Antibodies against ccr7 and uses thereof

Abstract

The application discloses an antibody against CCR7 and application thereof. The immunogen based on the N-terminal polypeptide of CD197 and the human Fc fragment is used to construct a flow antibody which can be applied to peripheral blood mononuclear cells (PBMC), and the flow antibody has good performance, high specificity and low preparation cost. It is verified that the flow antibody only combines with cells expressing CD197, does not combine with cells not expressing CD197, and can be effectively used in clinical real samples such as PBMC, and can be applied to diagnosis of diseases related to CD197 protein, immunological research and development of new treatment strategies and the like.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and in particular to an anti-CCR7 antibody and its application. Background Technology

[0002] CC-chemokine receptor 7 (CCR7), also known as CD197, is a G protein-coupled receptor encoded by the CCR7 gene, which is normally expressed in various lymphoid tissues. CCR7 can activate B lymphocytes and T lymphocytes, causing these cells to migrate to CCR7 ligands (CCL19 and CCL21), thereby facilitating its localization in secondary lymphoid organs (such as the spleen, lymph nodes, and Peyer's plaques). CCR7 can also be pathologically expressed by tumor cells and promote tumor growth and metastasis (especially to lymphoid organs). In this process, CCR7 forms constitutive expression with the two ligands CCL19 / CCL21.

[0003] CCR7 activity is associated with various disease states, such as chronic inflammation, atherosclerosis, HIV infection, and cancer. Related studies have found that CCR7 is expressed in a variety of tumor cells, including mantle cell lymphoma (MCL), follicular lymphoma, B-cell lymphoma, AIDS-related lymphoma, lymphoplasmacytic lymphoma, Burkitt lymphoma, B-cell acute lymphoblastic leukemia, Hodgkin's disease (lymphoreticular cell sarcoma), adult T-cell leukemia / lymphoma, mycosis fungoides, chronic myelodysplastic syndrome, myelodysplastic syndrome blast crisis, breast cancer, non-small cell lung cancer, melanoma, gastric cancer or head and neck squamous cell carcinoma, and colon cancer, especially B-cell chronic lymphocytic leukemia, non-Hodgkin's lymphoma, breast cancer cells, and malignant breast tumors. Furthermore, CCR7 has been found to play a role in lymph node metastasis in various cancers.

[0004] In preclinical studies, intervention on the CCL21 / CCR7 axis has emerged as a potential target for the treatment of tumor metastasis. Both downregulation and enhancement of the CCL21 / CCR7 axis can be used for anti-tumor therapy. On the one hand, the CCL21 / CCR7 axis can promote lymphatic metastasis of tumor cells; downregulation of the CCL21 / CCR7 axis can inhibit tumor cell metastasis. On the other hand, increasing CCL21 in the tumor microenvironment and reshaping the CCL21 concentration gradient between tumor tissue and tumor-associated lymphoid vessels can enhance the CCL21 / CCR7 axis. This can not only block CCR7-mediated tumor cell lymphatic migration but also recruit more immune cells at the tumor tissue to exert tumor-killing effects. Therefore, it can be determined that, based on the origin of CCR7, the role of the CCL21 / CCR7 axis in tumors is bidirectional: the binding of CCR7 expressed on tumor cells to CCL21 promotes tumor development, while the binding of CCR7 expressed on immune cells such as dendritic cells (DCs) and T cells to CCL21 inhibits tumor development. Specifically, the CCL21 / CCR7 axis promotes tumor cell proliferation by activating the ERK1 / 2 signaling pathway and upregulating the expression of matrix metalloproteinases (MMPs). It also upregulates the expression of vascular endothelial growth factor (VEGF), thereby inducing angiogenesis and promoting tumor metastasis. Furthermore, tumor cells expressing CCR7 can migrate into lymphatic vessels and metastasize along the path of increasing CCL21 concentration. However, when CCR7 expressed by immune cells binds to CCL21, it stimulates the immune response and inhibits tumor development by regulating the lymphatic migration and lymph node homing characteristics of immune cells such as T cells and dendritic cells (DCs).

[0005] Treatment based on anti-CCR7 antibodies can effectively reduce tumor cell migration and invasion. In related techniques, after subcutaneously or intravenously inoculating mice with human lymphoma cells, treatment with anti-CCR7 antibodies significantly delayed the appearance of subcutaneous tumors and inhibited tumor growth compared to untreated mice. Furthermore, compared to untreated mice, mice treated with anti-CCR7 antibodies showed a significantly reduced number of tumors metastasizing to distant lymphoid organs and a significantly increased number of apoptotic tumor cells. In an intravenous injection model, CCR7 antibody treatment significantly improved mouse survival. Therefore, CCR7 neutralization therapy has shown promising results in many preclinical models. In addition, related studies have shown that anti-CCR7 monoclonal antibodies have also demonstrated the ability to induce tumor cell death and reduce or avoid central nervous system diseases in a mouse model of T-cell lymphoblastic leukemia xenograft, and that single-chain anti-CCR7 antibodies successfully prevented T-cell acute lymphoblastic leukemia cells from crossing the blood-brain barrier in an in vitro model.

[0006] CCR7 (CD197) plays a crucial role in the fine segmentation of T cells during flow cytometry. However, compared to other members of the CD (differentiation cluster) family, CCR7 possesses unique structural characteristics, including seven transmembrane crossings and a shorter extracellular region. This makes antibody development more challenging, resulting in a limited range of available antibodies on the market. Adding to the difficulty, current CD197 antibodies require incubation at 37°C for flow cytometry detection, a requirement that hinders combination staining with other antibodies.

[0007] Therefore, there is an urgent need to provide a new anti-CCR7 antibody to overcome the shortcomings of existing CCR7 antibodies and provide a new tool for CCR7-related research. Summary of the Invention

[0008] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of this invention is to provide an anti-CCR7 antibody and its application. In this invention, the inventors constructed a CCR7 antigenic epitope peptide based on the N-terminal polypeptide of CCR7 (CD197) and the human Fc fragment, and obtained an anti-CCR7 antibody based on this peptide. This antibody can be effectively used in flow cytometry, and its screening effect is better than that of existing antibodies.

[0009] In a first aspect, the present invention provides an antibody against CCR7 or an antigen-binding fragment thereof, said antibody against CCR7 or an antigen-binding fragment thereof binding to an antigenic epitope peptide or protein comprising the sequence shown in SEQ ID NO: 1.

[0010] In this invention, "comprising," "containing," "having," or "including" means that at least the specified substance, component, element, or method step is present in the product, article, or method, but does not exclude the presence of other substances, components, elements, or method steps, even if the other such substances, components, elements, or method steps have the same function as the specified ones.

[0011] In some embodiments of the present invention, the antigenic epitope peptide or protein further includes a crystallizable segment (Fc) sequence linked to the sequence shown in SEQ ID NO: 1.

[0012] In this invention, the term "crystallizable segment (Fc)" refers to a fragment in an antibody molecule consisting of a constant region of the heavy chain, located at the carboxyl terminus (C-terminus) of the antibody, which is a non-antigen-binding region separated from the antibody after hydrolysis by papain.

[0013] In some embodiments of the present invention, the crystallizable segment (Fc) sequence is directly connected to or connected to the C-terminus (carboxyl terminus) via other sequences.

[0014] In some embodiments of the present invention, the crystallizable segment (Fc) sequence has the following amino acid sequence:

[0015] GPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 31).

[0016] In some embodiments of the present invention, the crystallizable segment (Fc) sequence further includes:

[0017] (1) A sequence having the same function as the sequence shown in SEQ ID NO: 31, but with one or more amino acid substitutions and / or deletions and / or additions; or

[0018] (2) A sequence that has at least 70% identity with SEQ ID NO: 31 and has the same function as the sequence shown in SEQ ID NO: 1.

