A nanobody targeting CCR7 and its preparation and application

Abstract

The present invention discloses a nanobody targeting CCR7 and its preparation and application. The nanobody targeting CCR7 or its antigen-binding fragment comprises a heavy-chain variable region, and 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. The nanobody targeting CCR7 of the present invention can specifically bind to CCR7 on the surface of tumor cells and in tumor tissues, and effectively block the migration of tumor cells and the internal signal pathway in tumors.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, and particularly to a nanobody targeting CCR7, its preparation and application. Background Art

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

[0003] In the prior art, there have been blocking antibodies for treating tumor metastasis and malignant hematological tumors by targeting CCR7. For example, the monoclonal antibody CAP100 (WO2017025569A1, WO2020127509A1) developed by Catapult therapeutics and the ADC drug JBH492 (WO2018142322A1, WO2021220199A1, US20200216548A1) developed by Novartis. CAP100 is a monoclonal antibody obtained by immunizing mice with polypeptides and using hybridoma technology. However, CAP100 has a large molecular weight and insufficient tissue penetration. JBH492 is an ADC drug obtained by immunizing mice with VLPs, obtaining a monoclonal antibody by hybridoma technology and then conjugating a small molecule toxin. This drug requires a high expression level of the antigen to exert effective endocytosis, which may result in poor therapeutic effects for patients with low expression levels of the target. Summary of the Invention

[0004] To solve the problems in the prior art that the antibody targeting CCR7 has a large molecular weight, insufficient tissue penetration or a high requirement for the expression level of the target, the present invention provides a nanobody targeting CCR7, its preparation and application. The nanobody targeting CCR7 of the present invention can be used to treat tumor metastasis and malignant hematological tumors.

[0005] 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 targeting CCR7 or an antigen-binding fragment thereof, which comprises a heavy-chain variable region, and 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 as shown in SEQ ID NO:5.

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

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

[0010] The term "variable region" refers to the domain in the heavy chain of an antibody that is involved in antibody binding to an antigen. VH contains four conserved framework regions (FRs) and three complementary determining regions (CDRs). Among them, the term "complementary 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 CDR residues. VH contains 3 CDR regions: CDR1, CDR2 and CDR3. Each VH is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0011] As is known in the art, the variable region of a heavy chain consists of four framework regions (FRs) connected by three complementarity-determining regions (CDRs) (also known as hypervariable regions) and contributes to the formation of the antigen-binding site of an antibody. If variants of the variable region of interest are desired, particularly those 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 variable region of interest with the variable regions of other antibodies containing CDR1 and CDR2 sequences of the same canonical type as the variable region of interest.

[0012] As used interchangeably herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion") refers 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 comprising one, two or more of the nanobodies or antigen-binding fragments thereof described herein.

[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 full 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 a camelid 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 CCR7 and CD3 simultaneously and can be formed by an antibody targeting CCR7 and an antibody targeting CD3.

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

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

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

[0028] In some embodiments, the CCR7 antibody is a nanobody. The nanobodies involved in the present invention, no matter in any form of genetic engineering modification, including but not limited to the following modifications: small molecule structure modification, humanization, bispecific nanobody modification, bivalent or multivalent nanobody modification, conjugation with other structural proteins or chemical reagents, etc., are all within the protection scope 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, heteroconjugate antibodies, single-chain (ScFv), their mutants, fusion proteins containing antibody parts (such as domain antibodies), humanized antibodies, and any other modified configurations of immunoglobulin molecules containing the antigen recognition sites with the required specificities, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibodies can be of murine, rat, human, or any other origin (including chimeric or humanized antibodies).

[0029] In some embodiments, the CCR7 conjugate is a fusion antibody, such as an Fc fusion protein, and in a specific embodiment, it is a nanobody 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 antibody (HcAb) is a novel antibody molecule that exists in camels and sharks and lacks the light chain naturally, consisting only of the heavy chain. Compared with ordinary antibodies, although the light chain is missing, the heavy chain antibody still retains the ability to bind antigens.

[0031] Single-chain antibody (scFv) is an antibody formed by connecting the variable region of the heavy chain and the variable region of the light chain of an antibody through a short peptide (linker) of 15-20 amino acids.

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

[0033] A fusion antibody, also known as an antibody fusion protein (Ig fusion protein), refers to a product obtained by fusing an antibody fragment with other bioactive proteins using genetic engineering techniques. Due to the different fusion proteins, this 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 characteristics 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 refer to antibodies that can simultaneously bind to two or more different epitopes or antigens.

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

[0036] Antibody conjugation refers to linking an antibody to other molecules or a solid surface. By leveraging the high specificity of the antibody to recognize antigens on tumor or other diseased cells, drugs or other molecules can then be delivered into the cells to exert a therapeutic effect. Alternatively, the antibody can be immobilized on a specific substrate surface to enable stable and effective recognition and binding of specific antigens, thereby facilitating the detection of immune responses. Common antibody conjugates include antibody-drug conjugates (ADCs) and antibody-oligonucleotide (Ab-oligo) conjugates.

[0037] A chimeric antigen receptor (CAR) is an engineered transmembrane protein that combines the specificity of an antigen-specific antibody with the function of a T cell receptor. Generally, a CAR includes an extracellular domain, a transmembrane domain, and an intracellular domain. In exemplary aspects, the extracellular domain of the CAR includes an antigen recognition region.

[0038] Specific binding means that an antibody binds to an antigen or an epitope within the antigen with a higher affinity than to other antigens or epitopes. Standard procedures can be used to measure the affinity EC 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, a human IgG Fc.

[0040] In some embodiments, the Fc fusion protein is, for example, a nanobody targeting CCR7 fused with 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 with the amino acid sequence shown in SEQ ID NO:7.

[0042] In some specific embodiments, the Fc fusion is at the C-terminus of the heavy chain variable region.

[0043] In some specific embodiments, the Fc is directly fused at 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 by the knob-into-hole method.

