Unnatural amino acid-modified anti-CCL5 nanobodies

Abstract

The present invention relates to the field of biomedicine, and in particular, to anti-CCL5 Nanobodies modified with non-natural amino acids, wherein one or more of positions 42, 81, and 86 of the Nanobodies are non-natural amino acids. The present invention also relates to bi-Nanobody constructs comprising the Nanobodies, nucleic acid molecules, vectors, and host cells encoding the same, and their use for the treatment of diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular, to anti-CCL5 nanobodies modified with non-natural amino acids, as well as double nanobody constructs comprising the same, nucleic acid molecules, vectors and host cells encoding the same, and their use in treating diseases. Background Art

[0002] CCL5 (CC motif chemokine ligand 5), also known as RANTES, is a chemokine that attracts and activates immune cells such as T cells, monocytes, and basophils. Antibodies targeting CCL5 can block the binding of CCL5 to its receptors (such as CCR1 and CCR5), thereby reducing the infiltration and activation of inflammatory cells and lowering the severity of the inflammatory response.

[0003] AIDS (Acquired Immunodeficiency Syndrome) is a serious immune system disorder caused by the human immunodeficiency virus (HIV), making it difficult for patients to fight infections and other diseases. Currently, approximately 40 million people are infected with HIV worldwide, and as of early 2022, the number of AIDS patients in my country was approximately 820,000. Current treatment primarily relies on antiretroviral therapy (ART), which uses a combination of antiviral drugs to suppress HIV replication and growth. CCL5, also known as RANTES (Regulated upon Activation, Normal T Cell Expressed and Secreted), belongs to the chemokine family. It plays a crucial role in HIV infection, attracting and activating T cells, monocytes, and other immune cells, participating in immune inflammatory responses. Research has shown that blocking the binding of CCL5 to its receptors (such as CCR5) can reduce HIV replication and spread within the host, thereby inhibiting viral pathology. Therefore, CCL5 antibodies or CCL5 receptor antagonists are considered as a possible new approach to treating AIDS (NCT01428986 Safety Study of Maraviroc's Effecton Human Osteoclasts; NCT03666871 CCR5-modified CD4+T Cells for HIV Infection).

[0004] Autoimmune diseases are disorders in which the immune system attacks its own tissues. Studies have shown that CCL5 plays a role in a variety of autoimmune diseases, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), inflammatory bowel disease, multiple sclerosis, psoriasis, ankylosing spondylitis, graft-versus-host disease, polyarticular juvenile idiopathic arthritis, Behçet's syndrome, and uveitis. By promoting the infiltration of immune cells and inflammatory responses, it may exacerbate the pathological process of the disease. Therefore, antibodies targeting CCL5 are considered potential biological agents for the treatment of autoimmune diseases (NCT00427934 Maraviroc in Rheumatoid Arthritis).

[0005] CCL5 can promote tumor growth, invasion, and metastasis in tumors. Therefore, antibodies targeting CCL5 may inhibit tumor growth and spread by blocking its effects in the tumor microenvironment. This makes antibodies targeting CCL5 a potential anti-tumor treatment strategy. Studies have shown that cancer forkhead box protein 3 (Cancer-FOXP3 or C-FOXP3) recruits regulatory T cells (Tregs) into solid tumors by upregulating CCL5, thereby promoting cancer immune evasion.

[0006] CCL5 also plays a role in the nervous system, participating in neuroinflammation and neurodegeneration. Some studies have suggested that targeting CCL5 may have a neuroprotective effect against neurodegenerative diseases such as Alzheimer's disease, although research in this area is still underway.

[0007] Currently available inhibitors targeting CCL5 are primarily small molecule compounds. Met-CCL5: This is a modified CCL5 molecule that can prevent it from binding to its receptor, thereby inhibiting its biological activity. Maraviroc: Although primarily used to treat HIV infection, Maraviroc has also been found to inhibit CCL5's receptor, CCR5, thereby affecting its signaling pathway. DAPTA: This is a CCL5 mimetic designed to competitively bind to the CCR5 receptor, preventing the actual CCL5 from binding to it, thereby reducing the inflammatory response. Plerixafor: It is primarily used to increase the migration of hematopoietic stem cells into the blood, but it can also indirectly affect the action of CCL5 by inhibiting the effect of CXCL12 (SDF-1) on the CCR5 receptor. Existing small molecule inhibitors have poor targeting and toxic side effects, limiting their clinical application. Nanobodies have excellent targeting and low immunogenicity. Developing antibody drugs with affinity to target CCL5 can effectively improve the targeting and safety of CCL5 inhibitors.

[0008] Nanobodies ( Nanobodies (VHH) are recombinant single-domain antibodies cloned from the variable region genes of heavy-chain antibodies. They are the smallest fragment known to date that can bind to antigens. In 1993, immunologist Hamers-Casterman first reported a new type of antibody from camelids - heavy-chain antibody (hcIgG) in Nature. Compared with traditional antibody IgG, hcIgG has no light chain and does not have the first constant CH1 domain in the heavy chain. It is composed only of the variable antigen-binding domain (VHH) and heavy chain CH2 and CH3. Nanobodies have the same structure as human immunoglobulin VH: 4 framework regions (FR1-4) and 3 complementary variable regions (CDR1-3). Antigen-antibody specific binding mainly depends on the CDR3 of nanobodies, while the antigen-antibody binding strength depends on CDR1 and CDR2. The average length of the CDR3 of nanobodies is 18 amino acids, which is much longer than the CDR3 length of human or mouse VH. The longer CDR3 also makes the structure of nanobodies more flexible, and after binding to the antigen, they can be fixed in a single structure. The disulfide bonds within the CDR region further enhance the stability of nanobodies. As a prominent representative of miniaturized antibodies, nanobodies are only 1 / 10 the size of full-length antibodies and possess excellent tissue penetration, structural stability, and resistance to high temperatures, pH, and osmotic pressure. Furthermore, VHHs have an amino acid sequence highly homologous to human VHs, making them less immunogenic and easier to humanize.