[0019] In some embodiments of the invention, the identity is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, or 70%.

[0020] In this invention, the phrase "having the same function" means having at least one of the following characteristics or functions: similar immunogenicity, binding activity, specificity, or screening effect. In some embodiments of this invention, the difference in said characteristics or functions is no greater than 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0021] In some embodiments of the present invention, the crystallizable segment (Fc) sequence is a humanized or fully human crystallizable segment sequence.

[0022] In this invention, the term "humanized crystallizable segment sequence" refers to a sequence in which part or all of a non-human crystallizable segment sequence is fused into a human crystallizable segment sequence using genetic engineering techniques, thereby reducing the immunogenicity caused by non-human Fc without affecting binding activity (including specificity). The term "fully human crystallizable segment sequence" refers to a crystallizable segment sequence composed entirely of human crystallizable segment sequences.

[0023] In some embodiments of the present invention, the antigenic epitope peptide or protein further includes at least one functional sequence.

[0024] In some embodiments of the present invention, the antigenic epitope peptide or protein further includes 1-10 functional sequences.

[0025] In some embodiments of the present invention, the functional sequence includes, but is not limited to, at least one of a tag sequence, a linker, and an enzyme cleavage site.

[0026] In some embodiments of the present invention, the tag sequence includes, but is not limited to: affinity tags, such as GST, His, MBP, etc.; epitope tags, such as HA, Myc, Flag, etc.; fluorescent tags, such as GFP, mCherry, etc.; and other tags, such as SNAP and Avi, etc.

[0027] In some embodiments of the present invention, the tag sequence is Avi.

[0028] In some embodiments of the present invention, the linker includes, but is not limited to: flexible linkers, such as linkers composed of repeating small molecule amino acids such as glycine (G) and serine (S), such as GGS, GGGS, etc.; rigid linkers, such as linkers containing α-helices or proline-enriched sequences; cleavable linkers, such as linkers containing specific enzyme cleavage sites (such as thrombin, furin protease) or chemically sensitive groups (such as pH-responsive groups); and self-assembling linkers, such as leucine zipper Ap1 / Bp1, etc.

[0029] In some embodiments of the present invention, the linker is a flexible linker.

[0030] In some embodiments of the present invention, the functional sequence is located between the sequence shown in SEQ ID NO: 1 and the Fc sequence, and / or at one end of the sequence shown in SEQ ID NO: 1 and the Fc sequence that is far from each other.

[0031] In some embodiments of the present invention, without affecting the functionality of the sequence (such as immunogenicity, binding activity, specificity, or screening effect), there is no limit to the number of functional sequences located at the ends of the sequence and Fc sequence shown in SEQ ID NO: 1 that are far from each other, and between the sequence and Fc sequence shown in SEQ ID NO: 1. Any number of functional sequences can be inserted based on the purpose and requirements of use.

[0032] In some embodiments of the present invention, the antigenic epitope peptide or protein contains a sequence as shown in SEQ ID NO: 2.

[0033] In some embodiments of the present invention, the antigenic epitope peptide or protein further includes:

[0034] (1) A sequence having one or more amino acid substitutions and / or deletions and / or additions to SEQ ID NO: 2, and having the same function as the sequence shown in SEQ ID NO: 2; or

[0035] (2) A sequence that has at least 70% identity with SEQ ID NO: 2 and has the same function as the sequence shown in SEQ ID NO: 2.

[0036] In some embodiments of the invention, the identity is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, or 70%.

[0037] In some embodiments of the present invention, the antigenic epitope peptide or protein further contains chemical modifications and / or is linked with functional groups.

[0038] In some embodiments of the present invention, the chemical modification includes, but is not limited to, at least one of methylation, acetylation, phosphorylation, and glycosylation.

[0039] In some embodiments of the present invention, the functional gene includes, but is not limited to, at least one of: reporter group, protein tag, carrier and lipid group.

[0040] In some embodiments of the present invention, the reporter group includes, but is not limited to, at least one of: chromogenic enzyme, fluorescent labeling group, chemiluminescent labeling group, isotope or magnetic group.

[0041] In some embodiments of the present invention, the chromogenic enzyme includes, but is not limited to, at least one of: peroxidase and alkaline phosphatase.

[0042] In some embodiments of the present invention, the fluorescent labeling group includes, but is not limited to, at least one of the following: fluorescent protein groups, rhodamine groups, fluorescein groups, anthocyanin dyes, cyanine dyes, Alexa Fluor dyes, and / or quantum dot fluorescent groups.

[0043] In some embodiments of the present invention, the isotopes include, but are not limited to, radioactive nuclides.

[0044] In some embodiments of the present invention, the radionuclides include, but are not limited to, those used in radioimmunoassay and radioimmunotherapy.

[0045] In some embodiments of the present invention, the magnetic group includes, but is not limited to, at least one of the groups that enable magnetic resonance imaging or alter relaxation efficiency.

[0046] In some embodiments of the present invention, the protein tags include, but are not limited to: affinity tags, such as GST, His, MBP, etc.; epitope tags, such as HA, Myc, Flag, etc.; fluorescent tags, such as GFP, mCherry, etc.; and other tags, such as SNAP and Avi, etc.

[0047] In some embodiments of the present invention, the carrier includes, but is not limited to, carrier proteins, such as hemocyanin, serum albumin, ovalbumin, etc.

[0048] In some embodiments of the present invention, the lipid groups include, but are not limited to, fatty acid chains (such as palmitic acid) to form lipopeptides.

[0049] In some embodiments of the present invention, the anti-CCR7 antibody or its antigen-binding fragment includes a light chain variable region complementarity-determining region (CDR) and a heavy chain variable region CDR.

[0050] In this invention, the term "variable region" refers to the functional region in an antibody or immunoglobulin molecule that is responsible for specifically recognizing and binding antigens.

[0051] In some embodiments of the present invention, the light chain variable region CDR and the heavy chain variable region CDR are defined by antibody numbering systems such as Kabat, AbM, Chothia, Contact, or IMGT.

[0052] For relevant antibody numbering systems, please refer to: Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997); Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, Department of Health and Human Services, National Institutes of Health (1987), etc.

[0053] In some embodiments of the present invention, the light chain variable region CDR and the heavy chain variable region CDR are defined by Kabat. It should be understood that although the scope of protection claimed by the present invention is based on the sequences shown in the Kabat definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of the present invention.

[0054] In some embodiments of the present invention, the variable region CDR of the light chain is shown as GGNNIGSKAVH, NDDERPS, and QLWDSSAGVV, respectively; and the variable region CDR of the heavy chain is shown as SFGVT, FINGNNNAYYANWARS, and AYGTFTPADRLDL, respectively.

[0055] In some embodiments of the present invention, in the light chain variable region, CDR1 is GGNNIGSKAVH; CDR2 is NDDERPS; CDR3 is QLWDSSAGVV.

[0056] In some embodiments of the present invention, in the heavy chain variable region, CDR1 is SFGVT; CDR2 is FINGNNNAYYANWARS; CDR3 is AYGTFTPADRLDL.

[0057] In some embodiments of the present invention, the light chain variable regions CDR are shown as QASQNIDSYLA (CDR1), KASTLAS (CDR2), and CQSHYYSGSTGYAHTF (CDR3), respectively; and the heavy chain variable regions CDR are shown as GFSLSYV (CDR1), SSGVP (CDR2), and CARGSTDSNYYGGYYFTLW (CDR3), respectively.

[0058] In some embodiments of the present invention, the light chain variable region CDR is shown as QASQSISSYLS (CDR1), DASNLET (CDR2), and CQSYYYSSSSSYGSGF (CDR3); the heavy chain variable region CDR is shown as GFSLSYV (CDR1), SSGVP (CDR2), and CARGSTDSNYYGGYYFTLW (CDR3).