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

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

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

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

[0050] In some embodiments, the Fab segment 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 the 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 with the amino acid sequence shown in SEQ ID NO:14, and / or, the amino acid sequence of the 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 with the amino acid sequence shown in SEQ ID NO:13.

[0052] In some specific embodiments, the amino acid sequence of the 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 with the amino acid sequence shown in SEQ ID NO:14, and / or, the amino acid sequence of the 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 with 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 with 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 with the amino acid sequence shown in SEQ ID NO:10. Sequences with at least 90% homology only involve changes in the framework region sequences in the variable region of the CD3 Fab segment and do not involve changes in the amino acid sequences of the CDR regions.

[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 with 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 with the amino acid sequence shown in SEQ ID NO: 22.

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

[0056] One aspect of 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 its antigen-binding fragment of the present invention or the CCR7 conjugate of the present invention;

[0058] (ii) The complementary sequence of the coding sequence in (i); and

[0059] (iii) A 15-50 bp fragment of any sequence 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 is known in the art, "nucleic acid" in the present invention refers to a nucleotide chain of any length and includes DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate capable of being incorporated into the chain by DNA or RNA polymerase.

[0064] One aspect of 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 expression of an mRNA, protein, polypeptide, or peptide by a host cell when the construct contains a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide and the vector is contacted with the cell under conditions sufficient for expression of the mRNA, protein, polypeptide, or peptide within the cell. The vectors of the present disclosure 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 nucleotide, including but not limited to the following DNA and RNA: which may be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and which may contain natural, non-natural or altered nucleotides. Suitable vectors include those designed for propagation 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 condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0068] One aspect of the present invention provides a transformant comprising the isolated nucleic acid 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 specific 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 cell is, for example, HEK293 or CHO-K1 cells.

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

[0073] An 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, transduction of retroviruses, viral transfection, gene editing (CRISPR-Cas system, ZFN system or TALEN system), transposons (Sleeping Beauty or PiggyBAC), gene gun, lipid-based transfection or other conventional techniques. In the case of protoplast fusion, the cells are cultivated in a medium and suitable activity is screened. Methods and conditions for culturing the resulting transfected cells and for recovering the resulting antibody molecules are known to those skilled in the art and can be varied or optimized based on the methods known from this specification and the prior art, depending on the specific expression vector and host cell used. Additionally, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of the transfected host cells. The markers can, for example, provide prototrophy to auxotrophic hosts, biocide resistance (e.g., antibiotics) or heavy metal (such as copper) resistance, etc. Selectable marker genes can be directly linked to the DNA sequence to be expressed or introduced into the same cells by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements can include splicing signals, as well as transcriptional promoters, enhancers and termination signals.

[0074] In one aspect of the present invention, there is provided a method for preparing the nanobody targeting CCR7 or its antigen-binding fragment or the CCR7 conjugate of the present invention, the method comprising culturing the transformant of the present invention to obtain a culture.

[0075] In some embodiments, the nanobody or its antigen-binding fragment or the CCR7 conjugate is purified from the culture.

[0076] In one aspect of the present invention, there is provided a pharmaceutical composition, the pharmaceutical composition comprising the nanobody or its antigen-binding fragment of the present invention, the CCR7 conjugate, 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 compositions or pharmaceutical formulations of the present invention comprise a suitable pharmaceutically acceptable carrier such as a pharmaceutical excipient, such as a pharmaceutical carrier and a pharmaceutical excipient known in the art, including buffers. As used herein, "pharmaceutically acceptable excipient" or "pharmaceutical excipient" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, absorption delaying agents, etc. The pharmaceutical carriers suitable for the present invention may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is the preferred carrier. Aqueous saline solutions and aqueous dextrose and glycerol solutions may also be used as liquid carriers, especially for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc. For the use and application of excipients, see also "Handbook of Pharmaceutical Excipients", 5th Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago. If desired, the composition may also contain small amounts of wetting agents or emulsifying agents, or pH buffering agents. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, etc. Oral formulations may contain standard pharmaceutical carriers and / or excipients, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin. The pharmaceutical formulations or pharmaceutical compositions described herein can be prepared by mixing an antibody or an antigen-binding fragment thereof of the present invention having 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 compositions or formulations of the present invention may also comprise more than one active ingredient, which is required for the particular indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other active ingredients, such as other antibodies, antiviral agents, small molecule drugs or immunomodulators, etc. The active ingredients are present in a suitable combination in an amount effective for the intended use. Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing an antibody or an antigen-binding fragment thereof of the present invention, the matrix being in the form of a shaped article, such as a film or a microcapsule.

[0079] The present invention provides, on the one hand: the use of the nanobody or antigen-binding fragment thereof, the CCR7 conjugate, the isolated nucleic acid, the recombinant expression vector, the transformant or the pharmaceutical composition of the present invention in the preparation of a drug for preventing and / or treating diseases or conditions related to CCR7 expression.

[0080] In some embodiments, the diseases or conditions related to CCR7 expression are tumors.

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

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

[0083] The present invention provides, on the one hand, a CCR7 detection agent or kit, which comprises the nanobody or antigen-binding fragment, the CCR7 conjugate, the isolated nucleic acid, the recombinant expression vector, the transformant or the pharmaceutical composition of the present invention.

[0084] The present invention provides, on the one hand: the use of the nanobody or antigen-binding fragment, the CCR7 conjugate, 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, detection agent or kit.

[0085] In some embodiments, the diagnostic agent or detection agent is used for diagnosing or detecting diseases mediated by CCR7.

[0086] In some embodiments, the kit is used for detecting CCR7 in a sample, evaluating the therapeutic effect of a drug or diagnosing cancer.

[0087] In some embodiments, the diseases mediated by CCR7 are tumors.

