Nano antibody targeting CCR7 as well as preparation and application thereof

Abstract

The invention discloses a CCR7-targeted nano antibody as well as preparation and application thereof. The CCR7-targeting nano antibody or the antigen binding fragment thereof comprises a heavy chain variable region, the heavy chain variable region comprises CDR1, CDR2 and CDR3, the CDR1 comprises an amino acid sequence as shown in SEQ ID NO: 1, the CDR2 comprises an amino acid sequence as shown in SEQ ID NO: 2, and the CDR3 comprises an amino acid sequence as shown in SEQ ID NO: 3. The CCR7-targeted nano antibody can be specifically combined with CCR7 on the surfaces of tumor cells and tumor tissues, and migration of the tumor cells and signal channels in tumors are effectively blocked.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, and in particular to a nano antibody targeting CCR7 and the preparation and application thereof. Background Art

[0002] CC chemokine receptor type 7 (CCR7) and its ligand CC chemokine ligand 19 / 21 (CCL19 / 21) play an important role in lymphatic metastasis. Under the action of ligands, tumor cells expressing CCR7 use chemotaxis to migrate and enter lymphatic vessels for lymphatic metastasis in a mechanism similar to lymphocyte homing. In addition, the tumor cell CCR7 signaling pathway can promote the growth and proliferation of tumor cells.

[0003] In the prior art, there are already blocking antibodies that target CCR7 to treat tumor metastasis and malignant hematological tumors, for example, the monoclonal antibody CAP100 developed by Catapult therapeutics (WO2017025569A1, WO2020127509A1) and the ADC drug JBH492 developed by Novartis (WO2018142322A1, WO2021220199A1, US20200216548A1). CAP100 is a monoclonal antibody obtained by hybridoma technology through immunization of mice with polypeptides. However, CAP100 has a large molecular weight and insufficient tissue penetration. JBH492 is an ADC drug obtained by coupling small molecule toxins to monoclonal antibodies obtained by immunization of mice with VLPs and hybridoma technology. This drug requires a high level of antigen expression to achieve effective internalization, which may not be effective for patients with low expression levels of the target. Summary of the invention

[0004] In order to solve the problems in the prior art that antibodies targeting CCR7 have large molecular weight, insufficient tissue penetration or high requirements for target expression levels, the present invention provides a nano antibody targeting CCR7 and its preparation and use. The nano antibody targeting CCR7 of the present invention can be used to treat tumor metastasis and malignant hematological tumors.

[0005] In order to solve the above technical problems, the present invention solves the above technical problems through the following technical solutions:

[0006] On the one hand, the present invention provides a nanobody or an antigen-binding fragment thereof targeting CCR7, which comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3, CDR1 comprises the amino acid sequence shown in SEQ ID NO:1, CDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and CDR3 comprises the amino acid sequence shown in SEQ ID NO:3.

[0007] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:5.

[0008] The term "nanoantibody" in this article, also known as "single-domain antibody", contains only one heavy chain variable region VHH, with a molecular weight of about 15kDa and a diameter of about 10 nanometers. It retains all antigen binding ability and is the smallest fragment that retains complete antigen binding.

[0009] In the present invention, the amino acid sequences of the above-listed CDRs are all shown according to the Kabat numbering system definition rules. It should be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or its region (e.g., variable region) should be understood to encompass the complementary determining region defined by any of the above-mentioned known schemes described by the present invention. Therefore, when it comes to limiting antibodies with specific CDR sequences defined by the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined by the present invention due to the application of different schemes (e.g., different assignment system rules or combinations). Although the scope of the present invention is based on the sequences shown in the Kabat numbering system definition rules, the amino acid sequences corresponding to the definition rules of other CDRs should also fall within the scope of protection of the present invention.

[0010] The term "variable region" refers to the domain of the antibody heavy chain that is involved in antibody binding to antigen. VH contains four conserved framework regions (FR) and three complementarity determining regions (CDR). Among them, the term "complementarity determining region" or "CDR" refers to the region within the variable domain that mainly contributes to antigen binding; "framework" or "FR" refers to the variable domain residues other than the CDR residues. VH contains three CDR regions: CDR1, CDR2 and CDR3. Each VH is composed of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0011] As is known in the art, the variable region of the heavy chain consists of four framework regions (FR) connected by three complementarity determining regions (CDRs) (also called hypervariable regions), and contributes to the formation of the antigen binding site of the antibody. If a variant of the subject variable region is desired, particularly with substitutions of amino acid residues outside the CDR regions (i.e., in the framework regions), appropriate amino acid substitutions, preferably conservative amino acid substitutions, can be identified by comparing the subject variable region with the variable regions of other antibodies containing CDR1 and CDR2 sequences in the same typical species as the subject variable region.

[0012] As used interchangeably herein, the terms "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply, "antibody portion") refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CCR7). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0013] In one aspect, the present invention provides a CCR7 binder, which comprises one, two or more Nanobodies or antigen-binding fragments thereof according to the present invention.

[0014] In some embodiments, the CCR7 binder further has any one of the following characteristics:

[0015] 1) The CCR7 binder is a heavy chain antibody;

[0016] 2) The CCR7 binder is a single-chain antibody;

[0017] 3) The CCR7 binder is a complete antibody;

[0018] 4) The CCR7 binder is a fusion antibody;

[0019] 5) The CCR7 binder is a bispecific antibody or a multispecific antibody;

[0020] 6) the CCR7 binder is an antibody conjugate; and,

[0021] 7) The CCR7 binder is a chimeric antigen receptor.

[0022] In some embodiments, the heavy chain antibody is an alpaca heavy chain antibody.

[0023] In some embodiments, the fusion antibody is an Fc fusion protein, a Fab fusion protein, or a single-chain fusion protein.

[0024] In some embodiments, the bispecific antibody is a CCR7×CD3 bispecific antibody, which can specifically bind to CCR7 and CD3 at the same time and can be formed by an antibody targeting CCR7 and an antibody targeting CD3.

[0025] The CD3 molecule is a universal marker on the surface of T cells. It is a protein complex structure composed of 6 peptide chains with non-covalent bonds, and consists of four constant chains: CD3γ, CD3δ, CD3ε, CD3ζ, and CD3ε. CD3γ, CD3δ, and CD3ζ are encoded by CD3E, CD3G, CD3D, and CD3Z genes, respectively, and are located on human chromosome 23.

[0026] The antibody targeting CD3 can be an antibody fragment, and in some specific embodiments, the antibody targeting CD3 derived from Mosunetuzumab-axgb (Lunsumio, Genentech, Inc.) is used. In some specific embodiments, CCR7×CD3 bispecific antibody can effectively mediate T cells in PBMC to kill CCR7-positive tumor cells JVM-3.

[0027] In some embodiments, the antibody conjugate is an antibody drug conjugate.

[0028] In some embodiments, the CCR7 antibody is a nanobody. The nanobody involved in the present invention, regardless of any genetic engineering form, includes but is not limited to the following modifications: small molecule structure modification, humanization, bispecific nanobody modification, bivalent or multivalent nanobody modification, coupling to other structural proteins or chemical reagents, etc., all within the scope of protection of the present invention. The antibodies that can be used in the present invention may include monoclonal antibodies, polyclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugated antibodies, single chains (ScFv), mutants thereof, fusion proteins comprising antibody portions (such as domain antibodies), humanized antibodies, and any other modified configurations of immunoglobulin molecules comprising antigen recognition sites of desired specificity, including antibody glycosylation variants, antibody amino acid sequence variants, and covalently modified antibodies. The antibody may be of mouse, rat, human, or any other origin (including chimeric or humanized antibodies).

[0029] In some embodiments, the CCR7 binding substance is a fusion antibody, such as an Fc fusion protein, and in a specific embodiment is a nano antibody Fc (VHH-Fc) antibody fusion protein targeting CCR7. In some embodiments, the antibody is a chimeric, humanized or human antibody. In a specific embodiment, the antibody is a human antibody.

[0030] Heavy chain antibodies (HcAb) are new antibody molecules that exist in camels and sharks, with naturally missing light chains and only heavy chains. Although heavy chain antibodies lack light chains compared to ordinary antibodies, they still retain the ability to bind to antigens.

[0031] A single-chain antibody fragment (scFv) is an antibody composed of the heavy chain variable region and the light chain variable region of an antibody connected by a short peptide (linker) of 15 to 20 amino acids.

[0032] A complete antibody has a complete Ig molecule structure, usually IgG or IgM. The antibody as a whole is composed of four polypeptide chains, two light chains (L chains) with the same structure and two heavy chains (H chains) with the same structure. The four chains form a typical "Y"-shaped antibody structure.

[0033] Fusion antibodies, also known as antibody fusion proteins (Ig fusion proteins), are products obtained by fusing antibody fragments with other biologically active proteins using genetic engineering technology. Due to the differences in fusion proteins, this type of antibody fusion protein has multiple biological functions, and the expressed recombinant protein neither affects the antigen binding ability of the single-chain antibody nor the biological properties of the protein fused with it.