[0059] In some embodiments of the present invention, the light chain variable regions CDR are shown as QASQNIDSYLA (CDR1), KASTLAS (CDR2), and CQGGVHDSDEFPF (CDR3), respectively; and the heavy chain variable regions CDR are shown as GFSLNSY (CDR1), YDDGS (CDR2), and CVRDSDYVGFGYNLW (CDR3), respectively.

[0060] In some embodiments of the present invention, the light chain variable region CDR is shown as QASEDIESYLA (CDR1), YASNLES (CDR2), and CQGYYYSGRTVAF (CDR3), respectively; the heavy chain variable region CDR is shown as GFSLSSY (CDR1), GSSGS (CDR2), and CARDSYGGDYAIGFDIW (CDR3), respectively.

[0061] In some embodiments of the present invention, the light chain variable regions CDR are shown as QSSESVYNKNWLA (CDR1), EASTLAS (CDR2), and CAGDYIYNSDNAF (CDR3), respectively; and the heavy chain variable regions CDR are shown as GFSLNTF (CDR1), FGSGD (CDR2), and CARYFNYGSYNLW (CDR3), respectively.

[0062] In some embodiments of the present invention, the light chain variable regions CDR are shown as QASQSINNQLS (CDR1), LASTLAS (CDR2), and CQSTHDITTYGIAF (CDR3), respectively; and the heavy chain variable regions CDR are shown as GIDLSSY (CDR1), WGGSA (CDR2), and CARNPGYSANLW (CDR3), respectively.

[0063] In some embodiments of the present invention, the light chain variable region CDR is shown as QASQSIGSYLS (CDR1), AASTLES (CDR2), and CQGGYYSSSMGAF (CDR3); the heavy chain variable region CDR is shown as GFSLSSY (CDR1), NTGDT (CDR2), and CARGANAMDFW (CDR3).

[0064] In some embodiments of the present invention, the light chain variable region CDR is shown as QSSKSVYSNNYLS (CDR1), QASRLAS (CDR2), and CLGGYDDDSDCAF (CDR3), respectively; the heavy chain variable region CDR is shown as GFSLSSY (CDR1), SSSGS (CDR2), and CARGYPGERDDKLW (CDR3), respectively.

[0065] In some embodiments of the present invention, the light chain variable region CDR is shown as QASQSISTALA (CDR1), YISNLAS (CDR2), and CQGAYYSGSTDTLSNNF (CDR3); the heavy chain variable region CDR is shown as GFSLTRY (CDR1), LKSDK (CDR2), and CARHSDSTTTVDIW (CDR3).

[0066] In some embodiments of the present invention, the light chain variable region CDR is shown as QASEDIYDLLA (CDR1), DTSDLAS (CDR2), and CQQGYTYSSGDNIF (CDR3), respectively; the heavy chain variable region CDR is shown as GFSLSSF (CDR1), NGNNN (CDR2), and CARAYGTFTPADRLDLW (CDR3), respectively.

[0067] In some embodiments of the present invention, the light chain variable region of the anti-CCR7 antibody or its antigen-binding fragment is:

[0068] (1) The sequence shown in SYELTQLPSVSVSLGQTARITCGGNNIGSKAVHWYQQKPGLAPGLLIYNDDERPSGVPDRFSGSNSGDTATLTISGAQAGDEADYYCQLWDSSAGVVFGGGTQLTVT (SEQ ID NO: 5);

[0069] (2) A sequence having one or more amino acid substitutions and / or deletions and / or additions to SEQ ID NO: 5, and having the same function as the sequence shown in SEQ ID NO: 5; or

[0070] (3) A sequence that has at least 70% identity with SEQ ID NO: 5 and has the same function as the sequence shown in SEQ ID NO: 5.

[0071] In some embodiments of the present invention, the light chain variable region of the anti-CCR7 antibody or its antigen-binding fragment is:

[0072] (1) A sequence as shown in any one of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25;

[0073] (2) A sequence having the same function as the sequence shown in any one of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 by substitution and / or deletion and / or addition of one or more amino acids; or

[0074] (3) A sequence that has at least 70% identity with any one of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 and has the same function as the sequence shown in any one of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25.

[0075] In some embodiments of the present invention, the number of amino acid substitutions and / or deletions and / or additions is 1 to 10, specifically: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are substituted and / or deleted and / or added.

[0076] In some embodiments of the invention, the identity is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, or 70%.

[0077] In some embodiments of the present invention, the heavy chain variable region of the anti-CCR7 antibody or its antigen-binding fragment is:

[0078] (1) The sequence shown in QSVRESEGGLFKPMDTLTLTCTASGFSLSSFGVTWVRQAPGKGLEYIGFINGNNNAYYANWARSRSTITRNTNENTVTLKMTSLTAADTATYFCARAYGTFTPADRLDLWGQGTLVAVSS (SEQ ID NO: 6);

[0079] (2) A sequence having the same function as the sequence shown in SEQ ID NO: 6, but with one or more amino acid substitutions and / or deletions and / or additions; or

[0080] (3) A sequence that has at least 70% identity with SEQ ID NO: 6 and has the same function as the sequence shown in SEQ ID NO: 6.

[0081] In some embodiments of the present invention, the heavy chain variable region of the anti-CCR7 antibody or its antigen-binding fragment is:

[0082] (1) A sequence as shown in any one of SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26;

[0083] (2) A sequence having the same function as the sequence shown in any one of SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 by substitution and / or deletion and / or addition of one or more amino acids; or

[0084] (3) A sequence that has at least 70% identity with any one of SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 and has the same function as the sequence shown in any one of SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26.

[0085] In some embodiments of the present invention, the number of amino acid substitutions and / or deletions and / or additions is 1 to 10, specifically: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are substituted and / or deleted and / or added.

[0086] In some embodiments of the invention, the identity is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, or 70%.

[0087] In some embodiments of the present invention, the anti-CCR7 antibody or its antigen-binding fragment further includes a constant region.

[0088] In this invention, the term "constant region" refers to a region in an antibody or immunoglobulin molecule where the amino acid sequence is highly conserved, typically located at the carboxyl terminus (C-terminus) of the heavy chain (CH) and light chain (CL) of the antibody.

[0089] In some embodiments of the invention, the constant region is connected to the variable region, either via a linking peptide or not.

[0090] In some embodiments of the present invention, the linker peptide is selected from rigid linker peptides or flexible linker peptides.

[0091] In some embodiments of the present invention, the flexible linker peptide is selected from (G3S). n (G4S) n Or (G) n Where n is an integer not less than 3. For example, n is 3-10 (such as 3, 4, 5, 6, 7, 8, 9 or 10).

[0092] In some embodiments of the present invention, the rigid linker peptide is (EAAAK). m Where m is an integer not less than 3. For example, m is 3 to 10 (such as 3, 4, 5, 6, 7, 8, 9 or 10).

[0093] In some embodiments of the present invention, the light chain of the anti-CCR7 antibody contains a sequence as shown in SEQ ID NO: 3.

[0094] In some embodiments of the present invention, the light chain of the anti-CCR7 antibody contains any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 linked to the sequence obtained by SEQ ID NO: 27.

[0095] In some embodiments of the present invention, the heavy chain of the anti-CCR7 antibody contains a sequence as shown in SEQ ID NO: 4.

[0096] In some embodiments of the present invention, the heavy chain of the anti-CCR7 antibody contains any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 linked to the sequence obtained by SEQ ID NO: 28.

[0097] In some embodiments of the present invention, the anti-CCR7 antibody or its antigen-binding fragment further contains chemical modifications and / or is linked with functional groups.