[0009] Bispecific / bivalent nanoantibodies can simultaneously recognize two different antigens or different epitopes of the same antigen, showing better specificity and lower off-target toxicity. They also show advantages such as dual-target signal blocking, low immune escape and low drug resistance during treatment. The traditional method of constructing bispecific nanoantibodies is to design the sequence through genetic engineering, and the corresponding bispecific nanoantibodies can be directly obtained through ordinary protein expression methods, but this method has considerable limitations. The N-terminus and C-terminus of each of the two different protein sequences are connected by a flexible peptide composed of GS repeating sequences, resulting in the possibility of a single overall sequence. The conformation of this bispecific nanoantibody often shows an adverse effect on the antigen-antibody binding affinity. This phenomenon may be due to the steric hindrance effect blocking the normal binding between the antigen and antibody.

[0010] Based on the method of site-specific insertion of non-natural amino acids, by introducing special chemical group reaction handles at artificially selected positions, the coupling of bispecific nanoantibodies can be carried out at any artificially selected site, and combined with computer-assisted prediction, bispecific nanoantibodies with better theoretical binding to antigens can be obtained. Summary of the Invention

[0011] After extensive research, the inventors of the present application have provided anti-CCL5 Nanobodies containing unnatural amino acids, and bi-Nanobody constructs comprising such Nanobodies, which have improved stability and affinity compared to unmodified Nanobodies. The following invention is thus provided.

[0012] Nanobodies

[0013] In a first aspect, the present invention provides a Nanobody that specifically binds to CCL5, comprising a CDR1 shown in SEQ ID NO: 2, a CDR2 shown in SEQ ID NO: 3, and a CDR3 shown in SEQ ID NO: 4, and comprising a substitution of an unnatural amino acid at one or more positions corresponding to positions 42, 81, and 86 of SEQ ID NO: 1; preferably, the CDR1, CDR2, and CDR3 are determined by the Kabat numbering system.

[0014] As used herein, the expression "the Nanobody at the positions corresponding to position 42, 81 and 86 of SEQ ID NO: 1" refers to the amino acid residues in the amino acid sequence of the Nanobody being compared that are at equivalent positions to amino acid residues 42, 81 and 86 of SEQ ID NO: 1 when the amino acid sequence of the Nanobody is optimally aligned with SEQ ID NO: 1, i.e. when the amino acid sequence of the Nanobody is aligned with SEQ ID NO: 1 to obtain the highest percentage identity.

[0015] Unless otherwise specified or clearly contradicted by the context, the meanings of other similar expressions herein shall be defined in a similar manner to the above.

[0016] In certain embodiments, one or more of positions 42, 81 and 86 of the Nanobody is an unnatural amino acid.

[0017] In certain embodiments, the Nanobody is a mutant of a wild-type Nanobody whose sequence is shown in SEQ ID NO: 1, and the Nanobody comprises a substitution of an unnatural amino acid at one or more positions 42, 81, or 86 compared to the sequence shown in SEQ ID NO: 1.

[0018] In certain embodiments, the non-natural amino acid contains one or more functional groups selected from the group consisting of a carbonyl group, a keto group, an aldehyde group, an azide group, an alkynyl group, an alkenyl group, and an amide group.

[0019] In certain embodiments, the unnatural amino acid is one or more selected from the group consisting of p-acetylphenylalanine (pAcF), p-azidopiperidinane (pAzF), acetylphenylalanine (pAF), and a cyclopropene derivative of lysine (CypK).

[0020] In certain embodiments, the unnatural amino acid is pAcF.

[0021] In certain embodiments, the Nanobody comprises any one sequence selected from SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: 20.

[0022] In a second aspect, the present invention provides a Nanobody that specifically binds to CCL5, wherein the Nanobody comprises any one sequence selected from SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 10, SEQ ID NO: 18, and SEQ ID NO: 12 and SEQ ID NO: 20.

[0023] Bis-nanobody constructs

[0024] In a third aspect, the present invention provides a double nanobody construct, which comprises a first VHH that specifically binds to a first antigen and a second VHH that specifically binds to a second antigen, wherein the first antigen is CCL5 and the second antigen is the same as or different from the first antigen; the first VHH is the nanobody described in the first or second aspect of the present invention.

[0025] In certain embodiments, the second antigen is CCL5, and the second VHH is the Nanobody described in the first or second aspect of the invention.

[0026] In certain embodiments, the first VHH and the second VHH are connected by a linker at the site of the non-natural amino acid substitution.

[0027] In certain embodiments, the amino acid substitution at any one of the positions corresponding to positions 42, 81, and 86 of SEQ ID NO: 1 of the first VHH is pAcF, and the amino acid substitution at any one of the positions corresponding to positions 42, 81, and 86 of the second VHH is pAcF.

[0028] In certain embodiments, the first VHH and the second VHH are connected by a linker at the site of substitution with pAcF.

[0029] In certain embodiments, the first VHH and the second VHH are linked by bicyclononyne and tetrazine.

[0030] In certain embodiments, the pAcF in the first VHH and the pAcF in the second VHH are linked via Bicyclononyne and Tetrazine.

[0031] In certain embodiments, the carbonyl group of pAcF in the first VHH and the amino group of pAcF in the second VHH are linked to the amino group of Bicyclononyne or Tetrazine via the following route 1:

[0032]

[0033] In certain embodiments, the cyclooctane group of the Bicyclononyne and the azide group of the Tetrazine are connected via the following route 2:

[0034]

[0035] In certain embodiments, (1) the first VHH comprises the sequence shown in SEQ ID NO: 8, and / or the second VHH comprises the sequence shown in SEQ ID NO: 12;

[0036] (2) the first VHH comprises the sequence shown in SEQ ID NO: 8, and / or the second VHH comprises the sequence shown in SEQ ID NO: 10; or

[0037] (3) the first VHH comprises the sequence shown in SEQ ID NO: 10, and / or the second VHH comprises the sequence shown in SEQ ID NO: 12;

[0038] (4) the first VHH comprises the sequence shown in SEQ ID NO: 16, and / or the second VHH comprises the sequence shown in SEQ ID NO: 20;

[0039] (5) the first VHH comprises the sequence shown in SEQ ID NO: 16, and / or the second VHH comprises the sequence shown in SEQ ID NO: 18; or

[0040] (6) The first VHH comprises the sequence shown in SEQ ID NO: 18, and / or the second VHH comprises the sequence shown in SEQ ID NO: 20.