[0034] Bispecific antibodies can be designed to bind to two different antigens, or to different epitopes of the same antigen. Multispecific antibodies are antibodies that can bind to two or more different epitopes or antigens at the same time.

[0035] Knob-into-hole: This technology was developed by Genentech. The specific method is to mutate the small threonine (T) at position 151 of the Fc region of one of the antibodies to a larger tryptophan (W), forming a protruding "Knobs" structure (T151W); at the same time, the serine T at position 151 of the Fc region of another antibody is mutated to serine S, the leucine L at position 153 is mutated to alanine A, and the amino acid at position 192 is mutated from tyrosine Y to valine V, forming a concave "holes" structure (T151S, L153A and Y192V); the steric hindrance effect of the "Knobs-into-holes" structure is used to achieve the correct assembly between the heavy chains of two different antibodies.

[0036] Antibody conjugation refers to connecting antibodies to other molecules or solid surfaces, using the high specificity of antibodies to recognize antigens on tumors or other disease cells, and then delivering drugs or other molecules into cells to exert therapeutic effects, or fixing antibodies on specific substrate surfaces so that they can stably and effectively recognize and bind to specific antigens, thereby achieving immune response detection. Common antibody conjugates include antibody-drug conjugates (ADC) and antibody-oligonucleotide (Ab-oligo) conjugates.

[0037] Chimeric antigen receptor (CAR) is an engineered transmembrane protein that combines the specificity of antigen-specific antibodies with T cell receptor functions. In general, CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In exemplary aspects, the extracellular domain of CAR comprises an antigen recognition region.

[0038] Specific binding refers to the ability of an antibody to bind to an antigen or an epitope within that antigen with a higher affinity than it does to other antigens or epitopes. Affinity EC can be measured using standard procedures. 50 Value, such as the method described in Example 6 of the present invention.

[0039] In some embodiments, the Fc in the Fc fusion protein is, for example, human IgG Fc.

[0040] In some embodiments, the Fc fusion protein is, for example, a nanobody targeting CCR7 fused to Fc (VHH-Fc).

[0041] In some specific embodiments, the Fc sequence is as shown in SEQ ID NO:7 or has at least 90%, 95%, 96% or 97% homology to the amino acid sequence shown in SEQ ID NO:7.

[0042] In some embodiments, the Fc is fused to the C-terminus of the heavy chain variable region.

[0043] In some embodiments, the Fc is fused directly to the C-terminus of the heavy chain variable region.

[0044] In some embodiments, one arm of the CCR7×CD3 bispecific antibody is the Fc fusion protein, which is an antibody targeting CCR7, and the other arm is a CD3 Fab-Fc fusion protein, which is an antibody targeting CD3.

[0045] In some embodiments, the CCR7×CD3 bispecific antibody is produced using a knob-into-hole approach.

[0046] In some embodiments, the amino acid sequence of the CD3 Fab-Fc fusion protein and / or the Fc of the Fc fusion protein comprises amino acid residue differences at one or more positions selected from position 151, position 153 and position 192 compared to SEQ ID NO:7, wherein the amino acid difference at position 151 may be T151W or T151S, the amino acid difference at position 153 may be L153A, and the difference at position 192 may be Y192V.

[0047] In some embodiments, the amino acid sequence of Fc of the CD3 Fab-Fc fusion protein comprises the difference of T151W compared with SEQ ID NO:7, wherein the smaller threonine (T) at position 151 of the Fc region of the antibody is mutated to a larger tryptophan (W), forming a protruding "Knobs" type structure (T151W); and the amino acid sequence of Fc of the Fc fusion protein comprises the differences of T151S, L153A and Y192V compared with SEQ ID NO:7, wherein the serine T at position 151 of the Fc region of the antibody is mutated to serine S, the leucine L at position 153 is mutated to alanine A, and the amino acid at position 192 is mutated from tyrosine Y to valine V, forming a concave "holes" type structure (T151S, L153A, Y192V).

[0048] In some embodiments, the amino acid sequence of Fc of the Fc fusion protein comprises the difference of T151W compared with SEQ ID NO:7, wherein the smaller threonine (T) at position 151 of the Fc region of the antibody is mutated to a larger tryptophan (W), forming a protruding "Knobs" type structure (T151W); and the amino acid sequence of Fc of the CD3 Fab-Fc fusion protein comprises the differences of T151S, L153A and Y192V compared with SEQ ID NO:7, wherein the serine T at position 151 of the Fc region of the antibody is mutated to serine S, the leucine L at position 153 is mutated to alanine A, and the amino acid at position 192 is mutated from tyrosine Y to valine V, forming a concave "holes" type structure (T151S, L153A, Y192V).

[0049] In some embodiments, the amino acid sequence of the CD3 Fab-Fc fusion protein and / or the Fc of the Fc fusion protein, compared with SEQ ID NO:7, further comprises differences in amino acid residues at one or more positions selected from position 5, position 24, position 82 and position 117, wherein the amino acid difference at position 5 may be A5C; the amino acid difference at position 24 may be D24S; the amino acid difference at position 82 may be N82G; and the amino acid difference at position 117 may be E117I.

[0050] In some embodiments, the Fab fragment of the CD3 Fab-Fc fusion protein comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3, H-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 15, H-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 16, and H-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 17, the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, L-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 18, L-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 19, and L-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 20.

[0051] In some specific embodiments, the amino acid sequence of Fc of the CD3 Fab-Fc fusion protein is as shown in SEQ ID NO: 14, or has at least 90%, 95%, 96% or 97% homology to the amino acid sequence shown in SEQ ID NO: 14, and / or the amino acid sequence of Fc of the Nanobody Fc fusion protein targeting CCR7 is as shown in SEQ ID NO: 13, or has at least 90%, 95%, 96% or 97% homology to the amino acid sequence shown in SEQ ID NO: 13.

[0052] In some specific embodiments, the amino acid sequence of Fc of the Nanobody Fc fusion protein targeting CCR7 is as shown in SEQ ID NO: 14, or has at least 90%, 95%, 96% or 97% homology to the amino acid sequence shown in SEQ ID NO: 14, and / or the amino acid sequence of Fc of the CD3 Fab-Fc fusion protein is as shown in SEQ ID NO: 13, or has at least 90%, 95%, 96% or 97% homology to the amino acid sequence shown in SEQ ID NO: 13.

[0053] In some embodiments, the amino acid sequence of the heavy chain variable region of the CD3 Fab-Fc fusion protein is as shown in SEQ ID NO: 9 or has at least 90% homology to the amino acid sequence shown in SEQ ID NO: 9, and / or the amino acid sequence of the light chain variable region of the CD3 Fab-Fc fusion protein is as shown in SEQ ID NO: 10 or has at least 90% homology to the amino acid sequence shown in SEQ ID NO: 10. The sequences with at least 90% homology only involve changes in the framework region sequence in the variable region of the CD3 Fab segment, and do not involve changes in the amino acid sequence of the CDR region.

[0054] In some embodiments, the amino acid sequence of the heavy chain constant region of the CD3 Fab-Fc fusion protein is as shown in SEQ ID NO:21, or has at least 90% homology to the amino acid sequence shown in SEQ ID NO:21, and / or the amino acid sequence of the light chain constant region of the CD3 Fab-Fc fusion protein is as shown in SEQ ID NO:22, or has at least 90% homology to the amino acid sequence shown in SEQ ID NO:22.

[0055] In some specific embodiments, the heavy chain amino acid sequence of the CD3 Fab-Fc fusion protein is as shown in SEQ ID NO: 23 or has at least 90% homology to the amino acid sequence shown in SEQ ID NO: 23, and / or the light chain amino acid sequence of the CD3 Fab-Fc fusion protein is as shown in SEQ ID NO: 24 or has at least 90% homology to the amino acid sequence shown in SEQ ID NO: 24. The sequences with at least 90% homology only involve changes in the framework region and / or constant region sequence in the variable region, and do not involve changes in the amino acid sequence of the CDR region.

[0056] In one aspect, the present invention provides an isolated nucleic acid having a sequence selected from any one of the following:

[0057] (i) the coding sequence of the Nanobody or antigen-binding fragment thereof of the invention or the CCR7 binder of the invention;

[0058] (ii) the complement of the coding sequence of (i); and

[0059] (iii) A fragment of 15-50 bp of any of the sequences in (i) or (ii).

[0060] In some embodiments, the fragment is a primer.

[0061] In some embodiments, the isolated nucleic acid comprises the nucleotide sequence shown in SEQ ID NO:4.

[0062] In some specific embodiments, the isolated nucleic acid further comprises the nucleotide sequence shown in SEQ ID NO:6.

[0063] As known in the art, "nucleic acid" in the present invention refers to a nucleotide chain of any length, and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into the chain by DNA or RNA polymerase.

[0064] In one aspect, the present invention provides a recombinant expression vector comprising the isolated nucleic acid of the present invention.

[0065] In some embodiments, the recombinant expression vector is a plasmid, cosmid, phage or viral vector.

[0066] In some specific embodiments, the backbone of the plasmid is, for example, pcDNA3.4.