[0098] In some embodiments of the present invention, the chemical modification includes, but is not limited to, at least one of methylation, acetylation, phosphorylation, and glycosylation.

[0099] In some embodiments of the present invention, the functional gene includes, but is not limited to, at least one of: reporter group, protein tag, carrier and lipid group.

[0100] In some embodiments of the present invention, the reporter group includes, but is not limited to, at least one of: chromogenic enzyme, fluorescent labeling group, chemiluminescent labeling group, isotope or magnetic group.

[0101] In some embodiments of the present invention, the chromogenic enzyme includes, but is not limited to, at least one of: peroxidase and alkaline phosphatase.

[0102] In some embodiments of the present invention, the fluorescent labeling group includes, but is not limited to, at least one of the following: fluorescent protein groups, rhodamine groups, fluorescein groups, anthocyanin dyes, cyanine dyes, Alexa Fluor dyes, and / or quantum dot fluorescent groups.

[0103] In some embodiments of the present invention, the isotopes include, but are not limited to, radioactive nuclides.

[0104] In some embodiments of the present invention, the radionuclides include, but are not limited to, those used in radioimmunoassay and radioimmunotherapy.

[0105] In some embodiments of the present invention, the magnetic group includes, but is not limited to, groups that enable magnetic resonance imaging or alter relaxation efficiency.

[0106] In some embodiments of the present invention, the protein tags include, but are not limited to: affinity tags, such as GST, His, MBP, etc.; epitope tags, such as HA, Myc, Flag, etc.; fluorescent tags, such as GFP, mCherry, etc.; and other tags, such as SNAP and Avi, etc.

[0107] In some embodiments of the present invention, the carrier includes, but is not limited to, carrier proteins, such as hemocyanin, serum albumin, ovalbumin, etc.

[0108] In some embodiments of the present invention, the lipid groups include, but are not limited to, fatty acid chains (such as palmitic acid) to form lipopeptides.

[0109] In some embodiments of the present invention, the antibody includes, but is not limited to, at least one of: monoclonal antibody (mAb), polyclonal antibody, chimeric antibody, multispecific antibody, antibody fragment (such as Fab fragment, F(ab')2 and single-domain antibody (VHH)) and conjugate antibody (such as antibody-drug conjugate (ADC)).

[0110] A second aspect of the present invention provides a biomaterial comprising at least one of the following (1)-(3):

[0111] (1) A nucleic acid molecule encoding an antibody against CCR7 as described above or an antigen-binding fragment thereof;

[0112] (2) Contains an expression unit of the nucleic acid molecule described in (1);

[0113] (3) Transformants containing the nucleic acid molecules described in (1) and / or the expression molecules described in (2);

[0114] In some embodiments of the present invention, the transformant does not involve plant or animal reproductive materials.

[0115] In some embodiments of the present invention, the expression term includes viruses, plasmids, and bacteriophages.

[0116] In some embodiments of the present invention, the transformants include bacteria, fungi, and plant and animal cells.

[0117] In some embodiments of the present invention, the construction of the expressants and transformants can be achieved based on any conventional techniques in the art.

[0118] A third aspect of the present invention provides a conjugate comprising: an anti-CCR7 antibody or an antigen-binding fragment thereof as described in the above aspects, and a conjugation portion.

[0119] In some embodiments of the present invention, the coupling portion comprises at least one of a detectable marker, a drug, a toxin, biotin, a spin label, an enzyme, a nanoparticle, or a magnetic nanoparticle.

[0120] In some embodiments of the present invention, radioactive isotopes include, but are not limited to, those mentioned above. 3 H, 14 C 15 N、 35 S, 90 Y、 99 Tc, 111 In、 125 I or 131 At least one of I.

[0121] In some embodiments of the present invention, the toxin includes, but is not limited to, at least one of diphtheria toxin, ricin, or cholera toxin.

[0122] In some embodiments of the present invention, the enzyme includes, but is not limited to, at least one of peroxidase, alkaline phosphatase, horseradish peroxidase, or acetylcholinesterase.

[0123] A fourth aspect of the present invention provides the use of the anti-CCR7 antibody or its antigen-binding fragment, biological material, or conjugate described above in at least one of the following (1)-(4):

[0124] (1) CCR7 testing for non-diagnostic purposes;

[0125] (2) Screening and identification of biological materials for non-diagnostic purposes;

[0126] (3) Prepare drugs for treating CCR7-mediated diseases;

[0127] (4) Prepare products that specifically target CCR7.

[0128] In some embodiments of the present invention, the biomaterial includes microorganisms, cells, tissues, and implants.

[0129] In some embodiments of the present invention, the screening and identification of the biological materials can be based on the anti-CCR7 antibodies or their antigen-binding fragments, biological materials, or conjugates described above, and can be achieved by any quantitative or qualitative biological or chemical method, including but not limited to: flow cytometry, enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, radioimmunoassay (RIA), chemiluminescent immunoassay, agglutination assay, precipitation reaction, antibody screening precipitation assay, neutralization assay, complement fixation assay, Western blotting, etc.

[0130] In some embodiments of the present invention, the product includes a drug, a solid-phase carrier, a detection chip, a detection reagent, or a test strip.

[0131] In some embodiments of the present invention, the CCR7-mediated related diseases include: autoimmune diseases such as multiple sclerosis (MS), rheumatoid arthritis (RA), and psoriasis; cancers such as hematologic malignancies (including chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), follicular lymphoma (FL), etc.) and lymph node metastases of solid tumors (such as breast cancer, pancreatic cancer, esophageal cancer, lung cancer, etc.); hematologic diseases such as B-type chronic lymphocytic proliferative disorder (B-CLPD); and infectious diseases such as HIV and EBV infection.

[0132] The beneficial effects of this invention are:

[0133] 1. This invention develops an immunogen (antigen) based on the N-terminal peptide of CD197 and the human Fc fragment, and constructs a flow cytometry antibody applicable to peripheral blood mononuclear cells (PBMCs). This antibody exhibits good performance, high specificity, and low preparation cost, requiring only conventional experimental subjects such as mice or rabbits for immunization. This antibody overcomes the problems of insufficient specificity, high development cost, and high price in existing technologies, and possesses true scalability.

[0134] 2. The antibody immunogen in this invention is the N-terminal sequence of the CD197 protein with a clearly defined epitope. Verification has shown that it binds only to cells expressing CD197 and does not bind to cells that do not express CD197. Moreover, it can be effectively used in real clinical samples such as PBMCs and can be applied to the diagnosis and immunological research of CD197 protein-related diseases, as well as the development of novel treatment strategies. Attached Figure Description

[0135] Figure 1 This is a plasmid map of the immunogen CD197-N-hFc recombinant vector.

[0136] Figure 2 This is an SDS-PAGE image of the purified protein from the immunogen CD197-N-hFc.

[0137] Figure 3 The results are the serum titer assessment results for CD197-N-hFc.

[0138] Figure 4 For immunizing rabbits with CD197 + IgG + Flow cytometry sorting results of cells.

[0139] Figure 5 The results are from SDS-polyacrylamide gel electrophoresis of the antibody.

[0140] Figure 6 This is a diagram of the plasmid structure of CD197-SGPF2_pcDNA3.1.

[0141] Figure 7 The results are obtained by flow cytometry using different test antibodies on HEK293F cells.

[0142] Figure 8 The results are obtained by flow cytometry using different test antibodies on HEK293F cells.

[0143] Figure 9 The results are obtained by flow cytometry using different test antibodies on PBMC cells.

[0144] Figure 10 The results are obtained by flow cytometry using different test antibodies on PBMC cells.