[0041] In certain embodiments, (1) the first VHH comprises the sequence shown in SEQ ID NO: 8, and the second VHH comprises the sequence shown in SEQ ID NO: 12; or

[0042] (2) The first VHH comprises the sequence shown in SEQ ID NO: 16, and the second VHH comprises the sequence shown in SEQ ID NO: 20.

[0043] Preparation of nanobodies or diabody constructs

[0044] In a fourth aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the Nanobody of the first or second aspect of the invention, or the dual Nanobody construct of the third aspect of the invention; in the nucleotide sequence, the codon corresponding to the position replaced by the non-natural amino acid is TAG.

[0045] In certain embodiments, the isolated nucleic acid molecule comprises any one sequence selected from SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:11, SEQ ID NO:19, SEQ ID NO:13, and SEQ ID NO:21.

[0046] In a fifth aspect, the present invention provides a vector (eg, a cloning vector or an expression vector) comprising the isolated nucleic acid molecule of the fourth aspect of the present invention. In certain embodiments, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, or the like.

[0047] In a sixth aspect, the present invention provides a host cell comprising an isolated nucleic acid molecule or vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as Escherichia coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.).

[0048] In a seventh aspect, a method for preparing a Nanobody or a bis-Nanobody construct of the invention is provided, comprising:

[0049] - co-transfecting a host cell with the isolated nucleic acid molecule or vector according to the fourth aspect of the present invention and a vector encoding an amber codon suppressor tRNA and an aminoacyl-tRNA synthetase specific for an unnatural amino acid;

[0050] - cultivating the host cell in a culture medium containing the unnatural amino acid.

[0051] In certain embodiments, the unnatural amino acid is pAcF.

[0052] In certain embodiments, the aminoacyl-tRNA synthetase specific for an unnatural amino acid is a pAcF-specific aminoacyl-tRNA synthetase (eg, Mj-TyrRS).

[0053] Conjugate

[0054] In an eighth aspect, the present invention further provides a conjugate comprising the Nanobody according to the first or second aspect of the invention, or the double Nanobody construct according to the third aspect of the invention, and a coupling moiety.

[0055] In certain embodiments, the Nanobody or diabody construct of the invention is conjugated to said coupling moiety, optionally via a linker.

[0056] In certain embodiments, the coupling moiety is selected from a protein tag. Such protein tags are well known in the art, and examples thereof include, but are not limited to, His, Flag, GST, MBP, HA, Myc, GFP, or biotin, and those skilled in the art know how to select an appropriate protein tag (e.g., a purification tag, a detection tag, or a tracing tag) according to the desired purpose.

[0057] In certain embodiments, the coupling moiety is selected from a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent material (e.g., a chemiluminescent material) or biotin. The detectable label of the present invention can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means. Such labels are well known in the art, and examples thereof include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent materials (e.g., chemiluminescent materials, such as acridinium ester compounds), magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above-mentioned labels. In certain embodiments, such labels can be suitable for use in immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, detectable labels as described above can be attached to the Nanobodies or Bis-Nanobody constructs of the invention via linkers of varying lengths to reduce potential steric hindrance.

[0058] In certain embodiments, the conjugated moiety is selected from a therapeutic agent, such as an anti-tumor drug, an anti-inflammatory drug, or an immunosuppressant.

[0059] In certain embodiments, the conjugation moiety is selected from additional biologically active polypeptides.

[0060] In a ninth aspect, the present invention provides an antibody-drug conjugate comprising the nanobody of the first or second aspect of the invention, or the double nanobody construct of the third aspect of the invention, and a coupling portion; wherein the coupling portion is a cytotoxic drug, and the coupling portion is connected to the nanobody or double nanobody construct via a linker.

[0061] In certain embodiments, the linker is a non-cleavable linker (e.g., SMCC), a disulfide linker, a hydrazone linker, or a protease-cleavable linker. In certain embodiments, the protease-cleavable linker is selected from a cathepsin B substrate linker (e.g., a dipeptide linker Val-Cit, a dipeptide linker Val-Ala, or a tetrapeptide linker Gly-Gly-Phe-Gly), a pyrophosphodiester linker, a PEG linker, a β-glucuronidase substrate linker, a β-galactosidase substrate linker, or a sulfatase substrate linker.

[0062] In certain embodiments, the cytotoxic drug is selected from the group consisting of: paclitaxel, tubulysins, duostatins, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxydione anthrax, maytansine or an analog or derivative thereof, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin, calicheamicin or an analog or derivative thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, dacarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, cisplatin and other platinum derivatives (e.g., carboplatin), duocarmycin A, duocarmycin SA, CC-1065 (also known as rachelmycin) or analogs or derivatives of CC-1065), dolastatin, auristatin, pyrrolo[2,1-c][1,4]benzodiazepine Drugs (PDB), indole benzodiazepines (IGN) or its analogs, antibiotics (e.g., dactinomycin (formerly known as actinomycin), bleomycin, daunorubicin (formerly known as daunorubicin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)), anti-mitotic agents (e.g., microtubule targeting agents), such as diphtheria toxin and related molecules (e.g., diphtheria A chain and active fragments and hybrid molecules thereof), ricin (e.g., ricin A or deglycosylated ricin A chain toxin), cholera toxin, Shiga-like toxins (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, Pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin, saponin, modeccin, gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, pokeweed proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotonin, saponin inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin toxin, antimicrobial / lytic peptides (e.g., CLIP, Magainin 2, melittin, cecropin, and P18), ribonuclease (RNase), DNase I, Staphylococcus enterotoxin-A, and Pseudomonas endotoxin.

[0063] Pharmaceutical composition

[0064] In the tenth aspect, the present invention provides a pharmaceutical composition comprising the nanobody described in the first or second aspect of the invention, the double nanobody construct described in the third aspect of the invention, the isolated nucleic acid molecule described in the fourth aspect of the invention, the vector described in the fifth aspect of the invention, the host cell described in the sixth aspect of the invention, the antibody-drug conjugate described in the ninth aspect of the invention, or the conjugate described in the eighth aspect of the invention, and a pharmaceutically acceptable carrier and / or excipient.

[0065] In certain embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent.

[0066] In certain embodiments, the additional pharmaceutically active agent is an anti-tumor drug, an anti-inflammatory drug, or an immunosuppressant.