[0067] The term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that permits the expression of mRNA, protein, polypeptide or peptide by a host cell when the construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide or peptide and the vector is contacted with a cell under conditions sufficient to allow the mRNA, protein, polypeptide or peptide to be expressed in the cell. The vectors disclosed herein are generally not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vectors of the present invention may contain any type of nucleotides, including but not limited to DNA and RNA: they may be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and they may contain natural, non-natural or altered nucleotides. Suitable vectors include those designed for expansion and amplification or for expression or both, examples of vectors include but are not limited to viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0068] In one aspect, the present invention provides a transformant, wherein the transformant comprises the isolated nucleic acid of the present invention or the recombinant expression vector of the present invention.

[0069] In some embodiments, the host cell of the transformant is a prokaryotic cell or a eukaryotic cell.

[0070] In some embodiments, the host cell is selected from insect cells, mammalian cells or other cells suitable for preparing Nanobodies or antigen-binding fragments.

[0071] In some more specific embodiments, the mammalian cells are, for example, HEK293 or CHO-K1 cells.

[0072] As used herein, the term "host cell" refers to any type of cell that can contain a nucleic acid or vector described herein. The host cell can be a eukaryotic cell, such as a plant, animal, fungus, or algae; or the host cell can be a prokaryotic cell, such as a bacterium or a protozoan.

[0073] The expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene editing (CRISPR-Cas system, ZFN system or TALEN system), transposon (Sleeping Beauty or PiggyBAC), gene gun, lipid-based transfection or other conventional techniques. In the case of protoplast fusion, the cell is cultivated in a culture medium and screened for suitable activity. The method and conditions for cultivating the transfected cells produced and for recovering the antibody molecules produced are known to those skilled in the art and can be based on the present specification and the methods known in the prior art, according to the specific expression vector used and the host cell change or optimization. In addition, one or more markers of the host cell transfected can be selected by introducing, and the cell that has been stably incorporated into its chromosome by DNA can be selected. The marker can, for example, provide prototrophy, biocidal resistance (for example, antibiotics) or heavy metal (such as copper) resistance to auxotrophic hosts. Selectable marker genes can be directly connected to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements may include splicing signals, as well as transcriptional promoters, enhancers, and termination signals.

[0074] In one aspect, the present invention provides a method for preparing the nanobody or antigen-binding fragment thereof targeting CCR7 or the CCR7 conjugate according to the present invention, the method comprising culturing the transformant according to the present invention to obtain a culture.

[0075] In some embodiments, the Nanobody or antigen-binding fragment thereof or CCR7 binder is purified from the culture.

[0076] In one aspect, the present invention provides a pharmaceutical composition, which comprises the nanobody or antigen-binding fragment thereof, the CCR7 binder, the isolated nucleic acid, the recombinant expression vector or the transformant of the present invention.

[0077] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0078] In some embodiments, the pharmaceutical composition or pharmaceutical preparation of the present invention comprises suitable pharmaceutically acceptable carriers such as pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers. As used in the present invention, "pharmaceutically acceptable excipients" or "pharmaceutical excipients" include any and all solvents, dispersion media, isotonic agents and absorption delay agents that are physiologically compatible, etc. Pharmaceutical carriers suitable for the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. For the use of excipients and their uses, see also "Handbook of Pharmaceutical Excipients", Fifth Edition, RC Rowe, PJ Eskey and SCOwen, Pharmaceutical Press, London, Chicago. If desired, the composition may also contain a small amount of wetting agent or emulsifier, or pH buffer. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, etc. Oral formulations may include standard pharmaceutical carriers and / or excipients, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin. The pharmaceutical preparation or pharmaceutical composition comprising the present invention may be prepared by mixing the antibody or antigen-binding fragment thereof of the present invention with the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. ed. (1980)), preferably in the form of a lyophilized formulation or an aqueous solution. The pharmaceutical composition or preparation of the present invention can also include more than one active ingredient, which is required for the specific indication to be treated, preferably with those active ingredients of complementary activity that will not adversely affect each other. For example, it is desirable to also provide other active ingredients, such as other antibodies, antiviral active agents, small molecule drugs or immunomodulators, etc. The active ingredient is suitably combined in an amount effective for the purpose of use. Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing antibodies of the present invention or their Fabs, and the matrix is ​​a formed article, such as a film or microcapsule form.

[0079] On the one hand, the present invention provides: the use of the nanoantibody or antigen-binding fragment thereof, the CCR7 binder, the isolated nucleic acid, the recombinant expression vector, the transformant of the present invention or the pharmaceutical composition of the present invention in the preparation of a medicament for preventing and / or treating a disease or condition associated with CCR7 expression.

[0080] In some embodiments, the disease or condition associated with CCR7 expression is a tumor.

[0081] In some embodiments, the tumor is a tumor metastasis or a hematological malignancy.

[0082] In some embodiments, the hematological malignancy is chronic lymphocytic leukemia.

[0083] In one aspect, the present invention provides a CCR7 detection agent or kit, which comprises the nanobody or antigen-binding fragment described in the present invention, the CCR7 binder, the isolated nucleic acid, the recombinant expression vector, the transformant described in the present invention or the pharmaceutical composition described in the present invention.

[0084] In one aspect, the present invention provides: use of the nanobody or antigen-binding fragment, the CCR7 binder, the isolated nucleic acid, the recombinant expression vector, the transformant or the pharmaceutical composition of the present invention in the preparation of a diagnostic agent, a detection agent or a kit.

[0085] In some embodiments, the diagnostic agent or detection agent is used to diagnose or detect a disease mediated by CCR7.

[0086] In some embodiments, the kit is used to detect CCR7 in a sample, evaluate the effect of drug treatment, or diagnose cancer.

[0087] In some embodiments, the disease mediated by CCR7 is a tumor.

[0088] In some embodiments, the tumor is a tumor metastasis or a hematological malignancy.

[0089] In some embodiments, the tumor is chronic lymphocytic leukemia.

[0090] In one aspect, the present invention provides a method for detecting CCR7, which comprises contacting the nanobody or antigen-binding fragment described in the present invention or the CCR7 conjugate described in the present invention with a sample to be tested.

[0091] In some embodiments, the detecting is for non-diagnostic purposes.

[0092] In a preferred embodiment of the present invention, the method is for non-diagnostic and / or therapeutic purposes, such as detecting the presence or absence of CCR7 in a laboratory; or competing with other antibodies targeting CCR7 for binding, detecting whether there is competition between antibodies, that is, whether the antigenic epitopes are the same or similar, and other application scenarios.

[0093] On the one hand, the present invention provides a method for preventing, ameliorating or treating diseases, disorders or conditions associated with CCR7 expression, the method comprising administering an effective amount of the nanobody or antigen-binding fragment of the present invention, the CCR7 binder or the pharmaceutical composition of the present invention to a subject in need thereof.

[0094] In some embodiments, the disease mediated by CCR7 is a tumor.

[0095] In some embodiments, the tumor is a tumor metastasis or a hematological malignancy.

[0096] In some embodiments, the tumor is chronic lymphocytic leukemia.

[0097] As used herein, the term "effective amount" means the amount of a drug or pharmaceutical agent that elicits a biological or pharmaceutical response in a tissue, system, animal or human that is sought, for example, by a researcher or clinician. In addition, the term "therapeutically effective amount" means an amount that causes improved treatment, cure, prevention or alleviation of a disease, condition or side effect, or an amount that reduces the rate of progression of a disease or condition, compared to a corresponding subject that has not received the amount. The term also includes within its scope an amount that is effective to enhance normal physiological function.

[0098] In one aspect, the present invention provides: use of the nanobody or antigen-binding fragment of the present invention, the CCR7 conjugate or the pharmaceutical composition of the present invention in preventing, ameliorating or treating diseases, disorders or conditions associated with CCR7 expression.

[0099] In some embodiments, the disease mediated by CCR7 is a tumor.

[0100] In some embodiments, the tumor is a tumor metastasis or a hematological malignancy.

[0101] In some embodiments, the tumor is chronic lymphocytic leukemia.

[0102] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0103] The reagents and raw materials used in the present invention are commercially available.

[0104] The positive and progressive effect of the present invention is that the single-domain antibody fused with the Fc end of human IgG1 can specifically bind to CCR7 on the surface of tumor cells and tumor tissues, effectively blocking the migration of tumor cells and the signal pathway inside the tumor. It is a new type of highly specific active antibody with CCR7 targeting function and anti-tumor activity.

[0105] The single-domain antibody targeting CCR7 of the present invention enhances the ADCC effect by fusing the Fc end to achieve a tumor killing effect. In in vitro experiments, compared with the competing product CAP100, its affinity and functional activity are comparable or even better.

[0106] The blocking antibody targeting CCR7 developed by the present invention is obtained by immunizing alpaca and screening phage libraries. Compared with the existing clinical drug CAP100 with the same target, the candidate molecule has a smaller molecular weight, better tissue penetration, good pharmacokinetic activity, and equivalent or better therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] Figure 1 FACS experiments verify that the anti-CCR7 antibody 2037 can effectively bind to the CCR7 overexpressing stable cell line and chronic lymphocytic leukemia tumor cell JVM-3; Part A, the antibody molecule is verified to bind to the 293T CCR7 overexpressing stable cell line under concentration gradient dilution; Part B, the antibody molecule is verified to bind to the CHO-K1 CCR7 overexpressing stable cell line under concentration gradient dilution; Part C, the antibody molecule is verified to bind to the tumor cell JVM-3 under concentration gradient dilution.