[0145] Figure 11 The results are obtained by flow cytometry using different test antibodies on PBMC cells. Detailed Implementation

[0146] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0147] Example 1: Design and Preparation of Immunogens

[0148] In this embodiment, the design concept and specific preparation method of the CCR7 (CD197) immunogen are as follows:

[0149] (1) Construction of recombinant expression vector:

[0150] The selected CD197-N-terminal peptide was fused with the human Fc fragment sequence (conducted by Beijing BGI Genomics), followed by codon optimization. The optimized sequence (CD197-N-hFc) was then inserted into a commercially available pCDNA3.4(+) vector. After sequencing verification, the recombinant expression vector was obtained (plasmid map shown). Figure 1 (As shown).

[0151] The CD197-N-terminal polypeptide is: QDEVTDDYIGDNTTVDYTLFESLCSKKDVRNFKAW (SEQ ID NO: 1).

[0152] The specific sequence of CD197-N-hFc is: GLNDIFEAQKIEWHEQDEVTDDYIGDNTTVDYTLFESLCSKKDVRNF KAWGGSENLYFQSGGPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 2).

[0153] (2) Obtaining recombinant proteins:

[0154] Take 300 μg of the recombinant expression vector, add 12.5 mL of OPM CD05 medium (purchased from OPMA) and 800 μL of 1 mg / mL polyethyleneimine (PEI) solution (purchased from POLYSCIENCE), and mix well. Incubate at room temperature for 0.5 h, then add to 250 mL of 3 × 10⁻⁶ medium. 6 HEK293F cells were added at a density of 1 cell / mL and cultured overnight on a shaker at 37°C, 8% CO2, and 110 rpm. The next day, HEK293 cells were fed with protein-free OPM (purchased from OPMA) and cultured for 7 days. The cell supernatant was then collected by centrifugation.

[0155] (3) Recombinant protein purification:

[0156] Add 2 mL of protein A packing material (purchased from Sangon Biotech) to the column (F506607 affinity chromatography column, purchased from Sangon Biotech), and then wash with 30 mL of PBS to equilibrate the packing material. Then add the cell supernatant obtained in step (2) after filtration through a 0.22 μm MCE aqueous microporous membrane (Sangon Biotech). Add 20 mL of PBS solution for washing, and then add 10 mL of 100 mM glycine solution (purchased from Sangon Biotech) at pH 3.0 for elution. Add 1 M Tris-HCl solution (pH 9.0) to the eluent to neutralize it to pH 7.0. Concentrate the neutralized eluent to 1 mL, dialyze into PBS to obtain purified recombinant protein, flash freeze in liquid nitrogen, and store at -80°C for later use.

[0157] The purified protein was characterized using SDS-polyacrylamide gel electrophoresis (with and without reducing agent), and the results are as follows: Figure 2 As shown.

[0158] The results showed that the purified CD197-N-hFc bands were in line with expectations. Under non-reducing conditions, the protein formed dimers through disulfide bonds, with a size of about 80 kDa. Under reducing conditions, the disulfide bonds opened, and the protein monomers were about 40 kDa in size. The purity was greater than 95%, which is suitable for subsequent immunoassays.

[0159] Example 2 Animal Immunization

[0160] In this embodiment, the purified CD197-N-hFc obtained in Example 1 above was used as an immunogen for animal immunization, and the animal used was a rabbit.

[0161] The specific experimental steps are as follows:

[0162] (1) 1 mL of blood was collected from the ear of the experimental rabbits, and the serum was used as a blank control. The experimental rabbits were divided into two groups. The immunization and blood collection cycles of the two groups were the same, but one group of experimental rabbits (Group 1, i.e. Figure 3 Rabbit #3 in the group received half the dose of the immunization drug each time (compared to rabbits in group 2, group 2...). Figure 3 (Rabbit number 4 in the text). The specific immunization (primary immunization) procedure for Group 2 was as follows: 2 mg of the purified CD197-N-hFc (diluted in 1.5 mL PBS) from the above examples was mixed with Freund's complete adjuvant and emulsified by shaking on a shaker for at least 15 minutes until it reached a water-in-oil state. Then, immunization was performed on the back of the rabbits at a dose of 1.5 mL of the emulsified solution per rabbit (approximately 1 mg of CD197-N-hFc).

[0163] (2) Two weeks after the initial immunization, a second immunization was performed (the immunization dose for Group 1 was halved). The specific immunization procedure for Group 2 (second immunization) was as follows: 1 mg of the purified CD197-N-hFc (diluted in 1 mL PBS) from the above examples was mixed with Freund's incomplete adjuvant and emulsified by shaking on a shaker for at least 15 minutes until it reached a water-in-oil state. Then, 1 mL of the emulsified solution per rabbit (approximately 0.5 mg CD197-N-hFc) was administered to the back of the rabbit for immunization.

[0164] (3) Two weeks after the second immunization, a third immunization was performed (the immunization dose for Group 1 was halved). The specific immunization procedure for Group 2 (the third immunization) was as follows: 1 mg of the purified CD197-N-hFc (diluted in 1 mL PBS) from the above examples was mixed with Freund's incomplete adjuvant and emulsified by shaking on a shaker for at least 15 minutes until it reached a water-in-oil state. Then, 1 mL of the emulsified solution per rabbit (approximately 0.5 mg CD197-N-hFc) was administered to the back of the rabbit for immunization.

[0165] (3) Two weeks after the third immunization, a fourth immunization was performed (the immunization dose for Group 1 was halved). The specific immunization procedure for Group 2 (the fourth immunization) was as follows: 1 mg of the purified CD197-N-hFc (diluted in 1 mL PBS) from the above examples was mixed with Freund's incomplete adjuvant and emulsified by shaking on a shaker for at least 15 minutes until it reached a water-in-oil state. Then, 1 mL of the emulsified solution per rabbit (approximately 0.5 mg CD197-N-hFc) was administered to the back of the rabbit for immunization.

[0166] (4) One week after the fourth immunization, 1 mL of blood was taken from the ear of each group of experimental rabbits, serum was separated, and antibody titer was detected by ELISA. If the titer reached 1:100,000 or higher, a pulse immunization was performed. The method of pulse immunization was the same as that of the first immunization, and the immunization dose was 1.5 mL of emulsified solution per rabbit (approximately 1 mg CD197-N-hFc).

[0167] (5) Three days after the shock immunization, blood was drawn from the ear again (5 mL) to test the antibody titer. After confirming the antibody titer, whole blood was collected in an anticoagulant tube and the serum was separated to obtain the antiserum.

[0168] In this embodiment, the ELISA method used for antibody titer detection is specifically as follows:

[0169] The purified CD197-N-hFc was diluted to 0.1 μg / mL with PBS and added to an ELISA plate (100 μL / well), and incubated overnight at 4°C. After incubation, the plate was washed three times with PBST (300 μL / well), and the residual liquid was aspirated. 300 μL of 2% BSA blocking buffer was added to each well, and the plate was incubated at 37°C for 1 h. The plate was washed three more times with PBST (300 μL / well), and the residual liquid was aspirated. Antiserum was diluted with PBS, and 100 μL of antiserum was added to each well, and the plate was incubated at 37°C for 1 h. The plate was washed six times with PBST (300 μL / well), and the residual liquid was aspirated. 100 μL of Anti-rabbit Antibody (labeled with HRP, purchased from Yiqiao) diluted with PBS was added to each well, and the plate was incubated at room temperature for 1 h. The plate was washed six times with PBST (300 μL / well), and the residual liquid was aspirated. Add 100 μL of TMB chromogenic solution (purchased from Beyotime) to each well and incubate in the dark for 10 min. Stop the chromogenic reaction by adding 50 μL of 1M HCl to each well, and read the OD450 value using a microplate reader.