[0067] In certain embodiments, in the pharmaceutical composition, the Nanobody, bis-Nanobody construct, isolated nucleic acid molecule, vector, host cell, antibody-drug conjugate or conjugate of the invention and the additional pharmaceutically active agent may be provided as separate components or as mixed components. Thus, the Nanobody, bis-Nanobody construct, isolated nucleic acid molecule, vector, host cell, antibody-drug conjugate or conjugate of the invention and the additional pharmaceutically active agent may be administered simultaneously, separately or sequentially.

[0068] In certain embodiments, the pharmaceutically acceptable carrier and / or excipient may comprise a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0069] The pharmaceutical compositions of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of a Nanobody, a bis-Nanobody construct, an isolated nucleic acid molecule, a vector, a host cell, an antibody-drug conjugate, or a conjugate as described herein. A "prophylactically effective amount" is an amount sufficient to prevent, arrest, or delay the onset of a disease. A "therapeutically effective amount" is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently.

[0070] Therapeutic applications

[0071] In an eleventh aspect, the present invention provides a method for preventing and / or treating a disease associated with CCL5 in a subject, comprising administering to a subject in need thereof the Nanobody of the first or second aspect of the present invention, the bi-Nanobody construct of the third aspect of the present invention, the isolated nucleic acid molecule of the fourth aspect of the present invention, the vector of the fifth aspect of the present invention, the host cell of the sixth aspect of the present invention, the antibody-drug conjugate of the ninth aspect of the present invention, the conjugate of the eighth aspect of the present invention, or the pharmaceutical composition of the tenth aspect of the present invention. The present invention also relates to the use of the Nanobody, bi-Nanobody construct, isolated nucleic acid molecule, vector, host cell, antibody-drug conjugate, conjugate or pharmaceutical composition for the preparation of a medicament for preventing and / or treating a disease associated with CCL5 in a subject.

[0072] In certain embodiments, the CCL5-associated disease is characterized by elevated CCL5 expression and / or excessive CCL5 activity.

[0073] In certain embodiments, the disease associated with CCL5 is a solid tumor, leukemia, AIDS, Alzheimer's disease, an inflammatory disease, or an autoimmune disease.

[0074] In certain embodiments, the solid tumor is selected from the group consisting of breast cancer, prostate cancer, lung cancer, ovarian cancer, melanoma, colon cancer, pancreatic cancer, gastric cancer, liver cancer, cervical cancer, and lymphoma.

[0075] In certain embodiments, the inflammatory or autoimmune disease is selected from the group consisting of rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), inflammatory bowel disease, multiple sclerosis, psoriasis, ankylosing spondylitis, graft-versus-host disease, polyarticular juvenile idiopathic arthritis, Behcet's syndrome, and uveitis.

[0076] In certain embodiments, the subject is a mammal, such as a human.

[0077] In certain embodiments, the Nanobody, diabody construct, isolated nucleic acid molecule, vector, host cell, antibody-drug conjugate, conjugate or pharmaceutical composition is used alone or in combination with another pharmaceutically active agent (e.g., an anti-tumor drug, an anti-inflammatory drug or an immunosuppressant).

[0078] The nanobodies, bi-nanobody constructs, isolated nucleic acid molecules, vectors, host cells, antibody-drug conjugates, conjugates or pharmaceutical compositions of the invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use.

[0079] A preferred dosage form is an injection. Such an injection can be a sterile injectable solution. For example, a sterile injectable solution can be prepared by the following method: incorporating the necessary dose of the nanobody or bi-nanobody construct of the invention into an appropriate solvent, and optionally, simultaneously incorporating other desired ingredients (including but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonicity agents, preservatives, diluents, or any combination thereof), followed by filtration sterilization. In addition, the sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powder can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0080] The nanobodies of the invention, the bis-nanobody constructs, the isolated nucleic acid molecules, the vectors, the host cells, the antibody-drug conjugates, the conjugates or the pharmaceutical compositions of the invention can be administered by any suitable method known in the art, including but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powders, ointments or drops), or nasal routes. However, for many therapeutic uses, the preferred route / mode of administration is parenteral administration (e.g., intravenous or bolus, subcutaneous, intraperitoneal, intramuscular). It will be understood by the skilled person that the route and / or mode of administration will vary depending on the intended purpose. In certain embodiments, the nanobodies of the invention, the bis-nanobody constructs, the isolated nucleic acid molecules, the vectors, the host cells, the antibody-drug conjugates or the conjugates or the pharmaceutical compositions of the invention are administered by intravenous or bolus injection.

[0081] Detection Application

[0082] In a twelfth aspect, the present invention provides a method for detecting the presence or content of CCL5 in a sample, which comprises using the nanobody described in the first or second aspect of the invention, the double nanobody construct described in the third aspect of the invention, or the conjugate described in the eighth aspect of the invention.

[0083] In certain embodiments, the method is an immunological assay, such as immunoblotting, an enzyme immunoassay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

[0084] In certain embodiments, the conjugate for use in the methods comprises a Nanobody, a diabody construct of the invention and a detectable label.

[0085] In certain embodiments, the Nanobody or diabody construct used in the methods is detectably labeled.

[0086] In certain embodiments, the nanobody or bis-nanobody construct used in the method does not have a detectable label. Thus, the method may also include detecting the nanobody or bis-nanobody construct using other reagents (such as a second antibody) with a detectable label.

[0087] In certain embodiments, the method comprises the following steps:

[0088] (1) contacting the sample with a Nanobody, a bis-Nanobody construct or a conjugate of the invention;

[0089] (2) detecting the formation of a complex between the Nanobody, diabody construct or conjugate and the antigen or detecting the amount of the complex.

[0090] The formation of the complex indicates the presence of the antigen or a cell expressing the antigen;

[0091] Wherein, the antigen is CCL5.

[0092] The methods may be used for diagnostic purposes, or for non-diagnostic purposes (eg, the sample is a cell sample rather than a sample from a patient).

[0093] In certain embodiments, the method is used to diagnose whether a subject suffers from a disease associated with CCL5. In such embodiments, the method may further include a step of comparing the amount of CCL5 in a sample from the subject with a reference value. The reference value may be the level of CCL5 in a sample from a subject (e.g., a healthy control) that is known not to have a disease associated with CCL5 (also referred to as a "negative reference value"). For example, if the amount of CCL5 in a sample from the subject increases relative to a negative reference value, it indicates that the subject suffers from a disease associated with CCL5.