[0108] Figure 2 FACS experiments were performed to verify that the 2037 antibody molecule specifically binds to the target protein CCR7, rather than other chemokine receptors.

[0109] Figure 3 FACS experiment verifies the species cross-reactivity of 2037 antibody; Part A, affinity diagram of 2037 antibody molecule with stable cell line expressing mouse CCR7 under concentration gradient dilution conditions; Part B, affinity diagram of 2037 antibody molecule with cynomolgus macaque PBMC under concentration gradient dilution conditions.

[0110] Figure 4 To verify the functional activity of the antibody; Part A, β-arrestin2 recruitment experiment based on NanoBiT; Part B, cAMP experiment; Part C, cell chemotaxis experiment.

[0111] Figure 5ADCC experiments were performed to determine the tumor killing effect of the 2037 antibody molecule under conditions of co-incubation with tumor cells JVM-3 and effector cells (Part A is NK92-CD16a, Part B is human PBMC).

[0112] Figure 6 Pharmacokinetics of anti-CCR7 antibody in C57 mice after single intravenous administration.

[0113] Figure 7 The tumor killing effect of CCR7xCD3 bispecific antibody under the co-culture conditions of tumor cells JVM-3 and effector cells (human PBMC). DETAILED DESCRIPTION

[0114] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0115] 293T culture medium: DMEM + 10% FBS + 1% Penicillin Streptomycin solution (10,000 U / mL) + 1% Sodium Pyruvate (100 mM) + 1% MEM NEAA (all from Gibco);

[0116] CHO-K1 medium: Ham's F-12K (Kaighn's) + 10% FBS + 1% Penicillin-Streptomycin solution (10,000 U / mL) (all from Gibco);

[0117] CAP-100 (a functional antibody targeting CCR7 produced by Catapult Therapeutics) was used as a positive control antibody.

[0118] Helper phage rescue library: Helper phage M13K07 is used to rescue phagemids and has kanamycin resistance. With its assistance, the phagemids can be replicated and expressed on the phage surface in a fusion form.

[0119] The present invention uses a combination of multiple antigens for animal immunization, and obtains single-domain antibody molecules that specifically bind to cells expressing CCR7 through enrichment and selection of phage immune libraries. The target antibody molecules are obtained by tandemly fusing the single-domain antibody molecules targeting CCR7 with the Fc end of human IgG1 with enhanced ADCC effect.

[0120] Example 1 Antigen preparation

[0121] 1.1 DNA preparation: Use pcDNA3.1 as a vector to construct a plasmid with NCBI nucleic acid ID NM_001838.4 (CCR7). (Plasmid production comes from the supplier GenScript)

[0122] 1.2 Peptide preparation: CCR7 protein (NCBI protein ID. NP_001829.1) N-terminal 32 amino acids (EDEVTDDYIGDNTTVDYTLFESLCSKKDVRNK, SEQ ID NO: 8) and tyrosine sulfonation modified peptide. (Peptide production source from supplier GenScript)

[0123] 1.3 Stable cell line: Using 293T cells as hosts, monoclonal cells that stably express the modified CCR7 target protein. (Cell line construction originated from the supplier Kangyuan Bochuang)

[0124] 1.4 Expression and purification of CCR7 (micelle) protein:

[0125] The human CCR7 DNA sequence (NM_001838.4) was cloned into the pFastBac vector (Thermo Fisher, #10360014) for expression in insect cells. The receptor CCR7 sequence was fused with enhanced green fluorescent protein to monitor expression, and then purified with a C-terminal 10-histidine tag. In the construction, a 3C protease cleavage site was introduced to cut the C-terminal fluorescent protein and the tag protein. The expression conditions of CCR7 were 27°C, using Sf900-III medium (Thermo Fisher), when the SF9 cell density was 2E6 / mL, the viability was >97%, and the average diameter was 17-17.5μM, the infection volume (VOI) was 0.1%-5% according to different virus strengths, and the expression was about 72 hours after infection, and the cells were collected and stored at -80°C. All purification steps of CCR7 protein were performed at 4°C. The cells were thawed in a low-salt buffer containing 10mM HEPES / NaOH pH7.5, 10mM MgCl2, 20mM KCl, and complete protease inhibitors (Roche) (1 tablet / 100mL), stirred at 300rpm, and the cell membrane was destroyed by dounce (homogenizer), separated by ultracentrifugation, washed once with low-salt buffer, washed twice with high-osmotic high-salt buffer (10mM HEPES / NaOH pH7.5, 10mM MgCl2, 20mM KCl, 1M NaCl, Roche protease inhibitor tablets (1 / 100mL)), and washed once with low-salt buffer, repeatedly resuspended and centrifuged to wash the membrane. The washed membrane with CCR7 receptor protein was resuspended in low-salt buffer, quickly frozen in liquid nitrogen, and stored at -80°C until the next use. On the day of purification, the membrane was thawed in room temperature water, 2 mg / mL iodoacetamide and complete protease inhibitor (Roche) (1 tablet / 50 mL) were added, and the membrane was incubated at 4°C for 1 hour. The membrane protein was extracted using 50mM HEPES / NaOH pH 7.5, 300mM NaCl, 20mM imidazole / HCl pH 7.5, 1% (w / v) DDM, 0.2% (w / v) CHS and complete protease inhibitor (1 tablet / 50 mL) as solvents, maintaining a ratio of 1:1.75 (biomass to final volume ratio) to dissolve, and the mixture was stirred at 4°C, 600rpm for 1 hour. The mixture was ultracentrifuged and the supernatant was separated for affinity chromatography.The supernatant was incubated with pre-equilibrated Talon Superflow resin at 4°C, 600 rpm for 1 hour. After binding, the resin was washed five times with 2 column volumes of wash buffer (50 mM HEPES / NaOH pH 7.5, 300 mM NaCl, 20 mM imidazole / HCl pH 7.5, 0.03 / 0.006% (w / v) DDM / CHS), followed by elution with elution solution (50 mM HEPES / NaOH pH 7.5, 300 mM NaCl, 300 mM imidazole / HCl pH 7.5, 0.03 / 0.006% (w / v) DDM / CHS). Finally, the protein buffer was replaced with 25 mM HEPES / NaOH pH 7.5, 150 mM NaCl, 0.03 / 0.006% (w / v) DDM / CHS by molecular sieve chromatography (SuperdexTM 200Increase10 / 300GL), and high-purity protein was isolated. The purified protein was collected and quickly frozen in liquid nitrogen and stored at -80°C.

[0126] Assembly and purification of 1.5 nanometer phospholipid disks:

[0127] Take out the purified micelle protein, membrane scaffold protein 1D1 (MSP1D1, SigmaAldrich), and POPC (1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine, Avanti Polar Lipids) and melt them on ice, then add cholic acid sodium salt (Anatrace) with a final concentration of 16mM to POPC. Mix micelle, MSP, and POPC at a molar ratio of 1:8:480, and place the mixture on ice for 45 minutes to 1 hour. Then use the pretreated Bio-beads to remove the detergent, add it to the mixture at a ratio of 0.8-1g / mL, rotate it overnight at 4℃, let it stand the next morning, collect the supernatant, wash it with PBS 2-3 times, and collect the washing liquid. Subsequently, affinity chromatography was used to remove the empty beads, and the collected solution was mixed with Ni-NTA beads, incubated with rotation at 4°C for 1 hour, the beads were collected, and the impurity proteins were eluted with washing buffer (PBS+20mM imidazole). Subsequently, 3C enzyme was added to the column for overnight digestion. The next day, the flow-through and the target protein eluted with the washing buffer were collected, concentrated, separated by molecular sieve chromatography (SuperdexTM 200Increase10 / 300GL), and the storage buffer (PBS) was replaced. The purified protein was collected and quickly frozen in liquid nitrogen and stored at -80°C.

[0128] Example 2 Alpaca Immunization

[0129] A healthy adult alpaca (ABLINK) was immunized with a combination of various antigens, including a fusion protein of the N-terminal peptide of CCR7 and hemocyanin (KLH), a stable cell line expressing CCR7, DNA of human CCR7, and a nanophospholipid disk. Five consecutive immunizations were performed, each with an interval of 3 weeks, using conventional methods, with multiple subcutaneous injections (cells 1x10 7 , protein 0.25mg). Immunization was performed with electroporation after DNA injection; Freund's adjuvant was used as an auxiliary for protein immunization. Peripheral blood was collected 1 week after each immunization for immune titer detection. After the immune titer stabilized, a large amount of blood was drawn, PBMC was isolated, lysed with Trizol (Invitrogen, 15596026), and stored at -80°C.