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

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

[0090] The present invention provides, on the one hand, a method for detecting CCR7, which comprises contacting the nanobody or antigen-binding fragment of the present invention or the CCR7 conjugate of the present invention with a sample to be tested.

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

[0092] In a preferred embodiment of the present invention, the method is for non-diagnostic and / or non-therapeutic purposes, such as detecting the presence or absence of CCR7 in a laboratory; or competing with other anti-CCR7 antibodies for binding to detect whether there is competition between the antibodies, i.e., 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 a disease, disorder or condition related to CCR7 expression, the method comprising administering to a subject in need an effective amount of the nanobody or antigen-binding fragment of the present invention, the CCR7 conjugate or the pharmaceutical composition of the present invention.

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

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

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

[0097] As used in the present invention, the term "effective amount" refers to the amount of a drug or agent that elicits a biological or pharmaceutical response of a tissue, system, animal or human being, such as that sought by a researcher or clinician. In addition, the term "therapeutically effective amount" means an amount that causes an improvement in the treatment, cure, prevention or alleviation of a disease, disorder or side effect, or a reduction in the rate of progression of a disease or condition, compared to a corresponding subject who has not received that amount. The term also includes within its scope an amount that effectively enhances normal physiological functions.

[0098] On the one hand, the present invention provides the 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 a disease, disorder or condition related to 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 malignant hematological tumor.

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

[0102] On the basis of conforming to the common general knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

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

[0104] The positive and progressive effects of the present invention are as follows: The single-domain antibody integrating the human IgG1 Fc terminus of the present invention can specifically bind to CCR7 on the surface of tumor cells and in tumor tissues, effectively blocking the migration of tumor cells and the internal signal pathway of tumors. It is a brand-new highly specific active antibody with the function of targeting CCR7 and anti-tumor activity.

[0105] The single-domain antibody targeting CCR7 of the present invention enhances the ADCC effect by fusing the Fc terminus to achieve the tumor killing effect. In in vitro experiments, compared with the competitor CAP100, its affinity and functional activity are equivalent or even more excellent.

[0106] The blocking antibody targeting CCR7 developed by the present invention is obtained by immunizing alpacas and screening from a phage library. The candidate molecule has a smaller molecular weight compared with the existing clinical drug CAP100 with the same target, has better tissue penetration, has good pharmacokinetic activity, and has equivalent or better therapeutic effects. Brief Description of the Drawings

[0107] Figure 1 For the FACS experiment to verify that the anti-CCR7 antibody 2037 can effectively bind to the CCR7-overexpressing stable cell line and the chronic lymphocytic leukemia tumor cell JVM-3; Part A, the binding verification of the antibody molecule to the 293T CCR7-overexpressing stable cell line under serial dilution; Part B, the binding verification of the antibody molecule to the CHO-K1 CCR7-overexpressing stable cell line under serial dilution; Part C, the binding verification of the antibody molecule to the tumor cell JVM-3 under serial dilution.

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

[0109] Figure 3 For the FACS experiment to verify the species cross-reactivity of the 2037 antibody; Part A, the affinity graph of the 2037 antibody molecule to the CCR7-expressing stable cell line of mice under serial dilution; Part B, the affinity graph of the 2037 antibody molecule to cynomolgus monkey PBMC under serial dilution.

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

[0111] Figure 5To determine the tumor killing effect of the 2037 antibody molecule by ADCC assay under the condition of co-incubation with tumor cells JVM-3 and effector cells (NK92-CD16a in part A and human PBMC in part B).

[0112] Figure 6 For the pharmacokinetics of the anti-CCR7 antibody after single-dose intravenous administration in C57 mice.

[0113] Figure 7 For the tumor killing effect of the CCR7xCD3 bispecific antibody under the condition of co-incubation with tumor cells JVM-3 and effector cells (human PBMC). Detailed implementation mode

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

[0115] 293T 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 from Catapult Therapeutics) was used as a positive control antibody.

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

[0119] The present invention uses a method of combining multiple antigens for animal immunization, enriches and screens through a phage immune library, obtains single-domain antibody molecules that specifically bind to CCR7-expressing cells, and fuses the single-domain antibody molecules targeting CCR7 in series with the Fc end of human IgG1 with enhanced ADCC effect to obtain the target antibody molecule.

[0120] Example 1 Antigen preparation

[0121] 1.1 DNA Preparation: Construct a plasmid with the NCBI nucleic acid ID NM_001838.4 (CCR7) using pcDNA3.1 as the vector. (The plasmid was produced by the supplier GenScript.)

[0122] 1.2 Peptide Preparation: A peptide segment consisting of the first 32 amino acids at the N-terminus of the CCR7 protein (NCBI protein ID NP_001829.1) (EDEVTDDYIGDNTTVDYTLFESLCSKKDVRNK, SEQ ID NO:8) with tyrosine sulfonation modification. (The peptide was produced by the supplier GenScript.)

[0123] 1.3 Stable Cell Line: A monoclonal cell line that stably expresses the modified CCR7 target protein using 293T cells as the host. (The cell line was constructed by the supplier Kangyuan Botech.)