[0094] In certain embodiments, the CCL5-related disease is characterized by elevated CCL5 expression and / or excessive CCL5 activity. In certain embodiments, the CCL5-related disease is a solid tumor, AIDS, Alzheimer's disease, an inflammatory disease, or an autoimmune disease. In certain embodiments, the inflammatory disease or autoimmune disease is selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, psoriasis, ankylosing spondylitis, graft-versus-host disease, polyarticular juvenile idiopathic arthritis, Behcet's syndrome, and uveitis.

[0095] In certain embodiments, the sample can be selected from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue associated cells (i.e., free cells), tissue (e.g., surgically resected tumor tissue, biopsy tissue, or fine needle aspirate tissue), histological preparations, and the like.

[0096] In certain embodiments, the CCL5 is human CCL5.

[0097] In the thirteenth aspect, there is provided the use of the nanobody described in the first or second aspect of the invention, the double nanobody construct described in the third aspect of the invention, or the conjugate described in the eighth aspect of the invention in the preparation of a detection reagent, wherein the detection reagent is used to detect the presence or level of CCL5 in a sample, or to diagnose whether a subject has a disease related to CCL5.

[0098] In certain embodiments, the conjugate used to prepare a detection reagent comprises a Nanobody, a diabody construct of the invention and a detectable label.

[0099] In certain embodiments, the Nanobody or diabody construct used to prepare the detection reagent is detectably labeled.

[0100] In certain embodiments, the nanobody or bi-nanobody construct used to prepare the detection reagent does not carry a detectable label. In such embodiments, the detection reagent may further comprise other reagents (such as a second antibody) capable of detecting the nanobody or bi-nanobody construct of the invention.

[0101] Definition of terms

[0102] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, procedures in cell culture, molecular biology, biochemistry, nucleic acid chemistry, immunology, and the like used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0103] When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0104] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0105] As used herein, the term "CCL5 (CC motif chemokine ligand 5)" refers to a chemokine that is expressed and secreted by T cells, monocytes, NK cells, epithelial cells, fibroblasts, and platelets. CCL5 can attract and activate immune cells such as T cells, monocytes, and basophils. The sequence of CCL5 is well known to those skilled in the art (see, for example, NCBI database Gene ID: 6352).

[0106] As used herein, the term "nanobody" has a meaning generally understood by those skilled in the art and refers to an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region), typically derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or a shark antibody). Typically, a nanobody consists of four framework regions and three complementarity determining regions, with a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A nanobody may be truncated at the N-terminus or C-terminus so that it comprises only a portion of FR1 and / or FR4, or lacks one or two of those framework regions, as long as it substantially maintains antigen binding and specificity. Nanobodies are also referred to as single-domain antibodies (sdAbs), and the two are used interchangeably.

[0107] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. In nanobodies, there are three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given Nanobody, a person skilled in the art will easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003).

[0108] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0109] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its targeted antigen. The strength or affinity of a specific binding interaction can be expressed as the equilibrium dissociation constant (KD) of the interaction. In the present invention, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0110] The specific binding properties between two molecules can be measured using methods well known in the art. One method involves measuring the speed of formation and dissociation of antigen binding sites / antigen complexes. Both "association rate constant" (ka or kon) and "dissociation rate constant" (kdis or koff) can be calculated by concentration and the actual rate of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis / kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). KD, kon and kdis values ​​can be measured by any effective method. In certain embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR). In addition, the dissociation constant can also be measured using bioluminescence interferometry or Kinexa.

[0111] As used herein, the term "nucleic acid" can be any polymer comprising deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA and RNA, and its length is not particularly limited. For nucleic acids used to construct recombinant constructs, it is preferably DNA because DNA is more stable and easier to handle than RNA.

[0112] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0113] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells. A host cell may include a single cell or a cell population.

[0114] As used herein, the term "identity" refers to the match between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match). Typically, two sequences are compared when they are aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0115] The twenty conventional amino acids referred to herein are compiled according to conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0116] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like.

[0117] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease or condition or symptom (e.g., a disease associated with CCL5) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in the extent of the disease, stabilization (i.e., no longer worsening) of the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relief of symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" may also refer to prolonging survival compared to the expected survival if not receiving treatment.

[0118] Advantageous Effects of the Invention

[0119] The present invention provides mutants of anti-CCL5 nanobodies with improved affinity and stability, and obtains a double nanobody construct by coupling the mutants of the anti-CCL5 nanobodies with each other, which has better specificity and lower off-target toxicity and has important clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0120] Figure 1 : The specific location of the mutation site in the nanobody.

[0121] Figure 2 : Schematic diagram of the plasmid pultra-CNFRS structure.

[0122] Figures 3A-3C : Chemical formula, in which, Figure 3A is p-acetylphenylalanine (pAcF), Figure 3B Tetrazine (TET) is the linker. Figure 3C The linker is Bicyclononyne (BCN).

[0123] Figures 4A-4B : 10C8 mutant nanobody expression and purification results, wherein, Figure 4A This is the expression and purification of group 10C8-S86. The bands from left to right are: M: marker, 1: whole bacterial solution, 2: supernatant, 3: precipitate, 4: penetration peak, 5-13: purified components; Figure 4B These are the nanoantibodies in each group and the negative control group. The strips from left to right are M: marker, 1: 10C8-WT, 2-7: expression of three groups of 10C8 mutants and their respective negative controls.

[0124] Figures 5A-5C : Mass spectrometry detection of 10C8 nanobody wild type and mutant types, among which, Figure 5A It is 10C8-P42, Figure 5B It is 10C8-S86, Figure 5C It is 10C8-WT.

[0125] Figure 6 : Schematic diagram of the construction principle of the double nanobody construct.

[0126] Figures 7A-7B : Double 10C8 nanobody connection, wherein, Figure 7A This is the size exclusion chromatogram of the P42+S86 group. Figure 7BThis is the electrophoresis diagram of the P42+S86 group connection. The bands from left to right are: M: marker; 1-5: 10C8-P42, 10C8-P42-Bcn, 10C8-S86, 10C8-S86-Tet, 10C8-P42+10C8-S86.