[0130] Example 3 VHH phage display library construction

[0131] Total RNA was extracted from peripheral blood mononuclear cells (PBMC) using Trizol reagent. TM RT Master Mix kit, total RNA was reverse transcribed into cDNA. The variable region (VHH) of heavy chain immunoglobulin was amplified using nested polymerase chain reaction (PCR). In the first step of PCR, 1 μg of cDNA template was mixed with primers. The DNA product from the first step of PCR reaction was separated by agarose gel electrophoresis. IgG and VHH bands were obtained, and after gel purification of the VHH bands, the DNA product of the first step of PCR was used as a template in the second step of PCR. A second round of PCR was performed using VHH-specific primers. The second round of PCR products were gel purified and enzymatically digested, and then inserted into a phagemid plasmid after internal optimization of the stuffer gene was changed based on the pComb3xss plasmid (Hunan Yada Fenghui New Materials Co., Ltd.). The recombinant plasmid with the VHH gene fragment was electrotransformed into Escherichia coli cells to produce a phage display VHH immune library. The library was scraped and suspended in 30% glycerol and then stored at -80°C.

[0132] Example 4 Phage Display Panning

[0133] Solid-state and cell panning were performed using the peptides in 1.2 and cells overexpressing CCR7 (stable cell lines in 1.3). The library was grown to the logarithmic growth phase, and then the library was rescued with M13KO7 helper phage and amplified overnight at 30°C in a shaker. The phage was precipitated with PEG, resuspended in PBS, and stored at -80°C with 50% glycerol. For solid-phase panning, the peptide dissolved in PBS was incubated in an immunotube at 4°C overnight, and the blocked phage particles were added to a blank immunotube for negative screening, and then added to the immunotube incubated with the peptide for positive screening. Non-specific phages were washed away with PBST, the bound phage particles were eluted with 100mM triethylamine (TEA), and the eluate was neutralized with 1M Tris HCl (pH7.4). Half of the eluate was then used to infect exponentially growing Escherichia coli, and the remaining eluate was used for output titration of bound phages. For cell panning, the blocked phage particles were added to 293T blank cells for negative screening, and then cells overexpressing CCR7 were added for positive screening. After washing the cells after positive screening with PBS, the washed cells were added to exponentially growing Escherichia coli for output titration. CCR7-specific clones were detected by phage flow cytometry fluorescence sorting technology (FACS). Single output phage clones were grown in 96-well deep-well plates and rescued overnight with M13KO7-assisted phage. 100 μL of phage supernatant and 293T blank cells or cells overexpressing CCR7 were incubated at 4°C for one hour, washed twice, and anti-M13 fluorescent secondary antibody (Progen61497) was added and incubated at 4°C for one hour. After washing twice, the fluorescence value was read on BD FACS Canto II to identify CCR7-specific clones. Multiple antibodies were obtained, and the antibody with the best specificity was selected and named 2037 antibody, and its antibody sequence was detected.

[0134] Example 5 Expression and purification of antibodies

[0135] The gene of 2037 antibody was synthesized by GenScript, and the plasmid vector was pcDNA3.4. After receiving the plasmid, the plasmid was amplified and then expressed using Expi293f cells (Thermo Fisher) at 37°C, 8% carbon dioxide concentration, and 110 rpm. Using Expi293f medium (Thermo Fisher), when the cell density was 2E6 / mL and the viability was >95%, transfection was performed. After transfection, expression was performed for about 120 hours, the cell supernatant was collected, and the culture supernatant was neutralized with 1M Tris-HCl, pH 8.0, and then incubated with balanced Protein A beads at 4°C for 2 hours, beads were collected, washed with PBS, and then eluted with 0.1M glycine buffer pH 3.0. After elution, the target protein used a desalting column to replace the protein buffer with PBS, and finally SDS-PAGE and SEC-HPLC purity analysis was performed. The high-purity protein (i.e., 2037 antibody) was aliquoted and stored at -80°C.

[0136] Example 6 Determination of affinity between antibody and antigen

[0137] Using CHO-K1 cells as hosts, the modified CCR7 target protein was stably expressed to obtain a monoclonal stable cell line of CHO-K1 overexpressing human CCR7.

[0138] Using flow cytometry fluorescence sorting technology (FACS), 293T (stable cell line in 1.3) and CHO-K1 monoclonal stable cell line overexpressing human CCR7, as well as tumor cells JVM-3 (CBP60618, Nanjing Kebai) were cultured to the logarithmic growth phase, the culture medium was discarded and rinsed with DPBS, and an appropriate amount of trypsin was added to digest the cells at 37°C for about 5 minutes. The digestion was terminated and the cells were collected with a culture medium containing 10% FBS. After centrifugation at 300g for 5 minutes, the culture medium was discarded, DPBS was added to resuspend the cells, and the cell density was counted by trypan blue staining. Take a 96-well plate and add 100 μL of a suspension containing 2E5 cells to each well. After centrifugation at 300g for 5 minutes, DPBS was discarded, and 100 μL of 5% BSA was added to each well and blocked at 4°C for 1 hour. After the blocked cells were centrifuged at 300g for 5 minutes, BSA was discarded. The cells were divided into two groups, one group was added with 2037 antibody, and the other group was added with CAP-100 as a positive control antibody. 100 μL of the antibody to be tested was added to each well, the cells were resuspended, and incubated at 4°C for 1 hour. After centrifugation at 300g for 5 minutes, the supernatant was discarded, 100 μL of FACS buffer (PBS containing 1% BSA) was added to each well, and washed twice. 100 μL of secondary antibody (Alexa Fluor 647AffiniPure TMGoat Anti-Human IgG, Jackson #109-605-098) resuspended cells, incubated at 4°C in the dark for 1 hour. After centrifugation at 300g for 5 minutes, discard the supernatant, add 100μL FACS buffer to each well and rinse three times. Finally, add 100μL FACS buffer to each well to resuspend cells, and read the fluorescence value on the BD FACS Canto II instrument. For details on antibody-cell binding flow fluorescence detection, see Figure 1 , affinity EC 50 The values ​​are shown in Table 1, indicating that the 2037 antibody molecule has a strong antigen affinity.

[0139] Table 1. Affinity parameters of 2037 and positive control antibodies for monoclonal cell lines and tumor cell lines overexpressing CCR7

[0140]

[0141] Example 7 Specificity detection of antibodies

[0142] Using 293T cells as hosts, stable cell lines overexpressing human CCR1 (CCDS2737.1), CCR6 (CCDS5298.1), CCR9 (CCDS2732.1), CXCR4 (CCDS33295.1) and CXCR6 (CCDS2735.1) were constructed. The specific methods are as follows:

[0143] Prepare expression vectors containing the target genes: pIRESpuro3-Flag-CCR1, pIRESpuro3-Flag-CCR6, pIRESpuro3-Flag-CCR9, pIRESpuro3-Flag-CXCR4 and pIRESpuro3-Flag-CXCR4 and pIRESpuro3-Flag-CXCR6 (gene synthesis and plasmid extraction were completed by GenScript).

[0144] Prepare transfection complex: 400 μL Opti-MEM, add 2 μg plasmid, mix well and add 2 μL LTX reagent, mix again, and let stand at room temperature for 5 minutes. Add 10 μL PLUS TMReagent, mix well and let stand at room temperature for 30 minutes. Digest the cells to be transfected and adjust the cell density to 4E5 cells / mL with culture medium. Take a 6-well plate, add 2mL of cell suspension and prepared transfection complex, mix well and place in the incubator for continued culture. The cells continue to be cultured for 24 hours, take some cells to detect the transfection efficiency, and continue to culture the remaining cells to 50-60% fusion. Selective antibiotics are added to screen cells expressing resistance genes to obtain stable cell lines. A monoclonal cell line overexpressing human CCR7 was used as a positive control cell. The binding of the 2037 antibody to the above cells was detected by flow cytometry fluorescence sorting technology. For details, see Figure 2 , indicating that the 2037 antibody molecule specifically binds to the target protein CCR7 and does not recognize other chemokine receptor family proteins.

[0145] Example 8 Detection of species cross-reactivity of antibodies

[0146] Mouse CCR7 protein sequence (Gene ID: 12775) was used to overexpress mouse CCR7 in 293T cells.

[0147] The binding of 2037 antibody and positive control (CAP-100) antibody to the above cells was detected by flow cytometry fluorescence sorting using a mouse CCR7 monoclonal stable cell line and cynomolgus monkey PBMC cells, respectively. For details, see Figure 3 In part A, the 2037 antibody molecule has a strong affinity to the stably transfected cell line expressing mouse CCR7 under the condition of concentration gradient dilution, and in part B, the 2037 antibody molecule has a strong affinity to cynomolgus macaque-derived PBMC under the condition of concentration gradient dilution. The affinity parameters are shown in Table 2, indicating that the 2037 antibody molecule can recognize the CCR7 protein of mouse and cynomolgus macaque origin.

