Plot a standard curve with standard concentration on the x-axis (Ng / mL) and optical density OD450 value on the y-axis. Results are as follows: Figure 18 As shown, this detection method exhibits good linearity in the range of 0.1-100 (unit: nanograms / mL), R0 2 It is 0.9997 ( Figure 18 ).

[0208] 3. Detection of SEMA3G secretion levels in cell line supernatant

[0209] Using a 96-well flat-bottom polystyrene ELISA plate as the solid-phase carrier, the monoclonal antibody 1C1A6 purified by Protein G affinity chromatography was diluted to 2 μg / mL with antibody coating buffer. 100 μL of the diluted antibody was added to each well of the ELISA plate, and the plate was sealed with sealing film and incubated overnight at 4°C. The supernatant was discarded, and each well was washed twice with 300 μL of PBST. Then, 200 μL of 3% bovine serum albumin (Sangon Biotech, Shanghai, China) prepared with phosphate buffer was added to each well for blocking, and the plate was incubated at 37°C for 90 min. After washing three times with PBST, 100 μL of supernatant from different cell lines was added to each well, and the plate was sealed and incubated at room temperature for 2 h. After washing five times with PBST, 100 μL of biotin-labeled antibody 15H1F5-Biotin working solution was added to each well, and the plate was sealed and incubated at room temperature for 1 h. Wash five times with PBST. Add 100 μL of Streptavidin-HRP (GenScript, Nanjing, China) detection working solution to the reaction wells, seal the plate, and incubate at room temperature for 45 minutes. Wash five times with PBST. Add 100 μL of chromogenic substrate TMB to the reaction wells, seal the plate, and incubate at room temperature in the dark for 15 minutes. Add 50 μL of stop solution to terminate the reaction. Measure the optical density at 450 nm using a microplate reader (Thermo Fisher Scientific, MA, USA). Calculate the SEMA3G protein content in the sample based on the plotted standard curve.

[0210] The test results are shown in Table 1 below:

[0211] Table 1. SEMA3G protein content in samples

[0212]

[0213] Example 8: Antibody sequencing and humanized antibody design, purification, and affinity identification

[0214] 1. Antibody sequencing

[0215] Freshly collected hybridoma cell pellets were sent to Suzhou Genewiz Biotechnology Co., Ltd. for hybridoma antibody sequencing. RNA was extracted using the Trizol method, cDNA was obtained through reverse transcription, and the heavy and light chain variable regions were amplified and sequenced. The sequencing results were then compared to an international immunogenetic database, and CDR1 / 2 / 3 information was extracted. The amino acid sequences of monoclonal antibodies 2A6D8, 1C1A6, and 15H1F5 are shown in Tables 2, 4, and 6, and the nucleotide sequences of monoclonal antibodies 2A6D8, 1C1A6, and 15H1F5 are shown in Tables 3, 5, and 7.

[0216] Table 2. Amino acid sequence of monoclonal antibody 2A6D8

[0217]

[0218] Table 3. Nucleotide sequence of monoclonal antibody 2A6D8

[0219]

[0220]

[0221] Table 4. Amino acid sequence of monoclonal antibody 1C1A6

[0222]

[0223] Table 5. Nucleotide sequence of monoclonal antibody 1C1A6

[0224]

[0225]

[0226] Table 6. Amino acid sequence of monoclonal antibody 15H1F5

[0227]

[0228] Table 7. Nucleotide sequence of monoclonal antibody 15H1F5

[0229]

[0230]

[0231] 2. Humanization design, expression, and purification of antibody 2A6D8

[0232] The monoclonal antibody 2A6D8 was humanized to obtain the amino acid sequence of the humanized 2A6D8 monoclonal antibody 7K3Q. The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:25, the amino acid sequence of its light chain is shown in SEQ ID NO:26, the nucleotide sequence of its heavy chain variable region is shown in SEQ ID NO:34, and the nucleotide sequence of its light chain variable region is shown in SEQ ID NO:35. The nucleotide sequence of the light chain variable region was inserted into pcDNA3.1-IgK, and the nucleotide sequence of the heavy chain variable region was inserted into pcDNA3.1-IgG1Fc to construct an expression vector. The expression vector construction was verified to be correct. The expression vector was transfected into 293 cells, and the supernatant was collected after 96 hours. The humanized antibody was purified using Protein A beads (Tiandi Renhe, Jiangsu, China). The amino acid sequence of the humanized antibody 7K3Q is shown in Table 8, and the nucleotide sequence of the humanized antibody 7K3Q is shown in Table 9.