[0127] Figures 8A-8B :Indirect ELISA was used to verify the affinity of 10C8 nanobody mutants, among which, Figure 8A This is the indirect ELISA curve. Figure 8B is the affinity data of each group.

[0128] Figures 9A-9B :Indirect ELISA verifies the affinity of bivalent 10C8 nanobody, among which, Figure 9A This is the indirect ELISA curve. Figure 9B is the affinity data of each group.

[0129] Sequence information

[0130] The information of the sequences involved in the present invention is described in the following table:

[0131] Table 1: Sequence description

[0132]

[0133]

[0134] Wherein, X represents pAcF. DETAILED DESCRIPTION

[0135] The invention will now be described in the following non-limiting examples.

[0136] Those skilled in the art will appreciate that the examples are provided to illustrate the present invention by way of example and are not intended to limit the scope of the invention. The experimental methods in the examples are conventional methods unless otherwise specified. Where specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional products.

[0137] Example 1: Expression and purification of nanobodies with non-natural amino acid insertion

[0138] The 3D structure of the nanoantibody 10C8 (anti-CCL5 nanoantibody, 10C8 VHH sequence is shown in SEQ ID NO: 1) was obtained by online modeling using Alphafold2, and the CDR region in the 10C8 nanoantibody was predicted through the CDR region online prediction website (http: / / aligncdr.labshare.cn / ). The wild-type nanoantibody 10C8 and the antigen CCL5 were subjected to MD simulations respectively. The 6-frame conformations after equilibrium of 10C8 were selected, and a certain amino acid in 10C8 was mutated to the non-natural amino acid pAcF. The 6-frame conformations were calculated using Rosetta Cartesian ddG to obtain the average Cartesian ddG of the 6-frame mutants, and ranked from low to high. The top-ranked mutants P42pAcF (SEQ ID NO: 8), Y81pAcF (SEQ ID NO: 10), and S86pAcF (SEQ ID NO: 12) were selected, and the three-dimensional structures of the specific positions of the mutation sites on 10C8 are shown as follows. Figure 1 shown.

[0139] When constructing the nanobody expression vector, the nucleotide sequences corresponding to the amino acids at the three mutation sites were replaced with TAG codons. The three mutant plasmids 10C8-P42, 10C8-Y81, and 10C8-S86 were synthesized by GeneWeizhi Biotechnology.

[0140] (1) Three groups of 10C8 mutant plasmids were combined with pULTRA-TSDV plasmid (in the plasmid pultra-CNFRS (as Figure 2 The archaeal tyrosyl-tRNA synthetase mutant (sequence see SEQ ID NO: 12 in CN202211225132.5) was inserted into the vector (as shown in the figure) and transformed into TransB competent cells (Quanshijin Company).

[0141] (2) Inoculate the bacterial solution into 5 mL of LB solution (lysogeny broth) containing 1:1000 Amp and 1:1000 spectinomycin at a ratio of 1:100 and culture overnight. The next day, add the bacterial solution to 200 mL of LB shake flask solution containing 1:1000 Amp and spectinomycin at a ratio of 1:100, and culture at 37°C and 220 rpm until the OD600 of the bacterial solution is 0.6-0.8. Add 200 μL of IPTG solution and 200 μL of pAcF (acetylphenylalanine) (as Figure 3A ) solution, and at the same time, only 200 μL IPTG (isopropyl-β-d-thiogalactoside) solution was added to the negative control group, and cultured at 16°C and 160 rpm for 16 h.

[0142] (3) Collect the cells by centrifugation at 4°C and 10,000 rpm for 10 min, discard the supernatant, and wash the sludge twice with PBS solution.

[0143] (4) The bacterial sludge was weighed and resuspended in PBS solution at a ratio of 1:10 to 1:15 (w / v). The sludge was placed in an ice water bath and ultrasonically disrupted using a cell ultrasonic disruptor. The setting conditions were 200 W, 35% power, 2 seconds of disruption and 3 seconds of rest, and the working time was 30 minutes.

[0144] (5) After ultrasonic disruption, centrifuge the bacterial suspension at 4°C and 10,000 rpm for 15 min. Aspirate the supernatant with a syringe and filter it through a 0.45 μm PES filter for later use.

[0145] (6) Purify the nanoantibody using AKTA prime plus protein purifier. The specific steps are as follows:

[0146] 1) Turn on the AKTA purification machine, open the SCG protein purification software on the computer, select the system flushing program, and use deionized water to clean the instrument pipeline.

[0147] 2) Install the HIS Trap™ prepacked column on the instrument and flush the column at a flow rate of 4 mL / min until the UV280 line and the conductivity line on the instrument are flat and close to zero. This requires approximately 50 mL of deionized water.

[0148] 3) Replace the input liquid from deionized water with 80 mM imidazole solution and flow it into the prepacked column at a flow rate of 4 mL / min for 8-10 column volumes to equilibrate the prepacked column.

[0149] 4) Load the supernatant prepared in the previous step and select an infusion pump for injection at a flow rate of 1 mL / min.

[0150] 5) After loading, flush the column with 80 mM imidazole solution at a flow rate of 1 mL / min for 10 min, then increase the flow rate to 4 mL / min until the UV280 line flattens.

[0151] 6) Set pump A to 80 mM imidazole solution and pump B to 500 mM imidazole solution. Set pump B from 0 to 100% and set the volume to 50 mL for gradient elution at a flow rate of 3 mL / min. Start collecting when the elution peak appears and continue collecting until the UV280 line flattens. Then, flush the line and column with deionized water until the line conductivity is essentially zero.

[0152] 7) Rinse the column and tubing with deionized water until the UV280 line and the conductivity line are flat and close to zero.

[0153] 8) Flush the system with 20% ethanol solution, flushing 50 mL to fill the tubing and chromatography column with 20% ethanol solution. Remove the HIS prepacked column, store it at 4°C, and turn off the instrument.

[0154] 9) Analyze the purified fractions by SDS-PAGE gel electrophoresis.

[0155] like Figures 4A-4B As shown, the molecular weight of 10C8 nanoantibody is about 15kDa. The purified protein band is close to it by comparison with the marker (scale ruler) in the electrophoresis result diagram. The mutant nanoantibodies are all successfully expressed, and no target protein is produced in the negative control group without adding pAcF. Based on this, the successful expression of the mutant 10C8 nanoantibody with the introduction of the unnatural amino acid pAcF is proved.