[0148] Table 2. Binding affinity parameters of antibodies to mouse and cynomolgus monkey CCR7

[0149]

[0150] Example 9 Determination of the functional activity of antibodies

[0151] 9.1 NanoBiT-based β-arrestin2 recruitment experiment

[0152] 293FT-LgBiT-arrb2-CCR7-SmBiT cell construction: using LTX transferred the pIREShyg3-LgBiT-arrb2 plasmid (ordered from GenScript) into 293FT cells, and the specific transfection steps were the same as in Example 7. After 48 hours of transfection, the cells were screened and cultured using a medium containing 150 μg / mL of hygromycin to obtain cells that stably expressed LgBiT-arrb2. The pIRESpuro3-CCR7-SmBiT plasmid (ordered from GenScript) was transfected into 293FT-LgBiT-arrb2 cells and screened using 1 μg / mL of puromycin to obtain a 293FT-LgBiT-arrb2-CCR7-smBiT stable cell line.

[0153] Take 20 μL of 293FT-LgBiT-arrb2-CCR7-SmBiT cells (1E6 / mL) in the logarithmic growth phase and plate them in a 384-well test plate coated with Poly-D-Lysine, and place them in a cell culture incubator at 37°C overnight. The next day, aspirate the complete medium in the test plate, then take 15 μL (2037 antibody or positive control antibody CAP-100) antibody dilution (final concentration 0.1, 1, 10, 100, 1000, 10000nM) and add it to the test wells. Incubate in a cell culture incubator for 30 minutes, then take it out and let it stand for 10 minutes to equilibrate to room temperature. Add 5 μL of the test solution (Promega, Nano- Live Cell Detection Kit):Nano After adding the live cell substrate + 95% LCS diluent) to the detection well, add 5 μL of 0.55 μM CCL19 diluent. Immediately read with a multifunctional microplate reader to detect the luminescence signal. For detailed experimental results, see Figure 4 Part A shows that based on NanoBiT validation, the 2037 antibody molecule effectively inhibits the recruitment of downstream β-arrestin2 protein induced by CCR7 and inhibits the active IC 50 The parameters are shown in Table 3, indicating that the 2037 antibody molecule can effectively inhibit the recruitment of β-arrestin2 protein and its mediated downstream signals.

[0154] 9.2 cAMP experiment

[0155] The Lance Ultra cAMP kit (PerkinElmer Cat#TRF0263) was used to detect the changes in intracellular cAMP levels. The specific steps are as follows: Take CHO-K1-CCR7 cells with good growth status, discard the culture medium and rinse with DPBS, add an appropriate amount of dissociation buffer and digest the cells into single cells at 37°C. Centrifuge at 275g for 5 minutes to collect the cells, discard the supernatant, resuspend the cells with HBSS and count them. Centrifuge at 275g for 5 minutes and discard HBSS. Resuspend the cells with stimulation buffer and adjust the cell density to 2E5 cells / mL. Take a white opaque 384-well plate, add 5μL cell suspension and 2.5μL test antibody (final antibody concentration is 0.1, 1, 10, 100, 1000, 10000nM) and 2.5μL CCL19 / forskolin (1.25μL 2μM CCL19 and 1.25μL 32μM forskolin) to each well, incubate for 30 minutes, add 5μLEU-cAMP tracer and 5μL ULight-anti-cAMP to each well, incubate at room temperature for 1 hour, and use a multifunctional microplate reader to detect TR-FRET signals. For detailed experimental results, see Figure 4 Part B shows that the cAMP experiment verifies that the 2037 antibody molecule effectively inhibits the production of cyclic adenosine monophosphate (cAMP) induced by CCR7 and inhibits the activity of IC 50 The parameters are shown in Table 3, indicating that the 2037 antibody molecule can effectively inhibit the production of cAMP and its mediated downstream signals.

[0156] 9.3 Cell chemotaxis assay

[0157] Take 50 μL of JVM-3 cells (4E6 cells / mL) in the logarithmic growth phase and transfer them to a 96-well culture plate. Mix well with 50 μL of gradiently diluted antibody diluent (final concentration 0.1, 1, 10, 100, 1000, 10000 nM) and incubate in a cell culture incubator for 30 minutes. After adding 600 μL of culture medium containing CCL19 to the lower chamber of the Transwell 24-well plate, gently place the upper chamber on the well plate. Take the incubated antibody and cell suspension and add them to the upper chamber of the Transwell. The volume of each well is 100 μL to avoid bubbles on the liquid surface. Cover the 24-well plate with a lid, and use a flow cytometer to count the cells in the lower chamber after culturing in a cell culture incubator for 6 hours. For detailed experimental results, see Figure 4 Part C shows that the cell migration experiment verifies that the 2037 antibody molecule effectively inhibits the migration of tumor cells JVM-3 induced by CCR7 ligands, inhibiting the activity of IC 50 The parameters are shown in Table 3, indicating that the 2037 antibody molecule can effectively inhibit the migration of CCR7-positive cells.

[0158] Table 3. Antibody function blocking activity parameters

[0159]

[0160] Example 10 Antibody-dependent cell-mediated cytotoxicity (ADCC) experiment

[0161] The 2037 antibody molecule was modified to enhance the ADCC effect at the Fc end (the C-terminus of the VHH with an amino acid sequence such as SEQ ID NO: 5 was directly fused to the N-terminus of the Fc end with an amino acid sequence such as SEQ ID NO: 7) to form an enhanced 2037 antibody, and the ADCC effect was verified by co-incubation of tumor cells and effector cells. The specific experimental steps are as follows:

[0162] 10.1 JVM-3 cells were used as target cells and NK92 cells were used as effector cells.

[0163] NK92 cells were NK92-CD16a cell line (Huabo Biopharmaceuticals).

[0164] The DELFIA EuTDA Cytotoxicity Detection kit (PerkinElmer) was used to detect cytotoxicity. The experimental detection methods, control settings and data processing methods were all referred to the kit instructions. The target cell JVM-3 was cultured in RPMI 1640 medium supplemented with 10% FBS. NK92-CD16a cells were cultured in MEMα (ThermoFisher) medium supplemented with 400μg / mL G418. JVM-3 cells were collected by centrifugation, and the cell concentration was adjusted to 1E6 cells / mL using assay medium (phenol red-free RPMI1640 + 5% FBS). 2μLDELFIA BATDA reagent was added to each 1mL JVM-3 cell suspension for labeling, incubated at 37°C for 20 minutes, and mixed once every 10 minutes. The labeled JVM-3 cells were washed 4 times with PBS and resuspended in assay medium to adjust to 1E5 cells / mL. The antibody (Fc-terminal ADCC-enhanced 2037 antibody or positive control CAP-100) was adjusted to 40 nM using assay medium, and then diluted 5-fold to make a 4× serial gradient dilution of the antibody (10 -4 , 10 -2 , 10 0 , 10 2nM). Collect effector cells NK92-CD16a and adjust to 8E5 cells / mL using assay buffer (RPMI 1640+5% FBS+1xPen-Strep). Add 100 μL JVM-3 cell suspension (1E4 cells per well), 50 μL antibody diluent, and 50 μL NK92-CD16a cell suspension (4E4 cells per well) to the 96-well plate in sequence. Incubate in an incubator at 37°C for 2 hours. Centrifuge at 500g for 5 minutes to collect the supernatant. Transfer 25 μL of supernatant to a flat-bottomed detection plate, add 200 μL Eu-solution, shake at 250rpm at room temperature for 15 minutes, and then detect the fluorescence intensity on an instrument (EnVision, PerkinElmer). The %ADCC calculation formula is as follows:

[0165]

[0166] *Sample: the signal generated by co-incubation of target cells, effector cells, and test antibodies; Ts: the background signal brought by the target cells themselves; Tm: the signal released when all target cells are lysed.

[0167] The experimental results are detailed in Figure 5 In Part A, the parameters of the antibody tumor killing inhibitory activity are shown in Table 4, indicating that the Fc-terminal ADCC-enhanced 2037 antibody molecule can effectively kill tumor cells with NK92-CD16a as effector cells.

[0168] 10.2 JVM-3 cells were used as target cells and human PBMC cells were used as effector cells.

[0169] The day before the experiment, the target cell JVM-3 was adjusted to 5E5 cells / mL using RPMI 1640 complete medium supplemented with 10% FBS and 1x Pen-Strep, and cultured in a T25 culture flask at 37°C overnight in an incubator. The day before the experiment, cryopreserved PBMCs were resuscitated according to the recommended resuscitation process (Lot 2302020014, Rubai), and the cell density was adjusted to 1E6 cells / mL using RPMI 1640 complete medium, and cultured in a T25 culture flask at 37°C overnight in an incubator. On the day of the experiment, the target cell JVM-3 was collected by centrifugation at 300g for 5 minutes, resuspended in assay buffer (RPMI 1640+5% FBS+1xPen-Strep) and the cell concentration was adjusted to 5E5 cells / ml. The test antibody (Fc-terminal ADCC enhanced 2037 antibody or positive control CAP-100) was diluted to 8nM using assay buffer, and then diluted 5-fold to make a 4× serial gradient dilution of the antibody (0.0001, 0.001, 0.01, 0.1, 1, 10, 100nM). 25μL JVM-3 cell suspension (1.25E4 cells) and 25μL 4× antibody dilution were co-incubated in a 96-well plate well in an incubator for 30 minutes. The effector cells PBMC were collected by centrifugation at 300g for 10 minutes, resuspended in assay buffer and the cell concentration was adjusted to 1E7 cells / mL. 50μLPBMC cell suspension (5E5 cells) was added to the 96-well plate well where the co-incubation was completed in the previous step, and cultured in an incubator for 4 hours. After the culture, the cytotoxicity was detected using the Cytotoxicity LDHAssay Kit-WST (CK12, DOJINDO). OD was measured using a microplate reader (Infinite, Tecan) 490 The control settings and data processing methods required for the experiment are all referred to the kit instructions. The %ADCC calculation formula is as follows:

[0170]

[0171] *ER: signal generated by co-incubation of target cells, effector cells, and test antibodies; ESR: background signal brought by the effector cells themselves; TSR: background signal brought by the target cells themselves; TMR: signal released when all target cells are lysed.