[0233] Table 8. Amino acid sequence of humanized antibody 7K3Q

[0234]

[0235] Table 9 Nucleotide sequence of humanized antibody 7K3Q

[0236]

[0237]

[0238] 3. Enzyme-linked immunosorbent assay (ELISA) demonstrated that the humanized antibody could bind to the SEMA3G protein.

[0239] SEMA3G protein at a concentration of 0.5 μg / mL was coated onto an ELISA plate (Costar, ME, USA) and incubated overnight at 4°C, discarding the supernatant. Each well was washed three times with 300 μL of PBST and then blocked with 3% bovine serum albumin (Sangon Biotech, Shanghai, China) prepared in 200 μL of PBS at 37°C for 90 min. After washing three times with PBST, serially diluted 7K3Q humanized antibody was added to each well and incubated at 37°C for 60 min. After washing five times with 300 μL of PBST, each well was incubated with specific anti-human Fc fragment antibody diluted in PBS (Jackson, PA, USA) at 37°C for 30 min. After washing with PBST 5 times, prepare TMB chromogenic solution (Sangon Biotech, Shanghai, China), 100 μL per well, and incubate in an incubator for 10 minutes. Then add 50 μL of stop solution (Sangon Biotech, Shanghai, China) to stop the reaction. Detect the optical density at a wavelength of 450 nm using an ELISA reader (Thermo Fisher Scientific, MA, USA).

[0240] like Figure 19 The dose-response curves for the binding of the 7K3Q humanized antibody to the purified SEMA3G protein are shown. The nonlinear fitting curve results show that the EC50 of the 2A6D8 humanized antibody 7K3Q to the SEMA3G antigen is 17.16 (unit: ng / mL). These results indicate that the humanized antibody 7K3Q and the murine antibody 2A6D8 have comparable affinity for SEMA3G.

[0241] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0242] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. An isolated antigen-binding protein, said antigen-binding protein binding to SEMA3G protein, said antigen-binding protein comprising a heavy chain variable region and a light chain variable region; said heavy chain variable region comprising HCDR1 as shown in any one of SEQ ID NO. 3, 11, 19, HCDR2 as shown in any one of SEQ ID NO. 4, 12, 20, and HCDR3 as shown in any one of SEQ ID NO. 5, 13, 21; said light chain variable region comprising LCDR1 as shown in any one of SEQ ID NO. 6, 14, 22, LCDR2 as shown in any one of SEQ ID NO. 7, 15, 23, and LCDR3 as shown in any one of SEQ ID NO. 8, 16, 24.

2. The antigen-binding protein according to claim 1, characterized in that, The antigen-binding protein has any of the following characteristics: 1) Including HCDR1 as shown in SEQ ID NO.3, HCDR2 as shown in SEQ ID NO.4, HCDR3 as shown in SEQ ID NO.5, LCDR1 as shown in SEQ ID NO.6, LCDR2 as shown in SEQ ID NO.7, and LCDR3 as shown in SEQ ID NO.8; 2) Including HCDR1 as shown in SEQ ID NO.11, HCDR2 as shown in SEQ ID NO.12, HCDR3 as shown in SEQ ID NO.13, LCDR1 as shown in SEQ ID NO.14, LCDR2 as shown in SEQ ID NO.15, and LCDR3 as shown in SEQ ID NO.16; 3) Including HCDR1 as shown in SEQ ID NO.19, HCDR2 as shown in SEQ ID NO.20, HCDR3 as shown in SEQ ID NO.21, LCDR1 as shown in SEQ ID NO.22, LCDR2 as shown in SEQ ID NO.23, and LCDR3 as shown in SEQ ID NO.

24.

3. The antigen-binding protein according to claim 1, characterized in that, The heavy chain variable region of the antigen-binding protein includes an amino acid sequence as shown in any one of SEQ ID NO. 1, 9, 17, 25, and the light chain variable region of the antigen-binding protein includes an amino acid sequence as shown in any one of SEQ ID NO. 2, 10, 18, 26. Preferably, the antigen-binding protein has any of the following characteristics: 1) Includes the heavy chain variable region as shown in SEQ ID NO.1 and the light chain variable region as shown in SEQ ID NO.2; 2) Includes the heavy chain variable region as shown in SEQ ID NO.9, and the light chain variable region as shown in SEQ ID NO.10; 3) Includes the heavy chain variable region as shown in SEQ ID NO.17 and the light chain variable region as shown in SEQ ID NO.18; 4) Includes the heavy chain variable region as shown in SEQ ID NO.25 and the light chain variable region as shown in SEQ ID NO.