[0124] 1.4 Expression and Purification of CCR7 (micelle) Protein:

[0125] Clone the human CCR7 DNA sequence (NM_001838.4) into the pFastBac vector (Thermo Fisher, #10360014) for expression in insect cells. Fuse the receptor CCR7 sequence with enhanced green fluorescent protein for monitoring expression, and then purify it with a 10-histidine tag at the C-terminus. In the construction, introduce the cleavage site of 3C protease for cleaving the C-terminal fluorescent protein and tag protein. The expression conditions of CCR7 are a temperature of 27 °C, using Sf900-III medium (Thermo Fisher). When the density of SF9 cells is 2E6 / mL, the viability > 97%, and the average diameter is 17 - 17.5 μM, according to different virus intensities, the infection volume (VOI) is 0.1% - 5%. After infection, express for about 72 hours, collect the cells, and store them at -80 °C. All purification steps of the CCR7 protein are carried out at 4 °C. Thaw the cells in a low-salt buffer containing 10 mM HEPES / NaOH pH7.5, 10 mM MgCl 2 , 20 mM KCl, and a complete protease inhibitor (Roche) (1 tablet / 100 mL), while stirring at 300 rpm. Disrupt the cell membrane through a dounce (homogenizer), separate by ultracentrifugation, wash once with the low-salt buffer, and use a high-osmotic high-salt buffer (10 mM HEPES / NaOH pH7.5, 10 mM MgCl 2, wash twice with 20 mM KCl, 1 M NaCl, Roche protease inhibitor tablets (1 / 100 mL) and once with low-salt buffer, resuspend and centrifuge repeatedly 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, thaw the membrane in water at room temperature, add 2 mg / mL iodoacetamide and complete protease inhibitor (Roche) (1 tablet / 50 mL), and incubate the membrane at 4 °C for 1 hour. Extract membrane proteins using 50 mM HEPES / NaOH pH 7.5, 300 mM NaCl, 20 mM imidazole / HCl pH 7.5, 1% (w / v) DDM, 0.2% (w / v) CHS and complete protease inhibitor (1 tablet / 50 mL) as the solvent, dissolve at a ratio of 1:1.75 (biomass to final volume), and stir the mixture at 4 °C and 600 rpm for 1 hour. Ultracentrifuge the mixture and separate the supernatant for affinity chromatography. Incubate the supernatant with pre-equilibrated Talon Superflow resin at 4 °C and 600 rpm for 1 hour, wash 5 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) after binding, and then elute 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, change the protein buffer to 25 mM HEPES / NaOH pH 7.5, 150 mM NaCl, 0.03 / 0.006% (w / v) DDM / CHS by size exclusion chromatography (SuperdexTM 200 Increase 10 / 300 GL), separate the high-purity protein, collect the purified protein, quickly freeze it in liquid nitrogen, and store it at -80 °C.

[0126] Assembly and purification of 1.5-nanometer phospholipid discs:

[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. Subsequently, add sodium cholate (cholic acid, sodium salt, Anatrace) with a final concentration of 16 mM to POPC. Mix micelle, MSP, and POPC together at a molar ratio of 1:8:480, and place the mixture on ice for 45 minutes to 1 hour. Subsequently, use pre-treated Bio-beads to remove the detergent, add it to the mixture at a ratio of 0.8 - 1 g / mL, rotate overnight at 4°C, let it stand the next morning to collect the supernatant, wash it 2 - 3 times with PBS, and collect the washing solution. Subsequently, use affinity chromatography to remove the empty ones. Mix the collected solution with Ni-NTA beads, incubate with rotation at 4°C for 1 hour, collect the beads, elute the miscellaneous proteins with the washing buffer (PBS + 20 mM imidazole), then add 3C enzyme to digest overnight on the column. The next day, collect the flow-through solution and the target protein eluted with the washing buffer, concentrate it, and separate the aggregates by size exclusion chromatography (SuperdexTM 200 Increase 10 / 300GL) and change the storage buffer (PBS). Collect the purified protein and quickly freeze it in liquid nitrogen, and store it at -80°C.

[0128] Example 2 Alpaca Immunization

[0129] Immunize 1 adult healthy alpaca (ABLINK) by means of multiple antigen combinations, including the fusion protein of the N-terminal polypeptide of CCR7 and keyhole limpet hemocyanin (KLH), the stable cell line expressing CCR7, the DNA of human CCR7, and nanolipoprotein discs. Immunize continuously five times at intervals of 3 weeks, and use the conventional method for subcutaneous multi-point injection (1x10 7 , protein 0.25 mg). Assist electroshock for immunization after DNA injection; when immunizing with protein, assist with Freund's adjuvant. Collect peripheral blood 1 week after each immunization for detection of immunization titer. After the immunization titer is stable, collect a large amount of blood, isolate PBMC, lyse it with Trizol (Invitrogen, 15596026), and freeze it at -80°C for storage.

[0130] Example 3 Construction of VHH Phage Display Library

[0131] Use Trizol reagent to extract total RNA from peripheral blood mononuclear cells (PBMC) separated. Use PrimeScript TMRT Master Mix kit was used to reverse transcribe total RNA into cDNA. The variable region of heavy-chain immunoglobulin (VHH) was amplified by nested polymerase chain reaction (PCR). In the first-step PCR, 1 μg of cDNA template was mixed with primers. The DNA products from the first-step PCR reaction were separated by agarose gel electrophoresis. IgG and VHH bands were obtained. After gel purification of the VHH band, the DNA products of the first-step PCR were used as templates in the second-step PCR. The second-round PCR was performed using VHH-specific primers. The second-round PCR products were gel purified and digested with enzymes, and then inserted into a phagemid plasmid which was internally optimized by modifying the stuffer gene 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 generate 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-phase and cell panning were performed using the polypeptides in 1.2 and cells overexpressing CCR7 (the stable cell line in 1.3), respectively. 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 added. For solid-phase panning, the polypeptide dissolved in PBS was incubated in an immunotube overnight at 4 °C. After negative selection by adding the blocked phage particles to a blank immunotube, the phage particles were then added to the immunotube incubated with the polypeptide for positive selection. Nonspecific phage was washed off with PBST, and the bound phage particles were eluted with 100 mM triethylamine (TEA), and the eluate was neutralized with 1 M Tris HCl (pH 7.4). Then, half of the eluate was used to infect exponentially growing Escherichia coli, and the remaining eluate was used to detect the output titer of the bound phage. For cell panning, after negative selection by adding the blocked phage particles to 293T blank cells, cells overexpressing CCR7 were added for positive selection. After washing the cells after positive selection with PBS, the washed cells were added to exponentially growing Escherichia coli for output titer determination. CCR7-specific clones were detected by phage fluorescence-activated cell sorting (FACS). Single output phage clones were grown in 96-well deep-well plates and rescued overnight with M13KO7 helper phage. 100 μL of phage supernatant was incubated with 293T blank cells or cells overexpressing CCR7 at 4 °C for one hour, washed twice, and then incubated with anti-M13 fluorescent secondary antibody (Progen 61497) at 4 °C for one hour. After washing twice, the fluorescence value was read on a BD FACS Canto II to identify CCR7-specific clones. Multiple antibodies were obtained, and the antibody with the best specificity was selected and named antibody 2037, and its antibody sequence was detected.