[0156] Example 2: Mass spectrometry validation of nanobodies inserted with unnatural amino acids

[0157] After the amino acid at the selected site on Nanobody 10C8 was mutated to pAcF, its molecular weight changed compared to wild-type 10C8. The molecular weight of the mutant 10C8 was detected by MALDI-TOF mass spectrometry to confirm the successful insertion of the unnatural amino acid. The detection steps are as follows:

[0158] (1) Concentrate the nanobody using an ultrafiltration tube (3000 MWCO), centrifuge at 4°C and 5000 rpm to 1 mL, add 9 mL of deionized water, and concentrate again to 1 mL. Repeat three times to replace the nanobody into deionized water.

[0159] (2) HCCA (α-cyano-4-hydroxycinnamic acid) was selected as the detection matrix with a matrix concentration of 10 mg / mL. The concentration of the nanoantibody was adjusted so that the molar ratio of the matrix to the nanoantibody was 1000:1. 1 μL of sample was mixed with 1 μL of matrix and spotted on the target well. After it was dried naturally, it was placed in the target well.

[0160] (3) Select the linear cation detection mode, the acceleration voltage is 15,000 V, the delay time is 300 ns, and 30 points are randomly selected in the instrument interface for sampling.

[0161] The obtained data were processed using Data Analysis 4.1 software to obtain the mass spectrometry peak graph corresponding to the nanobody.

[0162] In order to more accurately confirm the insertion of unnatural amino acids, the relative molecular weight of the obtained 10C8 nanobody was accurately measured using MALDI-TOF mass spectrometry. The molecular weight of pAcF was 207 Da. The mass spectrometry detection results of the 10C8 mutant and 10C8 wild type were compared to confirm the molecular weight change caused by the amino acid replacement. 10C8-P42 and 10C8-S86 were selected for mass spectrometry molecular weight detection. The mass spectrometry results are shown in the figure. Figures 5A-5C shown.

[0163] The theoretical molecular weight of 10C8-WT was 14.89 kDa, and the detected molecular weight was 14.938 kDa; the theoretical molecular weight of 10C8-P42 was 15.02 kDa, and the detected molecular weight was 15.015 kDa; and the theoretical molecular weight of 10C8-S86 was 14.95 kDa, and the detected molecular weight was 14.965 kDa. These data indicate that the molecular weights detected for the mutant nanobodies are consistent with the calculated theoretical molecular weights after replacing the original amino acids with pAcF, further demonstrating the successful introduction of unnatural amino acids into the 10C8 nanobody.

[0164] Example 3: Nanobody conjugation based on unnatural amino acids

[0165] Example 1 successfully obtained a 10C8 mutant nanobody with an unnatural amino acid pAcF introduced at a designated site. Two different groups of mutant monomers were selected to construct an anti-CCL5 double nanobody construct based on a pre-designed coupling strategy. The principle is as follows: Figure 6 As shown, the coupling strategy is as follows:

[0166] (1) Taking the construction of the 10C8-P42+10C8-S86 group as an example, the nanobody monomer purified in 4.1 was concentrated using an ultrafiltration tube (3000 MWCO), centrifuged at 4°C and 5000 rpm to 1 mL, and 9 mL of 50 mM sodium acetate buffer solution (pH = 4.5) was added, and then concentrated to 1 mL. This was repeated three times to replace the nanobody into a weakly acidic buffer environment.

[0167] (2) The concentrations of the two nanobodies were measured using a Nanodrop ultra-micro spectrophotometer (Thermo). The concentration was adjusted to 1 mg / mL using sodium acetate solution. One group was added with a linker, Bicyclononyne (BCN) (e.g. Figure 3C ), and the other group added the linker Tetrazine (TET) (such as Figure 3B ), the molar ratio of the linker added to the nanobody was 50:1, and the reaction was allowed to proceed at 30°C for 24h.

[0168] (3) After 24 h, the reaction solution in step 2 was added to an ultrafiltration tube, and the solution was ultrafiltered and replaced into a PBS solution (pH = 7.4) according to step 1. The concentrations of the two groups of nanobody-linker complexes after the reaction were measured using Nanodrop and the nanobody concentration was adjusted to 2 mg / mL. The two different complexes were mixed in equal volumes, and the mixed solution was placed at 30°C for reaction for 24 h.

[0169] (4) After 24 hours, the reaction solution was concentrated to 600-1000 μL using an ultrafiltration tube (10,000 MWCO) and the unconjugated nanobody monomers and small molecule linkers in the solution system were removed by size exclusion chromatography. The size exclusion purification steps are as follows:

[0170] 1) Turn on the machine, open the SCG protein purification software on the computer, select the system flushing program, and use deionized water to clean the instrument pipeline.

[0171] 2) Superdex TM A 75increase 10 / 300GL prepacked column was installed on the instrument. The flow rate was set to 0.4 mL / min, the upper pressure limit was set to 2.0 MPa, and 1 to 2 column volumes of deionized water were flowed through the tubing until the baseline was flat.

[0172] 3) Replace the solution with PBS solution and flow 1 to 2 column volumes of solution through the tubing until the UV280 line is flat. At the same time, use a 1 mL syringe to draw up the PBS solution and inject it into the sample loop injection port. Repeat the cleaning five times.

[0173] 4) Fill the sample volume to 1 mL with PBS solution and inject it into the sample loop from the sample loop injection port. Select the sample loop injection mode at the injection flow rate of 0.4 mL / min and the injection volume of 2 mL.

[0174] 5) When the instrument shows that the UV line is rising, start collecting samples and stop collecting when the first peak ends and the peak corresponding to the second monomer does not rise significantly.

[0175] 6) Replace the solution with deionized water and clean the tubing with 1 to 2 column volumes.

[0176] 7) Replace the solution with 20% ethanol, adjust the flow rate to 0.25 mL / min, and flow 30 mL of solution to protect the tubing and column. Remove the column and store at 4°C.

[0177] 8) Analyze the collected fractions by SDS-PAGE gel electrophoresis, and concentrate the bivalent nanobody into one tube using an ultrafiltration tube.