[0172] The experimental results are detailed in Figure 5 In part B, the parameters of the antibody tumor killing inhibitory activity are shown in Table 4, indicating that the Fc-terminal ADCC-enhanced 2037 antibody molecule can effectively kill tumor cells with human PBMCs as effector cells.

[0173] Table 4. Parameters of antibodies in ADCC tumor killing experiments

[0174]

[0175]

[0176] Example 11 Pharmacokinetics of anti-CCR7 antibody after single-dose intravenous administration in C57 mice

[0177] A total of 2 groups of C57 mice (4-6 weeks, weighing less than 20g, purchased from Beijing Sibeifu; n=6, 3 mice were alternately blood-collected at different time points) received a single tail vein injection of 1 mg / kg or 10 mg / kg of the enhanced 2037 antibody molecule (VHH-Fc), and the solvent was normal saline. Mouse blood samples were collected at the following time points: before administration, 0.25 hours, 2 hours, 6 hours, 24 hours, 48 ​​hours, 96 hours, 168 hours, 240 hours, 336 hours, 432 hours and 504 hours. For detailed experimental results, see Figure 6 , the relevant pharmacokinetic parameters are shown in Table 5.

[0178] Table 5. Pharmacokinetic parameters of enhanced 2037 antibody molecules after intravenous administration at 1 mg / kg and 10 mg / kg

[0179] dose Tmax(hours) AUC(h*ng / mL) Cmax(ng / mL) <![CDATA[T 1 / 2 (hours)]]> 1mg / kg 0.25 1112705 19534 199.92 10mg / kg 0.25 12222291 201007 121.43

[0180] Example 12 Expression and purification of CCR7×CD3 bispecific antibody

[0181] One arm of the CCR7×CD3 bispecific antibody is the 2037 antibody Fc fusion protein sequence, and the other arm is the CD3 Fab-Fc fusion protein sequence. The CD3 Fab-Fc is from Mosunetuzumab-axgb (Lunsumio, Genentech, Inc.), the heavy chain of CD3 Fab-Fc is shown in SEQ ID NO: 23, and the light chain of CD3 Fab-Fc is shown in SEQ ID NO: 24. The CCR7×CD3 bispecific antibody is produced by knob-into-hole method.

[0182] The sequence gene of CCR7×CD3 bispecific antibody was synthesized by GenScript, and the plasmid vector was pcDNA3.4. After receiving the plasmid, the plasmid was amplified and then expressed with Expi293f cells (Thermo Fisher) at 37°C, 8% carbon dioxide concentration, and 110rpm. Using Expi293f medium (Thermo Fisher), when the cell density was 2E6 / mL and the viability was >95%, transfection was performed. After transfection, expression was performed for about 120 hours, the cell supernatant was collected, and the culture supernatant was neutralized with 1M Tris-HCl, pH 8.0, and then incubated with balanced Protein A beads at 4°C for 2 hours, beads were collected, washed with PBS, and then eluted with 0.1M glycine buffer pH3.0. After elution, the target protein used a desalting column to replace the protein buffer with PBS, and finally SDS-PAGE and SEC-HPLC purity analysis was performed. The high-purity protein (i.e., CCR7×CD3 specific antibody) was aliquoted and stored at -80°C.

[0183] Example 13 Functional Activity Determination of CCR7×CD3 Bispecific Antibody

[0184] The functional activity of the CCR7×CD3 bispecific antibody was evaluated by T cell-mediated cytotoxicity (TDCC) assay, in which JVM-3 cells were used as target cells and human PBMC cells were used as effector cells.

[0185] The day before the experiment, cryopreserved PBMCs (Lot#2303210035, Rubai) were revived according to the recommended resuscitation process, and the cell density was adjusted to 1E6 cells / mL using RPMI 1640 complete medium, and cultured in a T25 culture flask at 37°C overnight in an incubator. The target cell JVM-3 was adjusted to 5E5 cells / mL using RPMI 1640 complete medium supplemented with 10% FBS and 1x Pen-Strep, and cultured in a T25 culture flask at 37°C overnight in an incubator. On the day of the experiment, the target cell JVM-3 was collected by centrifugation at 300g for 5 minutes, resuspended in the experimental buffer (RPMI 1640+5% FBS) and the cell concentration was adjusted to 8E5 cells / ml. The CCR7xCD 3 bispecific antibody or negative control iso-hIgG (GenScript) was diluted to 80nM using the experimental buffer, and then diluted 10 times to prepare a 4× serial gradient dilution of the antibody (0.02, 0.2, 2, 20nM). Take 50μL JVM-3 cell suspension (4E4 cells) and 50μL 4× antibody dilution in a 96-well plate and incubate in an incubator for 30 minutes. Collect the effector cells PBMC by centrifugation at 300g for 10 minutes, resuspend in assay buffer and adjust the cell concentration to 8E6 cells / mL. Add 100μL PBMC cell suspension (8E5 cells) to the 96-well plate well where the co-incubation was completed in the previous step, and culture in an incubator for 24 hours. After the culture, use the Cytotoxicity LDH Assay Kit-WST (CK12, DOJINDO) to detect cytotoxicity. Use a microplate reader (Infinite, Tecan) to measure OD 490 The control settings and data processing methods required for the experiment are all referred to the kit instructions. The cytotoxicity (%) calculation formula is as follows:

[0186] *ER: signal generated by co-incubation of target cells, effector cells, and test antibodies; ESR: background signal brought by the effector cells themselves; TSR: background signal brought by the target cells themselves; TMR: signal released when all target cells are lysed.

[0187] The experimental results are detailed in Figure 7 This indicates that the CCR7×CD3 bispecific antibody constructed based on the 2037 antibody can effectively mediate T cells in PBMCs to kill CCR7-positive tumor cells JVM-3.

[0188] Sequence information:

[0189] VHH CDR1 (SEQ ID NO: 1):

[0190] INAMG

[0191] VHH CDR2(SEQ ID NO:2):

[0192] AIHSGGSTNYADSVKG

[0193] VHH CDR3(SEQ ID NO:3):

[0194] EKWDFELGPNPKIDRY

[0195] VHH(SEQ ID NO:4):

[0196] CAGGTGCAGCTGGTGGAATCCGGCGGAGGCCTCGTGCAGGCCGGCGGCAGCCTGAGGCTCTCCTGTGCCGCCAGCGGCAACATGTTTTCCATTAACGCCATGGGCTGGTACAGGCAGGTGAGCGGTAAACAGAGGGGGCTCGTGGCCGCCATCCACAGCGGCGGGTCCACTAATTATGCAGATAGTGTTAAGGGTCGCTTCACTATCTCCCGTGATAACGCCAAGAATACCGTCTATCTGCAGATGAATAGCCTCAAACCAGAGGATACCGCTGTTTACTACTGTGCTGCCGAGAAATGGGATTTTGAACTGGGCCCTAACCCCAAAATTGATCGGTACTGGGGTCAGGGTACTCAGGTGACTGTGAGCGCC

[0197] VHH(SEQ ID NO:5):

[0198] QVQLVESGGGLVQAGGSLRLSCAASGNMFSINAMGWYRQVSGKQRGLVAAIHSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEKWDFELGPNPKIDRYWGQGTQVTVSA

[0199] Fc(SEQ ID NO:6):

[0200] GAGCCCAAATCTGCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGGACGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCGAGGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

[0201] Fc(SEQ ID NO:7):

[0202] EPKSADKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0203] CD3 Fab-Fc VH(SEQ ID NO:9):

[0204] EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQGLEWIGWIYPGDGNTKYNEKFKGRATLTADTSTSTAYLELSSLRSEDTAVYYCARDSYSNYYFDYWGQG TLVTVSS

[0205] CD3 Fab-Fc VL(SEQ ID NO:10):

[0206] DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIK

[0207] 2037-Fc CH3(SEQ ID NO:11):

[0208] GQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0209] CD3 Fab-Fc CH3(SEQ ID NO:12):

[0210] GQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0211] 2037-Fc Fc(SEQ ID NO:13):

[0212] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0213] CD3 Fab-Fc Fc(SEQ ID NO:14):

[0214] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0215] H-CDR1(SEQ ID NO:15):

[0216] NYYIH

[0217] H-CDR2(SEQ ID NO:16):

[0218] WIYPGDGNTKYNEKFKG

[0219] H-CDR3(SEQ ID NO:17):

[0220] DSYSNYYFDY

[0221] L-CDR1(SEQ ID NO:18):

[0222] KSSQSLLNSRTRKNYLA

[0223] L-CDR2(SEQ ID NO:19):

[0224] WASTRES

[0225] L-CDR3(SEQ ID NO:20):

[0226] TQSFILRT

[0227] CD3 Fab-Fc CH(SEQ ID NO:21):

[0228] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0229] CD3 Fab-Fc CL(SEQ ID NO:22):

[0230] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0231] CD3 Fab-Fc H(SEQ ID NO:23):

[0232] EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQGLEWIGWIYPGDGNTKYNEKFKGRATLTADTSTSTAYLELSSLRSEDTAVYYCARDSYSNYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0233] CD3 Fab-Fc L(SEQ ID NO:24):

[0234] DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. A nanobody or an antigen-binding fragment thereof targeting CCR7, comprising a heavy chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3, characterized in that: CDR1 comprises the amino acid sequence shown in SEQ ID NO:1, CDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and CDR3 comprises the amino acid sequence shown in SEQ ID NO:

3.