[0134] Example 5 Expression and Purification of Antibodies

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

[0136] Example 6 Affinity Determination of Antibody and Antigen

[0137] Using CHO-K1 cells as the host, a monoclonal stable transfected cell line overexpressing human CCR7 was obtained by stably expressing the modified CCR7 target protein.

[0138] Using fluorescence-activated cell sorting (FACS), 293T (the stable transfected cell line in 1.3), the monoclonal stable transfected cell line of CHO-K1 overexpressing human CCR7, and the tumor cell JVM-3 (CBP60618, Nanjing Kebai) were respectively cultured to the logarithmic growth phase. After discarding the medium, the cells were rinsed once 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 with medium containing 10% FBS, and the cells were collected. After centrifugation at 300g for 5 minutes, the medium was discarded, and the cells were resuspended with DPBS, and the cell density was counted by trypan blue staining. A 96-well plate was taken, and 100 μL of cell suspension containing 2E5 cells was added to each well. After centrifugation at 300g for 5 minutes, the DPBS was discarded, and 100 μL of 5% BSA was added to each well and blocked at 4°C for 1 hour. After centrifugation at 300g for 5 minutes for the blocked cells, the BSA was discarded. The cells were divided into two groups, one group was added with the 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 to resuspend the cells, and incubated at 4°C for 1 hour. After centrifugation at 300g for 5 minutes, the supernatant was discarded, and 100 μL of FACSbuffer (PBS containing 1% BSA) was added to each well and washed twice. 100 μL of the secondary antibody (Alexa 647 AffiniPure TMResuspend the cells with Goat Anti-Human IgG, Jackson#109-605-098), incubate at 4 °C in the dark for 1 hour. After centrifugation at 300 g for 5 minutes, discard the supernatant, and wash each well three times with 100 μL of FACS buffer. Finally, resuspend the cells in 100 μL of FACS buffer per well and read the fluorescence values on a BD FACS Canto II instrument. For details of the flow cytometry fluorescence detection of the antibody-cell binding, see Figure 1 , affinity EC 50 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 antibody with monoclonal cell lines and tumor cell lines overexpressing CCR7

[0140]

[0141] Example 7 Detection of antibody specificity

[0142] Using 293T cells as the host, stably transfected cell lines overexpressing human CCR1 (CCDS2737.1), CCR6 (CCDS5298.1), CCR9 (CCDS2732.1), CXCR4 (CCDS33295.1) and CXCR6 (CCDS2735.1) were constructed respectively. The specific method is 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 400 μL of transfection complex. Add 2 μg of plasmid to Opti-MEM, mix well and then add 2 μL of LTX reagent, mix again, and let stand at room temperature for 5 minutes. Add 10 μL of PLUS TMReagents, 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 the medium. Take a 6-well plate, add 2 mL of the cell suspension and the prepared transfection complex, mix well and place it in the incubator for further culture. The cells are cultured for another 24 hours. Take some cells to detect the transfection efficiency. When the remaining cells reach 50 - 60% confluence, add selective antibiotics to screen for cells expressing the resistance gene to obtain a stable transfected cell line. Use a monoclonal cell line overexpressing human CCR7 as a positive control cell. By flow cytometry fluorescence-activated cell sorting technology, detect the binding of the 2037 antibody to the above cells respectively. 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 the Antibody

[0146] Use the mouse CCR7 protein sequence (Gene ID: 12775) and overexpress mouse CCR7 in 293T cells.

[0147] Use the monoclonal stable transfected cell line overexpressing mouse CCR7 and cynomolgus monkey PBMC cells respectively. By flow cytometry fluorescence-activated cell sorting technology, detect the binding of the 2037 antibody and the positive control (CAP-100) antibody to the above cells respectively. See Figure 3 In part A of , the 2037 antibody molecule has a strong affinity for the stable transfected cell line expressing mouse CCR7 under the condition of concentration gradient dilution. In part B, the 2037 antibody molecule has a strong affinity for cynomolgus monkey-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 mouse and cynomolgus monkey-derived CCR7 proteins.

[0148] Table 2. Binding Affinity Parameters of the Antibody to Mouse and Cynomolgus Monkey CCR7

[0149]