[0170] Potency test results as follows Figure 3 As shown.

[0171] The results showed that the CD197-N-hFc obtained in the above examples could be used as an antigen for immunization, and the immunization effect was excellent, with antibody titers greater than 10 after immunization.-7 .

[0172] Example 3: Modification of CD197-N-hFc

[0173] In order to be used in subsequent screening steps, CD197-N-hFc is modified as follows in this embodiment:

[0174] (1) Remove hFc tags:

[0175] In the embodiment of this invention, the CD197-N-hFc sequence contains a TEV restriction site. Therefore, His-TEV (Beyotime) was used to digest the purified CD197-N-Fc to remove the hFc tag.

[0176] The specific steps are as follows:

[0177] Take 500 μg of the purified CD197-N-hFc from the above examples, add 10 μL of His-TEV, and incubate overnight at 4°C. The next day, take an empty centrifuge column (Beyotime, FCL108), add 100 μL of equilibrated HisTrap FF packing material (Cytiva), then add the protein that was incubated overnight, let stand for 2 min, centrifuge at 6000 rpm for 2 min at 4°C, and collect the flow-through. Take a new empty centrifuge column again, add 100 μL of equilibrated protein A packing material (Essential Science), add the flow-through, let stand for 2 min, centrifuge at 6000 rpm for 2 min at 4°C, and collect the flow-through. This yields the purified CD197-N protein after the hFc tag has been removed.

[0178] (2) Biotinylation modification:

[0179] Based on the presence of an avi-tag in CD197-N, site-specific biotinylation modification can be performed. Biotinylation modification was performed according to the Avidity BirA500 instruction manual, with incubation at 37°C for 1 hour, followed by dialysis and reconstitution with PBS to obtain the modified CD197-N protein (CD197-N-Bio), which was then used for cell sorting.

[0180] Example 4: Immunizing rabbits with CD197 + IgG + Cell sorting

[0181] Spleen cells from the immunized rabbits described in the above examples were ground and PBMC cells were isolated. The PBMC cells were then incubated with biotin-CD4 antibody (Bio-Rad), biotin-CD8 antibody (Bio-Rad), biotin-IgM antibody (BDPharmingen), and biotin-CD11b antibody (STEMCELL Technologies). Streptavidin magnetic beads (Miltenyi Biotec) were then added for further incubation. After adsorption using a magnet, the supernatant was collected to obtain B cells.

[0182] Take 1×10 6 The B cells obtained by the above method were added to the CD197-N-Bio sample obtained in the above examples and incubated on ice for 30 min. After washing three times with PBS, Goat Anti-Rabbit IgG H&L Alexa was added. Antibodies were prepared with 555 (Invitrogen) and APC Streptavidin (Biolegend) and incubated on ice for 30 min. The cells were washed three times with PBS, and CD197 were separated using flow cytometry. + IgG + Single cells were collected, and CD197 was recovered. + IgG + Single cell.

[0183] Flow sorting results are as follows Figure 4 As shown in the image. It can be observed that the recovered CD197... + IgG + The proportion of single cells was 0.5%.

[0184] Example 5: Screening, Expression, and Purification of Antibodies

[0185] The cells recovered in Example 4 were sent to Mozhuo for single-cell transcriptome sequencing. The antibody sequences obtained from the sequencing were screened and classified according to the repetition frequency. Then, the light / heavy chain sequences of the antibodies were synthesized and the codons were optimized. The optimized light / heavy chain sequences were inserted into the pCDNA3.4(+) vector respectively. After sequencing verification, the recombinant vector was obtained.

[0186] Take 300 μg of the recombinant vector (a 1:1 molar mixture of the light chain and heavy chain recombinant expression vectors), add 12.5 mL of OPM CD05 medium (purchased from OPMA) and 800 μL of 1 mg / mL polyethyleneimine (PEI) solution (purchased from POLYSCIENCE), and mix thoroughly. Incubate at room temperature for 0.5 h, then add to 250 mL of 3 × 10⁻⁶ medium. 6Cells were cultured in HEK293F cells at a density of 1 / mL on a shaker overnight at 37°C, 8% CO2, and 110 rpm. The next day, HEK293 cells were fed with protein-free OPM (purchased from OPMA) and cultured for 7 days. The cell supernatant was then collected by centrifugation.

[0187] Add 2 mL of protein A packing material (purchased from Sangon Biotech) to the column (F506607 affinity chromatography column, purchased from Sangon Biotech), and then wash with 30 mL of PBS to equilibrate the packing material. Then add the cell supernatant obtained in step (2) after filtration through a 0.22 μm MCE aqueous microporous membrane (Sangon Biotech). Add 20 mL of PBS solution for washing, and then add 10 mL of 100 mM glycine solution (purchased from Sangon Biotech) at pH 3.0 for elution. Add 1 M Tris-HCl solution (pH 9.0) to the eluent to neutralize it to pH 7.0. Concentrate the neutralized eluent to 1 mL, dialyze into PBS to obtain purified recombinant protein, flash freeze in liquid nitrogen, and store at -80°C for later use.

[0188] The purified light / heavy chain sequences were characterized using SDS-polyacrylamide gel electrophoresis, and the results are as follows: Figure 5 As shown. Ab1-3 are patented antibodies 1-3, derived from CAP-100 in CN115210259A, US2018237529, and R707 in US20150344580, respectively. 22, 121, 125, 146, 172, 550, 824, 1264, 2198, 60, 60-599, and 772 are antibodies obtained through the methods described in the above examples.

[0189] It can be observed that under non-reducing conditions, the protein forms a dimer through disulfide bonds, with the antibody size around 150 kDa. Under reducing conditions, the disulfide bonds open, and the antibody light chain size is approximately 25 kDa, while the heavy chain size is approximately 50 kDa. The antibody size is as expected, and the purity is good.

[0190] Example 6: Flow cytometry assay based on HEK293F cells

[0191] (1) Construction of CD197-SGFP2 transiently transfected cells:

[0192] The CD197 sequence was ligated to the sGFP green fluorescent protein reporter gene (conducted by Beijing BGI Genomics), followed by codon optimization. The optimized sequence (CD197-sGFP2) was then inserted into the commercially available pCDNA3.1(+) vector. After sequencing verification, the recombinant expression vector (CD197-SGPF2_pcDNA3.1, plasmid structure as shown) was obtained. Figure 6 (As shown).

[0193] The sequence of CD197 is:

[0194] MDLGKPMKSVLVVALLVIFQVCLCQDEVTDDYIGDNTTVDYTLFESLCSKKDVRNFKAWFLPIMYSIICFVGLLGNGLVVLTYIYFKRLKTMTDTYLLNLAVADILFLLTLPFWAYSAAKSWVFGVHFCKLIFAIYKMSFFSGMLLLLCISIDRYVAIVQAVSAHRHRARVLLISKLSCVGIWILATVLSIPELLYSDLQRSSSEQAMRCSLITEHVEAFITIQVAQMVIGFLVPLLAMSFCYLVIIRTLLQARNFERNKAIKVIIAVVVVFIVFQLPYNGVVLAQTVANFNITSSTCELSKQLNIAYDVTYSLACVRCCVNPFLYAFIGVKFRNDLFKLFKDLGCLSQEQLRQWSSCRHIRRSSMSVEAETTTTFSP(SEQ ID NO: 29);

[0195] The sequence of CD197-sGFP2 is:

[0196] (SEQ ID NO: 30).