2. The Nanobody or antigen-binding fragment thereof according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

5.

3. A CCR7 binder, characterized in that: The CCR7 binder comprises one, two or more Nanobodies or antigen-binding fragments thereof as claimed in claim 1 or 2; Preferably, the CCR7 binder further has any one of the following characteristics: 1) The CCR7 binder is a heavy chain antibody; for example, an alpaca heavy chain antibody; 2) The CCR7 binder is a single-chain antibody; 3) The CCR7 binder is a complete antibody; 4) The CCR7 binder is a fusion antibody; for example, an Fc fusion protein, a Fab fusion protein or a single-chain fusion protein; 5) The CCR7 binder is a bispecific antibody or a multispecific antibody; for example, the bispecific antibody is a CCR7×CD3 bispecific antibody; 6) the CCR7 binder is an antibody conjugate, such as an antibody drug conjugate; and, 7) The CCR7 binder is a chimeric antigen receptor.

4. The CCR7 binding substance according to claim 3, characterized in that The Fc in the Fc fusion protein is human IgGFc; Preferably, the amino acid sequence of the Fc is as shown in SEQ ID NO:7 or has at least 90%, 95%, 96% or 97% homology with the amino acid sequence shown in SEQ ID NO:7; and / or, the Fc is fused to the C-terminus of the heavy chain variable region, for example by direct fusion.

5. The CCR7 binding substance according to claim 3 or 4, characterized in that: One arm of the CCR7×CD3 bispecific antibody is the Fc fusion protein, and the other arm is the CD3 Fab-Fc fusion protein. Preferably, the CCR7×CD3 bispecific antibody is produced by a knob-into-hole method; Preferably: The amino acid sequence of the CD3Fab-Fc fusion protein and / or the Fc of the Fc fusion protein comprises, compared with SEQ ID NO:7, a difference in amino acid residues at one or more positions selected from position 151, position 153 and position 192, wherein the amino acid difference at position 151 may be T151W or T151S, the amino acid difference at position 153 may be L153A, and the amino acid difference at position 192 may be Y192V; preferably, the amino acid sequence of the Fc of the CD3Fab-Fc fusion protein comprises the difference of T151W compared with SEQ ID NO:7, and the amino acid sequence of the Fc of the Fc fusion protein comprises the differences of T151S, L153A and Y192V compared with SEQ ID NO:7, or, the amino acid sequence of the Fc of the Fc fusion protein comprises the difference of T151W compared with SEQ ID NO:7, and the amino acid sequence of the Fc of the CD3Fab-Fc fusion protein comprises the differences of T151S, L153A and Y192V compared with SEQ ID NO:

7. NO:7 contains differences of T151S, L153A and Y192V; and / or, The Fab segment of the CD3Fab-Fc fusion protein comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3, H-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 15, H-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 16, and H-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 17, the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, L-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 18, L-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 19, and L-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 20; Better: The amino acid sequence of Fc of the CD3Fab-Fc fusion protein is as shown in SEQ ID NO: 14, or has at least 90%, 95%, 96% or 97% homology to the amino acid sequence shown in SEQ ID NO: 14, and / or, the amino acid sequence of Fc of the Fc fusion protein is as shown in SEQ ID NO: 13, or has at least 90%, 95%, 96% or 97% homology to the amino acid sequence shown in SEQ ID NO: 13; or, the amino acid sequence of Fc of the Fc fusion protein is as shown in SEQ ID NO: 14, or has at least 90%, 95%, 96% or 97% homology to the amino acid sequence shown in SEQ ID NO: 14, and / or, the amino acid sequence of Fc of the CD3Fab-Fc fusion protein is as shown in SEQ ID NO: 13, or has at least 90%, 95%, 96% or 97% homology to the amino acid sequence shown in SEQ ID NO: 13; and / or, The amino acid sequence of the heavy chain variable region of the CD3Fab-Fc fusion protein is as shown in SEQ ID NO:9 or has at least 90% homology to the amino acid sequence shown in SEQ ID NO:9, and / or the amino acid sequence of the light chain variable region of the CD3Fab-Fc fusion protein is as shown in SEQ ID NO:10 or has at least 90% homology to the amino acid sequence shown in SEQ ID NO:

10.

6. An isolated nucleic acid, characterized in that The isolated nucleic acid has a sequence selected from any one of the following: (i) the coding sequence of the Nanobody or antigen-binding fragment thereof according to claim 1 or 2, or the CCR7 binder according to any one of claims 3 to 5; (ii) the complement of the coding sequence of (i); and (iii) a 15-50 bp fragment of any of the sequences in (i) or (ii), such as a primer; Preferably, the isolated nucleic acid comprises the nucleotide sequence shown in SEQ ID NO:4, and preferably further comprises the nucleotide sequence shown in SEQ ID NO:

6.

7. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the isolated nucleic acid of claim 6; Preferably, the recombinant expression vector is a plasmid, cosmid, phage or virus vector; the backbone of the plasmid is, for example, pcDNA3.

4.

8. A transformant, characterized in that: The transformant comprises the isolated nucleic acid of claim 6 or the recombinant expression vector of claim 7; Preferably, the host cell of the transformant is a prokaryotic cell or a eukaryotic cell; More preferably, the host cell is selected from insect cells, mammalian cells or other cells suitable for preparing nanobodies or antigen-binding fragments; the mammalian cells are, for example, HEK293 or CHO-K1 cells.

9. A method for preparing the nanobody or antigen-binding fragment thereof according to claim 1 or 2, or the CCR7 conjugate according to any one of claims 3 to 5, characterized in that: The method comprises culturing the transformant according to claim 8 to obtain a culture; Preferably, the Nanobody or antigen-binding fragment thereof or CCR7 binder is purified from the culture.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the Nanobody or antigen-binding fragment thereof as claimed in claim 1 or 2, the CCR7 binder as claimed in any one of claims 3 to 5, the isolated nucleic acid as claimed in claim 6, the recombinant expression vector as claimed in claim 7, or the transformant as claimed in claim 8; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

11. Use of a Nanobody or antigen-binding fragment thereof as claimed in claim 1 or 2, a CCR7 binder as claimed in any one of claims 3 to 5, an isolated nucleic acid as claimed in claim 6, a recombinant expression vector as claimed in claim 7, a transformant as claimed in claim 8 or a pharmaceutical composition as claimed in claim 10 in the preparation of a medicament for preventing and / or treating a disease or condition associated with CCR7 expression, wherein the disease or condition associated with CCR7 expression is preferably a tumor; Preferably, the tumor is tumor metastasis or malignant hematological tumor; More preferably, the malignant hematological tumor is chronic lymphocytic leukemia.

12. A CCR7 detection agent or kit, characterized in that: The CCR7 detection agent or kit comprises the nanobody or antigen-binding fragment as described in claim 1 or 2, the CCR7 binder as described in any one of claims 3-5, the isolated nucleic acid as described in claim 6, the recombinant expression vector as described in claim 7, the transformant as described in claim 8 or the pharmaceutical composition as described in claim 10.

13. Use of a nanobody or antigen-binding fragment as claimed in claim 1 or 2, a CCR7 binder as claimed in any one of claims 3 to 5, an isolated nucleic acid as claimed in claim 6, a recombinant expression vector as claimed in claim 7, a transformant as claimed in claim 8 or a pharmaceutical composition as claimed in claim 10 in the preparation of a diagnostic agent, a detection agent or a kit; Preferably, the diagnostic agent or detection agent is used to diagnose or detect a disease mediated by CCR7; and / or, the kit is used to detect CCR7 in a sample, evaluate the effect of drug treatment or diagnose cancer; More preferably, the disease mediated by CCR7 is a tumor, preferably tumor metastasis or a malignant hematological tumor, such as chronic lymphocytic leukemia.

14. A method for detecting CCR7, characterized in that: The method comprises contacting the Nanobody or antigen-binding fragment as described in claim 1 or 2 or the CCR7 binder as described in any one of claims 3 to 5 with a sample to be tested; Preferably, the detection is for non-diagnostic purposes.

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