[0150] Example 9 Determination of the Functional Activity of the Antibody

[0151] 9.1 NanoBiT-Based β-arrestin2 Recruitment Assay

[0152] Construction of 293FT-LgBiT-arrb2-CCR7-SmBiT cells: Using LTX transfected the pIREShyg3-LgBiT-arrb2 plasmid (ordered from GenScript) into 293FT cells. The specific transfection steps were the same as those in Example 7. After 48 hours of transfection, screening culture was carried out using a medium containing 150 μg / mL hygromycin to obtain cells stably expressing LgBiT-arrb2. The pIRESpuro3-CCR7-SmBiT plasmid (ordered from GenScript) was transfected into 293FT-LgBiT-arrb2 cells and screened using 1 μg / mL puromycin to obtain the 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 seed them in a 384-well detection plate coated with Poly-D-Lysine, and culture them overnight in a cell culture incubator at 37 °C. The next day, aspirate the complete medium in the detection plate, and then add 15 μL (2037 antibody or positive control antibody CAP-100) of antibody diluent (final concentrations of 0.1, 1, 10, 100, 1000, 10000 nM) to the detection wells. Incubate in a cell culture incubator for 30 minutes and then take it out and let it stand for 10 minutes to equilibrate to room temperature. Add 5 μL of detection solution ((Promega, Nano- Live Cell Detection Kit): Nano Live Cell Substrate + 95% LCS diluent) to the detection wells and then add 5 μL of 0.55 μM CCL19 diluent. Immediately read the Luminescence signal using a multifunctional microplate reader. The experimental results are shown in Figure 4 , Part A shows that based on NanoBiT verification, the 2037 antibody molecule effectively inhibits the recruitment of the downstream β-arrestin2 protein induced by the ligand of CCR7, and the inhibitory activity 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 intracellular cAMP change level was detected using the Lance Ultra cAMP kit (PerkinElmer Cat#TRF0263). The specific steps were as follows: CHO-K1-CCR7 cells in good growth state were taken. After discarding the culture medium, they were rinsed once with DPBS, and an appropriate amount of dissociation buffer was added to digest the cells into single-cell state at 37°C. The cells were collected by centrifugation at 275g for 5 minutes, the supernatant was discarded, and the cells were resuspended with HBSS and counted. Then centrifuged at 275g for 5 minutes again, and the HBSS was discarded. The cells were resuspended with stimulation buffer and the cell density was adjusted to 2E5 cells / mL. A white opaque 384-well plate was taken, and 5 μL of cell suspension and 2.5 μL of the antibody to be tested (the final concentration of the antibody was 0.1, 1, 10, 100, 1000, 10000 nM) were added to each well, 2.5 μL of CCL19 / forskolin (1.25 μL of 2 μM CCL19 and 1.25 μL of 32 μM forskolin). After incubation for 30 minutes, 5 μL of LEU-cAMP tracer and 5 μL of ULight-anti-cAMP were added to each well, and incubated at room temperature for 1 hour. The TR-FRET signal was detected using a multifunctional microplate reader. The experimental results are shown in 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 the ligand of CCR7, and the inhibition activity 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 experiment

[0157] 50 μL of JVM-3 cells in the logarithmic growth phase (4E6 cells / mL) were taken into a 96-well culture plate, mixed with 50 μL of gradient-diluted antibody diluent (final concentration 0.1, 1, 10, 100, 1000, 10000 nM), and incubated in a cell culture incubator for 30 minutes. After adding 600 μL of medium containing CCL19 to the lower chamber of the Transwell in a 24-well plate, the upper chamber was gently placed on the well plate. The incubated antibody and cell suspension were added to the upper chamber of the Transwell, with a volume of 100 μL per well, avoiding the presence of air bubbles on the liquid surface. The lid of the 24-well plate was covered, and after culturing in a cell culture incubator for 6 hours, the cells in the lower chamber were counted using a flow cytometer. The experimental results are shown in 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 the ligand of CCR7, and the inhibition activity 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) assay

[0161] The 2037 antibody molecule was modified by enhancing the ADCC effect on the Fc region (directly fusing the C-terminus of VHH with the amino acid sequence shown in SEQ ID NO: 5 to the N-terminus of the Fc region with the amino acid sequence shown in SEQ ID NO: 7) to form the enhanced 2037 antibody. The ADCC effect was verified by co-incubating tumor cells with effector cells. The specific experimental steps are as follows:

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

[0163] NK92 cells are the NK92-CD16a cell line (Huabo Biotech).

[0164] Use the DELFIA EuTDA Cytotoxicity Detection kit (PerkinElmer) to detect cytotoxicity. The experimental detection method, control settings, and data processing methods all refer to the kit instructions. Target cells JVM-3 are cultured in RPMI 1640 medium supplemented with 10% FBS. NK92-CD16a cells are cultured in MEMα (ThermoFisher) medium supplemented with 400 μg / mL G418. Centrifuge to collect JVM-3 cells, and adjust the cell concentration to 1E6 cells / mL using assay medium (phenol red-free RPMI1640 + 5% FBS). Add 2 μL of DELFIA BATDA reagent to each 1 mL of JVM-3 cell suspension for labeling, incubate at 37 °C for 20 minutes, and mix once every 10 minutes. The labeled JVM-3 cells are washed 4 times with PBS and resuspended in assay medium, adjusted to 1E5 cells / mL. Adjust the antibody (Fc-region ADCC-enhanced 2037 antibody or positive control CAP-100) to 40 nM using assay medium, and then make a 4× serial dilution series of the antibody by 5-fold dilution (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 + 1x Pen-Strep). Sequentially add 100 μL of JVM-3 cell suspension (1E4 cells per well), 50 μL of antibody diluent, and 50 μL of NK92-CD16a cell suspension (4E4 cells per well) into a 96-well plate. Incubate in an incubator at 37 °C for 2 hours. Centrifuge at 500 g for 5 minutes to collect the supernatant. Transfer 25 μL of the supernatant to a flat-bottom detection plate, add 200 μL of Eu-solution, shake at 250 rpm at room temperature for 15 minutes, and then detect the fluorescence intensity on the machine (EnVision, PerkinElmer). The % ADCC calculation formula is as follows:

[0165]

[0166] *Sample: The signal generated by co-incubating target cells, effector cells, and the test antibody; Ts: The background signal brought by the target cells themselves; Tm: The signal released under the condition of complete lysis of the target cells.