[0197] The recombinant expression vector (CD197-SGPF2_pcDNA3.1 plasmid) was transfected into HEK293F cells. Specifically, 30 μg of the recombinant expression vector was added to 1.25 mL of OPM CD05 medium (purchased from OPMA) and 80 μL of 1 mg / mL polyethyleneimine (PEI) solution (purchased from POLYSCIENCE), and mixed thoroughly. The mixture was incubated at room temperature for 0.5 h, and then added to 25 mL of 3×10⁻⁶ plasmid. 6 We placed the cells in a shaker and cultured them for 2 days at 37°C, 8% CO2, and 110 rpm to obtain CD197-SGFP2 transiently transfected cells.

[0198] (2) Flow cytometry efficacy test of antibodies:

[0199] Collect the CD197-SGFP2 transiently transfected cells obtained in the above steps, count them, and then divide them into groups of 1×10⁻⁶. 6Prepare individual cells / tubes, centrifuge at 300g for 5 min, discard the supernatant, and resuspend in 100 μL of DPBS buffer containing 1% BSA + 1 mM EDTA. Then add 100 μL of primary antibody (i.e., the antibody to be tested, final concentration 10 μg / mL) diluted in PBS containing 3% BSA, and incubate at 4°C for 1 h. Wash three times with 500 μL of cold PBS. Then add 100 μL of goat anti-rabbit secondary antibody (labeled APC, dilution 1:200, Invitrogen) diluted in DPBS buffer containing 1% BSA and 1 mM EDTA, and incubate at 4°C in the dark for 30 min. Wash three times with 500 μL of cold PBS. Resuspend in 200 μL of PBS and perform flow cytometry.

[0200] In this embodiment, the antibodies to be tested include: anti-CD197 antibodies (named 60-599) prepared in the above embodiments, anti-CD197 antibodies (named 22, 125, 146, 172, 550, 772, 824, 1264, 2198, and 60, respectively) prepared using different CD197 antigen fragments according to the methods in the above embodiments; and commercially available antibodies (BD Pharmaceuticals). TM APCMouse Anti-Human CCR7 (CD197), as the PC, antibody information can be found at https: / / www.bdbiosciences.com / en-ca / products / reagents / flow-cytometry-reagents / research-reagents / single-color-antibodies-ruo / apc-mouse-anti-human-ccr7-cd197.566763?tab=product_details), patented antibody 1 (Ab1, from CN115210259A), patented antibody 2 (Ab2, from CAP-100 in US2018237529), and patented antibody 3 (Ab3, from R707 in US20150344580). A blank control was also included.

[0201] The results are as follows Figure 7 and Figure 8 As shown.

[0202] like Figure 7 and Figure 8 As shown, the vertical axis represents CD197 antigen expression, and the horizontal axis represents CD197 antibody binding. In this figure, the specific binding signal should be in the Q2 region. It can be seen that the 60-599 antibody in this embodiment of the invention specifically recognizes only the CD197 antigen, and its specificity is better than that of commercially available antibodies and patented antibodies.

[0203] Sequencing of antibody 60-599 revealed the following full-length amino acid sequence of its light chain:

[0204] The italicized portions represent the light chain variable regions, while the bolded and underlined portions represent the antibody's three light chain complementarity-determining regions (CDRs).

[0205] Its full-length amino acid sequence of heavy chain is as follows:

[0206] The italicized parts are the heavy chain variable regions, and the bolded and underlined parts are the antibody's three heavy chain complementarity-determining regions (CDRs).

[0207] The antibodies numbered 22, 125, 146, 172, 550, 772, 824, 1264, 2198, and 60 were sequenced, and the relevant sequence information is shown below. The italicized and bolded parts are CDRs. twenty two:

[0209] The amino acid sequence of the light chain variable region is as follows:

[0210] The amino acid sequence of the heavy chain variable region is as follows: 125:

[0212] The amino acid sequence of the light chain variable region is as follows:

[0213] The amino acid sequence of the heavy chain variable region is as follows: 146:

[0215] The amino acid sequence of the light chain variable region is as follows:

[0216] The amino acid sequence of the heavy chain variable region is as follows: 172:

[0218] The amino acid sequence of the light chain variable region is as follows:

[0219] The amino acid sequence of the heavy chain variable region is as follows: 550:

[0221] The amino acid sequence of the light chain variable region is as follows:

[0222] The amino acid sequence of the heavy chain variable region is as follows: 772:

[0224] The amino acid sequence of the light chain variable region is as follows:

[0225] The amino acid sequence of the heavy chain variable region is as follows: 824:

[0227] The amino acid sequence of the light chain variable region is as follows:

[0228] The amino acid sequence of the heavy chain variable region is as follows: 1264:

[0230] The amino acid sequence of the light chain variable region is as follows:

[0231] The amino acid sequence of the heavy chain variable region is as follows: 2198:

[0233] The amino acid sequence of the light chain variable region is as follows:

[0234] The amino acid sequence of the heavy chain variable region is as follows: 60:

[0236] The amino acid sequence of the light chain variable region is as follows:

[0237]

[0238] The amino acid sequence of the heavy chain variable region is as follows:

[0239] The light chain constant region of all the above antibodies is: RDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGT TQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO: 27);

[0240] The heavy chain constant regions are: GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSV RQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLR GKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO: 28).

[0241] Example 7: Flow cytometry test based on PBMC cells

[0242] (1) Recovery of PBMC cells:

[0243] Remove PBMC cells (Ausnutria Biotech) from the liquid nitrogen container and quickly place them in a 37°C water bath, shaking occasionally, to thaw them completely within 1-2 minutes. Then, pipette the thawed PBMC cells into a 15mL centrifuge tube containing 5mL of 1640 medium (containing 10% FBS) and centrifuge at 400g for 10 minutes. Discard the supernatant and add 5mL of PBS or commercially available antibody diluent (BDPharmingen). TM Cells were washed with Stain Buffer (BSA, BD, 554657) and centrifuged at 400g for 5 min. The supernatant was discarded to obtain revived PBMC cells.

[0244] (2) Flow cytometry efficacy test of antibodies:

[0245] Resuscitate PBMC cells with 2 mL of commercially available antibody diluent and add 100 μL (5E5 cells per well) to each well of a 96-well plate. Add 100 μL of primary antibody (the antibody to be tested, final concentration 10 μg / mL) diluted in PBS containing 3% BSA and incubate at 4°C for 1 h. Wash three times with 500 μL of cold PBS. Then add 100 μL of goat anti-rabbit secondary antibody (labeled APC, dilution 1:200, Invitrogen) diluted in DPBS buffer containing 1% BSA and 1 mM EDTA and incubate at 4°C in the dark for 30 min. Wash three times with 500 μL of cold PBS. Resuspend in 200 μL of PBS and perform flow cytometry.

[0246] In this embodiment, the antibodies to be tested include: anti-CD197 antibodies (60-599, 22, 125, 146, 172, 550, 772, 1264 and 2198) prepared in the above embodiments, and commercially available antibodies (BD Pharmaceuticals). TM APC Mouse Anti-Human CCR7 (CD197) was used as a PC, along with patented antibody 1 (Ab1, from CN115210259A), patented antibody 2 (Ab2, from CAP-100 in US2018237529), and patented antibody 3 (Ab3, from R707 in US20150344580). A blank control was also included.

[0247] The results are as follows Figure 9-11 As shown.

[0248] like Figure 9-11 As shown, the antibody in this embodiment of the invention and the commercial antibody can both bind to cells in PBMCs. Moreover, compared with several patented antibodies, its ability to bind cell populations is more consistent with PC, thus demonstrating that the antibody in this embodiment of the invention can be applied to real samples.