26.

4. The antigen-binding protein according to claim 1, characterized in that, The antigen-binding protein includes an antibody or an antigen-binding fragment; preferably, the antibody is selected from monoclonal antibodies, polyclonal antibodies, chimeric antibodies, or humanized antibodies; and / or, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv, scFv, and dsFv fragments.

5. A chimeric antigen receptor, said chimeric antigen receptor comprising the antigen-binding protein as described in any one of claims 1-4.

6. A polynucleotide encoding an antigen-binding protein as described in any one of claims 1-4, or a chimeric antigen receptor as described in claim 5.

7. A vector comprising the polynucleotide as described in claim 6.

8. A host cell containing an antigen-binding protein as described in any one of claims 1-4, or a vector as described in claim 7, or having a polynucleotide as described in claim 6 integrated into its genome.

9. A method for preparing an antigen-binding protein or a chimeric antigen receptor, comprising culturing a host cell containing the vector of claim 7 or a host cell whose genome has integrated an exogenous polynucleotide of claim 6, under conditions allowing expression of the antigen-binding protein or chimeric antigen receptor, and recovering the antigen-binding protein or chimeric antigen receptor from the cultured host cell culture.

10. A pharmaceutical composition comprising an antigen-binding protein as described in any one of claims 1-4, a chimeric antigen receptor as described in claim 5, a polynucleotide as described in claim 6, a carrier as described in claim 7, or a host cell as described in claim 8.

11. A kit comprising an antigen-binding protein as described in any one of claims 1-4, or a chimeric antigen receptor as described in claim 5, or a polynucleotide as described in claim 6, or a carrier as described in claim 7, or a host cell as described in claim 8; preferably, the antigen-binding protein is labeled with a marker; more preferably, the marker is selected from enzymes, biotin, chemiluminescent groups, or isotopic groups.

12. A method for detecting SEMA3G protein content, comprising using an antigen-binding protein as described in any one of claims 1-4, or a kit as described in claim 10, to detect SEMA3G protein content using an immunoassay technique; preferably, the immunoassay technique is a double-antibody sandwich ELISA; preferably, the detection method is a detection method for non-disease diagnosis and treatment purposes.

13. Use of the antigen-binding protein as described in any one of claims 1-4, or the chimeric antigen receptor as described in claim 5, or the polynucleotide as described in claim 6, or the vector as described in claim 7, or the host cell as described in claim 8, or the kit as described in claim 11 in the preparation of immunoassay products or products for detecting SEMA3G protein content.

14. Use of the antigen-binding protein of any one of claims 1-4, or the chimeric antigen receptor of claim 5, or the polynucleotide of claim 6, or the carrier of claim 7, or the host cell of claim 8, or the pharmaceutical composition of claim 10 in the preparation of a product that blocks the binding activity of SEMA3G protein to NRP1.

15. Use of the antigen-binding protein of any one of claims 1-4, or the chimeric antigen receptor of claim 5, or the polynucleotide of claim 6, or the carrier of claim 7, or the host cell of claim 8, or the pharmaceutical composition of claim 10, or the kit of claim 11, in the preparation of a product, wherein the product is used for any one or more of the following: modulating immune responses; tumor diagnosis; tumor prevention; and tumor treatment; Preferably, the immune response is selected from autoimmune diseases, immunodeficiency diseases, allergic reactions, tumor immune escape, neurodegenerative diseases, cardiovascular diseases, diabetes, and infectious diseases; Preferably, the tumor is selected from ovarian cancer, breast cancer, lung cancer, colorectal cancer, melanoma, liver cancer, pancreatic cancer, sarcoma, nervous system tumors, leukemia, urothelial carcinoma, cervical cancer, prostate cancer, esophageal cancer, thymoma, squamous cell carcinoma, lymphoma, gastric cancer, testicular cancer, thyroid cancer, endometrial cancer, head and neck cancer, kidney cancer, adrenocortical carcinoma, bile duct cancer, mesothelioma, pheochromocytoma, and paraganglioma.

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