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

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

[0169] One day before the experiment, the target cells JVM-3 were adjusted to 5E5 cells / mL using RPMI 1640 complete medium supplemented with 10% FBS and 1x Pen-Strep, and cultured overnight at 37°C in a T25 culture flask in an incubator. One day before the experiment, the cryopreserved PBMC (Lot 2302020014, Rubei) was thawed according to the recommended thawing procedure, and the cell density was adjusted to 1E6 cells / mL using RPMI 1640 complete medium, and cultured overnight at 37°C in a T25 culture flask in an incubator. On the day of the experiment, the target cells JVM-3 were collected by centrifugation at 300g for 5 minutes, resuspended in assay buffer (RPMI 1640 + 5% FBS + 1x Pen-Strep), and the cell concentration was adjusted to 5E5 cells / ml. The test antibody (Fc-end ADCC-enhanced 2037 antibody or positive control CAP-100) was diluted to 8 nM using assay buffer, and then serially diluted 5-fold to prepare a 4× series of antibody dilution solutions (0.0001, 0.001, 0.01, 0.1, 1, 10, 100 nM). Take 25 μL of the JVM-3 cell suspension (1.25E4 cells) and 25 μL of the 4× antibody dilution solution and incubate them together in the wells of a 96-well plate 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. Add 50 μL of the PBMC cell suspension (5E5 cells) to the wells of the 96-well plate that had completed the co-incubation in the previous step, and place it in an incubator for 4 hours. After the incubation, the cytotoxicity was detected using the Cytotoxicity LDH Assay Kit-WST (CK12, DOJINDO). The OD was measured using a microplate reader (Infinite, Tecan). 490 . All the control settings required for the experiment and the data processing methods refer to the kit instructions. The formula for calculating %ADCC is as follows:

[0170]

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

[0172] For the experimental results, see Part B of Figure 5 . The antibody tumor killing inhibitory activity parameters are shown in Table 4, indicating that the Fc-end ADCC-enhanced 2037 antibody molecule can effectively kill tumor cells using human PBMC as effector cells.

[0173] Table 4. Parameters of the antibody in the ADCC tumor killing experiment

[0174]

[0175]

[0176] Example 11 Pharmacokinetic Performance of Anti-CCR7 Antibody after Single-Dose Intravenous Administration in C57 Mice

[0177] A total of 2 groups of C57 mice (4 - 6 weeks old, body weight less than 20 g, purchased from Beijing Speyford; n = 6, 3 mice were bled alternately at different time points) were respectively given 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 dosing, 0.25 hours, 2 hours, 6 hours, 24 hours, 48 hours, 96 hours, 168 hours, 240 hours, 336 hours, 432 hours, and 504 hours. The experimental results are shown in Figure 6 , and the relevant pharmacokinetic parameters are shown in Table 5.

[0178] Table 5 Pharmacokinetic Parameters of 1 mg / kg and 10 mg / kg Enhanced 2037 Antibody Molecule after Intravenous Administration

[0179] Dose Tmax (hours) AUC (h*ng / mL) Cmax (ng / mL) <![CDATA[T 1 / 2 (hours)]]> 1 mg / kg 0.25 1112705 19534 199.92 10 mg / 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. Among them, CD3 Fab-Fc is from Mosunetuzumab-axgb (Lunsumio, Genentech, Inc.), the heavy chain of CD3 Fab-Fc is shown as SEQ ID NO:23, and the light chain of CD3 Fab-Fc is shown as SEQ ID NO:24. The CCR7×CD3 bispecific antibody is produced by the knob-into-hole method.

[0182] The sequence gene of the CCR7×CD3 bispecific antibody was synthesized by GenScript Corporation. The plasmid vector was pcDNA3.4. After receiving the plasmid, plasmid amplification was carried out, and then expression was performed using Expi293f cells (Thermo Fisher Scientific) at a temperature of 37°C, a carbon dioxide concentration of 8%, and a speed of 110 rpm. Expi293f medium (Thermo Fisher Scientific) was used. When the cell density reached 2E6 / mL and the viability was >95%, transfection was carried out. After transfection, expression was carried out for approximately 120 hours, and the cell supernatant was collected. The medium supernatant was neutralized using 1M Tris-HCl, pH 8.0, and then incubated with pre-equilibrated Protein A beads at 4°C with rotation for 2 hours. The beads were collected, washed with PBS, and then eluted with 0.1M glycine buffer pH 3.0. After elution, the target protein was subjected to changing the protein buffer to PBS using a desalting column. Finally, SDS-PAGE and SEC-HPLC purity analysis were carried out, and the high-purity protein (i.e., the CCR7×CD3 specific antibody) was aliquoted and stored at -80°C.

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

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

[0185] One day before the experiment, thaw the cryopreserved PBMC (Lot#2303210035, Rubei) according to the recommended thawing procedure. Adjust the cell density to 1E6 cells / mL using RPMI 1640 complete medium, and culture overnight at 37°C in a T25 culture flask. Adjust the target cell JVM-3 to 5E5 cells / mL using RPMI 1640 complete medium supplemented with 10% FBS and 1x Pen-Strep, and culture overnight at 37°C in a T25 culture flask. On the day of the experiment, centrifuge the target cell JVM-3 at 300g for 5 minutes, resuspend it in the experimental buffer (RPMI 1640 + 5% FBS), and adjust the cell concentration to 8E5 cells / ml. Dilute the CCR7xCD3 bispecific antibody or the negative control iso-hIgG (GenScript) to 80 nM using the experimental buffer, and then make a 10-fold serial dilution to prepare a 4× series of antibody dilution solutions (0.02, 0.2, 2, 20 nM). Take 50 μL of the JVM-3 cell suspension (4E4 cells) and 50 μL of the 4× antibody dilution solution and incubate them together in a 96-well plate well for 30 minutes in an incubator. Centrifuge the effector cell PBMC at 300g for 10 minutes, resuspend it in the assay buffer, and adjust the cell concentration to 8E6 cells / mL. Add 100 μL of the PBMC cell suspension (8E5 cells) to the 96-well plate well that has completed the co-incubation in the previous step, and place it in the incubator for 24 hours. After the culture, use the Cytotoxicity LDH Assay Kit-WST (CK12, DOJINDO) to detect cytotoxicity. Measure the OD using a microplate reader (Infinite, Tecan). 490 For all control settings required for the experiment and the data processing method, refer to the kit instructions. The formula for calculating cytotoxicity (%) is as follows:

[0186] *ER: Signal generated by co-incubation of target cells, effector cells, and test antibody; ESR: Background signal brought by effector cells themselves; TSR: Background signal brought by target cells themselves; TMR: Signal released when target cells are completely lysed.