[0249] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An antibody against CCR7 or its antigen-binding fragment, characterized in that, The anti-CCR7 antibody or its antigen-binding fragment binds to an antigenic epitope peptide or protein containing the sequence shown in SEQ ID NO: 1; Preferably, the antigenic epitope peptide or protein further includes a crystallizable segment (Fc) sequence linked to the sequence shown in SEQ ID NO: 1; Preferably, the antigenic epitope peptide or protein further includes at least one functional sequence; Preferably, the functional sequence includes at least one of a tag sequence, a linker, and an enzyme cleavage site; Preferably, the functional sequence is located between the sequence shown in SEQ ID NO: 1 and the Fc sequence, and / or at the end of the sequence shown in SEQ ID NO: 1 and the Fc sequence that is far away from each other.

2. The anti-CCR7 antibody or its antigen-binding fragment according to claim 1, characterized in that, The antigenic epitope peptide or protein contains a sequence as shown in SEQ ID NO: 2; Preferably, the antigenic epitope peptide or protein further includes: (1) A sequence having the same function as the sequence shown in SEQ ID NO: 2, but with one or more amino acid substitutions and / or deletions and / or additions; or (2) A sequence that has at least 70% identity with SEQ ID NO: 2 and has the same function as the sequence shown in SEQ ID NO:

2.

3. The anti-CCR7 antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The anti-CCR7 antibody or its antigen-binding fragment includes the light chain variable region (CDR) and heavy chain variable region (CDR) as shown below: The light chain variable regions CDRs are shown as GGNNIGSKAVH, NDDERPS, and QLWDSSAGVV, respectively. The heavy chain variable region CDR is shown as SFGVT, FINGNNNAYYANWARS and AYGTFTPADRLDL, respectively. or The light chain variable regions (CDRs) are shown as QASQNIDSYLA, KASTLAS, and CQSHYYSGSTGYAHTF, respectively. The heavy chain variable regions CDR are shown as GFSLSYV, SSGVP and CARGSTDSNYYGGYYFTLW respectively; or The light chain variable regions (CDRs) are shown as QASQSISSYLS, DASNLET, and CQSYYYSSSSSYGSGF, respectively. The heavy chain variable regions CDR are shown as GFSLSYV, SSGVP and CARGSTDSNYYGGYYFTLW respectively; or The light chain variable regions CDRs are shown as QASQNIDSYLA, KASTLAS, and CQGGVHDSDEFPF, respectively. The heavy chain variable regions CDR are shown as GFSLNSY, YDDGS and CVRDSDYVGFGYNLW respectively; or The light chain variable regions CDRs are shown as QASEDIESYLA, YASNLES, and CQGYYYSGRTVAF, respectively. The heavy chain variable regions CDRs are shown as GFSLSSY, GSSGS, and CARDSYGGDYAIGFDIW, respectively. or The light chain variable regions (CDRs) are shown as QSSESVYNKNWLA, EASTLAS, and CAGDYIYNSDNAF, respectively. The heavy chain variable regions CDRs are shown as GFSLNTF, FGSGD, and CARYFNYGSYNLW, respectively. or The light chain variable regions CDRs are shown as QASQSINNQLS, LASTLAS, and CQSTHDITTYGIAF, respectively. The heavy chain variable region CDR is shown as GIDLSSY, WGGSA and CARNPGYSANLW respectively; or The light chain variable regions CDRs are shown as QASQSIGSYLS, AASTLES, and CQGGYYSSSMGAF, respectively. The heavy chain variable region CDR is shown as GFSLSSY, NTGDT and CARGANAMDFW respectively; or The light chain variable regions CDR are shown as QSSKSVYSNNYLS, QASRLAS, and CLGGYDDDSDCAF, respectively. The heavy chain variable regions CDR are shown as GFSLSSY, SSSGS, and CARGYPGERDDKLW, respectively. or The light chain variable regions (CDRs) are shown as QASQSISTALA, YISNLAS, and CQGAYYSGSTDTLSNNF, respectively. The heavy chain variable region CDR is shown as GFSLTRY, LKSDK and CARHSDSTTTVDIW respectively; or The light chain variable regions CDRs are shown as QASEDIYDLLA, DTSDLAS, and CQQGYTYSSGDNIF, respectively. The heavy chain variable regions CDRs are shown as GFSLSSF, NGNNN, and CARAYGTFTPADRLDLW, respectively.

4. The anti-CCR7 antibody or its antigen-binding fragment according to claim 3, characterized in that, The variable region of the light chain of the anti-CCR7 antibody or its antigen-binding fragment is: (1) A sequence as shown in any of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25; (2) A sequence having the same function as the sequence shown in any one of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 by substitution and / or deletion and / or addition of one or more amino acids; or (3) A sequence that has at least 70% identity with any one of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25 and has the same function as the sequence shown in any one of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25.

5. The anti-CCR7 antibody or its antigen-binding fragment according to claim 3, characterized in that, The heavy chain variable region of the anti-CCR7 antibody or its antigen-binding fragment is: (1) A sequence such as any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26; (2) A sequence having the same function as the sequence shown in any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 by substitution and / or deletion and / or addition of one or more amino acids; or (3) A sequence that has at least 70% identity with any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 and has the same function as the sequence shown in any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26.

6. The anti-CCR7 antibody or its antigen-binding fragment according to any one of claims 1 or 2, characterized in that, The anti-CCR7 antibody or its antigen-binding fragment further includes a constant region; Preferably, the light chain of the anti-CCR7 antibody contains the sequence shown in SEQ ID NO: 3, or the sequence obtained by linking any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 to SEQ ID NO: 27; Preferably, the heavy chain constant region of the anti-CCR7 antibody contains the sequence shown in SEQ ID NO: 28; Preferably, the heavy chain of the anti-CCR7 antibody contains the sequence shown in SEQ ID NO: 4, or the sequence obtained by linking any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 to SEQ ID NO:

28.

7. The anti-CCR7 antibody or its antigen-binding fragment according to any one of claims 3-5, characterized in that, The anti-CCR7 antibody or its antigen-binding fragment also contains chemical modifications and / or is linked with functional groups; Preferably, the chemical modification includes at least one of methylation, acetylation, phosphorylation, and glycosylation; Preferably, the functional gene includes at least one of a reporter group, a protein tag, a carrier, and a lipid group; Preferably, the reporter group includes at least one of chromogenic enzyme, fluorescent labeling group, chemiluminescent labeling group, isotope or magnetic group; Preferably, the magnetic group includes at least one of the groups that enable magnetic resonance imaging or alter relaxation efficiency.

8. A biomaterial, characterized in that, The biomaterial includes at least one of the following (1)-(3): (1) A nucleic acid molecule encoding an antibody against CCR7 as described in any one of claims 1-7 or an antigen-binding fragment thereof; (2) Contains an expression unit of the nucleic acid molecule described in (1); (3) Transformants containing the nucleic acid molecules described in (1) and / or the expression molecules described in (2); The transformant does not involve plant or animal reproductive materials; Preferably, the expression term includes viruses, plasmids, and bacteriophages; Preferably, the transformants include bacteria, fungi, and plant and animal cells.

9. A coupling, characterized in that, It comprises: the anti-CCR7 antibody or its antigen-binding fragment as described in any one of claims 1-7, and the conjugated portion; Preferably, the coupling portion comprises at least one of a detectable marker, a drug, a toxin, biotin, a spin label, an enzyme, a nanoparticle, or a magnetic nanoparticle.

10. The use of the anti-CCR7 antibody or its antigen-binding fragment according to any one of claims 1-7, the biomaterial according to claim 8, or the conjugate according to claim 9 in at least one of the following (1)-(4): (1) CCR7 testing for non-diagnostic purposes; (2) Screening and identification of biological materials for non-diagnostic purposes; (3) Prepare drugs for treating CCR7-mediated diseases; (4) Prepare products that specifically target CCR7; Preferably, the product includes a drug, a solid-phase carrier, a detection chip, a detection reagent, or a test strip.

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