[0187] For the experimental results, see Figure 7 , indicating that the CCR7×CD3 bispecific antibody constructed based on the 2037 antibody can effectively mediate the killing of CCR7-positive tumor cells JVM-3 by T cells in PBMC.

[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 targeting CCR7, comprising a heavy chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3, characterized in that: The amino acid sequence of CDR1 is shown in SEQ ID NO:1, the amino acid sequence of CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

3.

2. The Nanobody 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 the Nanobody as claimed in claim 1 or 2; The CCR7 binder also has any one of the following characteristics: 1) The CCR7 binder is a heavy chain antibody; 2) the CCR7 binder is an Fc fusion protein; and, 3) The CCR7 binder is a CCR7×CD3 bispecific antibody.

4. The CCR7 binding substance according to claim 3, characterized in that The Fc in the Fc fusion protein is human IgGFc; the Fc is fused to the C-terminus of the heavy chain variable region.

5. The CCR7 binding substance according to claim 4, characterized in that The amino acid sequence of Fc 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.

6. The CCR7 binding substance according to claim 4, characterized in that The Fc was fused to the C-terminus of the heavy chain variable region by direct fusion.

7. The CCR7 binding substance according to any one of claims 3 to 6, 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.

8. The CCR7 binding substance according to claim 7, characterized in that The CCR7×CD3 bispecific antibody is produced by a knob-into-hole method.

9. The CCR7 binding substance according to claim 7, characterized in that: The amino acid sequence of the CD3 Fab-Fc fusion protein and / or the Fc of the Fc fusion protein comprises a difference in amino acid residues at one or more positions selected from position 151, position 153 and position 192 compared with SEQ ID NO:

7.

10. The CCR7 binding substance according to claim 9, characterized in that The amino acid sequence of Fc satisfies one or more of the following conditions compared with SEQ ID NO: 7: 1) The difference in amino acid position 151 is T151W or T151S, 2) The difference in amino acid at position 153 is L153A, and 3) The difference in amino acid at position 192 is Y192V.

11. The CCR7 binding substance according to claim 10, characterized in that The amino acid sequence of Fc of the CD3 Fab-Fc fusion protein comprises the difference of T151W compared with SEQ ID NO:7, 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; alternatively, the amino acid sequence of Fc of the Fc fusion protein comprises the difference of T151W compared with SEQ ID NO:7, 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.

12. The CCR7 binding substance according to claim 7, characterized in that The Fab segment 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, the amino acid sequence of H-CDR1 is shown in SEQ ID NO: 15, the amino acid sequence of H-CDR2 is shown in SEQ ID NO: 16, and the amino acid sequence of H-CDR3 is shown in SEQ ID NO: 17, the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, the amino acid sequence of L-CDR1 is shown in SEQ ID NO: 18, the amino acid sequence of L-CDR2 is shown in SEQ ID NO: 19, and the amino acid sequence of L-CDR3 is shown in SEQ ID NO:

20.

13. The CCR7 binding substance according to claim 12, characterized in that: 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 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 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; and / or, 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.

14. An isolated nucleic acid, characterized in that The isolated nucleic acid is selected from the sequence of any one of the following: (i) a Nanobody according to claim 1 or 2, or a coding sequence of a CCR7 binder according to any one of claims 3 to 13; and, (ii) The complement of the coding sequence.

15. The isolated nucleic acid of claim 14, comprising the nucleotide sequence shown in SEQ ID NO:

4.

16. The isolated nucleic acid of claim 15, further comprising the nucleotide sequence shown in SEQ ID NO:

6.

17. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the isolated nucleic acid according to any one of claims 14-16.

18. The recombinant expression vector according to claim 17, characterized in that The recombinant expression vector is a plasmid, cosmid, phage or virus vector.

19. The recombinant expression vector according to claim 18, characterized in that The backbone of the plasmid is pcDNA3.

4.

20. A transformant, characterized in that: The transformant comprises the isolated nucleic acid according to any one of claims 14 to 16 or the recombinant expression vector according to any one of claims 17 to 19.

21. The transformant according to claim 20, characterized in that The host cell of the transformant is a prokaryotic cell or a eukaryotic cell.

22. The transformant according to claim 21, characterized in that The host cell is an insect cell or a mammalian cell.

23. The transformant according to claim 22, characterized in that The mammalian cells are HEK293 or CHO-K1 cells.

24. A method for preparing the Nanobody according to claim 1 or 2, or the CCR7 binder according to any one of claims 3 to 13, characterized in that: The method comprises culturing the transformant according to any one of claims 20 to 23 to obtain a culture.

25. The method of claim 24, wherein: The Nanobody or CCR7 binder is purified from the culture.

26. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the Nanobody as described in claim 1 or 2, the CCR7 binder as described in any one of claims 3-13, the isolated nucleic acid as described in any one of claims 14-16, the recombinant expression vector as described in any one of claims 17-19, or the transformant as described in any one of claims 20-23.

27. The pharmaceutical composition according to claim 26, characterized in that The pharmaceutical composition also includes pharmaceutically acceptable excipients.

28. Use of a Nanobody as described in claim 1 or 2, a CCR7 binder as described in any one of claims 3-13, an isolated nucleic acid as described in any one of claims 14-16, a recombinant expression vector as described in any one of claims 17-19, a transformant as described in any one of claims 20-23, or a pharmaceutical composition as described in claim 26 or 27 in the preparation of a medicament for the prevention and / or treatment of chronic lymphocytic leukemia.

29. A CCR7 detection agent or kit, characterized in that: The CCR7 detection agent or kit comprises the Nanobody as described in claim 1 or 2 or the CCR7 binder as described in any one of claims 3-13.

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