[0178] For the three mutant nanobodies obtained above, according to the connection strategy described above, a two-step click chemistry reaction was used to construct a double nanobody construct based on the mutants P42 and S86 with the best affinity. The size exclusion chromatogram and gel electrophoresis diagram are shown in Figure 2. Figures 7A-7B As shown, the double nanobody construct flows out first in the chromatography column, corresponding to the peak on the left in the chromatogram, and the peak on the right is the monovalent nanobody that flows out later. The P42+S86 group was verified by the peak diagram of size exclusion chromatography and the SDS-PAGE gel electrophoresis diagram, and finally the double nanobody construct after the two were coupled was obtained. The electrophoresis result diagram shows that the two nanobody monomers have a slight change in molecular weight after being connected to their respective linkers. The corresponding double nanobody construct band successfully appeared in the 5th lane, proving that the two different nanobody monomers were successfully coupled through the above two-step click reaction. This result also confirmed the feasibility of the nanobody coupling strategy based on non-natural amino acids, and established an experimental basis for the subsequent coupling between different types of nanoantibodies.

[0179] Example 4: ELISA test of nanobody binding affinity

[0180] Indirect ELISA was used to detect the changes in affinity of the mutated nanobodies and the constructed double nanobody constructs to the antigen CCL5.

[0181] Recombinant human RANTES protein (Active) (ab269212) (Abcam) was used as an antigen to perform ELISA testing of the nanobody. The testing steps are as follows:

[0182] (1) The antigen concentration was determined using Nandrop. The coating buffer was used to adjust the antigen concentration to 20 μg / mL. 100 μL was added to each well of a 96-well plate. Each test group was repeated three times in three columns. At the same time, a column of bovine serum albumin solution (BSA) of the same concentration was set as a negative control group. The 96-well plate was placed at 4°C overnight.

[0183] (2) Discard the coating solution, add 200 μL of PBST solution to each well, and wash three times, each time for 5 minutes.

[0184] (3) Add 100 μL of freshly prepared blocking solution to each well and transfer to a 37°C incubator for 1 hour.

[0185] (4) Discard the blocking solution and add 200 μL of PBST solution to each well. Wash three times, each time for 5 minutes.

[0186] (5) Prepare gradient concentrations of nanoantibodies, with the highest concentration of the nanoantibody monomer group being 100 μg / mL and the highest concentration of the double nanoantibody construct group being 400 μg / mL. Perform 4-fold gradient dilutions, with a total of 10 gradient concentrations. Set up a zero concentration control group (PBS) and incubate in a 37°C incubator for 2 h.

[0187] (6) Discard the liquid in the wells, add 200 μL of PBST solution to each well, and wash 5 times, each time for 5 minutes.

[0188] (7) HRP-labeled mouse anti-HIS monoclonal antibody was diluted 1:10,000, 100 μL was added to each well, and incubated in a 37°C incubator for 1 h.

[0189] (8) Discard the liquid in the wells, add 200 μL of PBST solution to each well, and wash 5 times, each time for 5 minutes.

[0190] (9) Add 100 μL of pre-prepared TMB colorimetric solution to each well and incubate at room temperature in the dark for 10–15 min.

[0191] (10) Add 100 μL of stop solution to each well and measure the OD450 value of each well using a microplate reader.

[0192] After confirming the successful introduction of pAcF into the nanoantibody, the affinity of the three groups of mutant 10C8 nanoantibodies obtained was tested by indirect ELISA to confirm that the introduction of pAcF would not hinder the binding between the nanoantibody and the antigen. BSA was used as the negative control of the antigen, the OD450 value measured in the final reaction was used as the vertical coordinate, and the gradient logarithmic concentration after antibody dilution was used as the horizontal coordinate. The S-shaped curve after fitting was drawn, and the corresponding EC50 value was calculated by Graphpad Prism software to obtain the affinity of antigen-antibody binding. Graphpad Prism 8.0 (Graphpad Software Inc., San Diego, CA, USA) and SPSS22.0 (IBM Corporation., Armonk, NY, USA) software were used to perform statistical analysis and graph drawing on the experimental data. One-way ANOVA was used to compare the differences between the groups, and when p < 0.05, it was considered statistically significant. The results are shown in the figure. Figures 8A-8B As shown, the horizontal axis is the molar concentration of the nanobody and the vertical axis is the OD450 absorbance.

[0193] BSA serves as a negative control. As the nanobody concentration increases, the OD450 values ​​in the BSA group do not significantly increase, demonstrating that each 10C8 mutant exhibits virtually no nonspecific binding. Analysis software calculated the affinity of each group: 10C8-WT 5.164nM; 10C8-P42 1.369nM; 10C8-S86 1.764nM; and 10C8-Y81 3.99nM. Some mutants exhibited improved affinities compared to the wild-type, demonstrating that the insertion of pAcF does not significantly impede antigen-antibody binding. This suggests that these mutant monomers can be used to construct bioactive, multivalent, or multispecific nanobodies.

[0194] The affinity of the obtained 10C8-P42+10C8-S86 dual-nanobody construct was tested by ELISA, and the experimental data were statistically analyzed and graphed using Graphpad Prism 8.0 (Graphpad Software Inc., San Diego, CA, USA) and SPSS22.0 (IBM Corporation., Armonk, NY, USA) software. One-way ANOVA was used to compare the differences between the groups, and p < 0.05 was considered statistically significant. The ELISA results are shown in the figure below. Figures 9A-9B As shown, the calculation results show that the affinity of the double nanobody construct is 0.574nM, which shows that the constructed double nanobody construct has a high affinity, further demonstrating the feasibility of the multivalent / multispecific nanobody coupling strategy based on non-natural amino acid coupling.

[0195] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A nanobody that specifically binds to CCL5, wherein: The sequence of the nanobody is shown in SEQ ID NO: 8 or SEQ ID NO:

16.

2. A double nanobody construct, which consists of a first VHH that specifically binds to a first antigen, a second VHH that specifically binds to a second antigen, and a linker connecting the first VHH and the second VHH; in, The first antigen is CCL5, and the first VHH is the Nanobody of claim 1; the second antigen is CCL5, and the sequence of the second VHH is shown in SEQ ID NO: 12 or SEQ ID NO: 20; The pAcF of the first VHH and the pAcF of the second VHH are connected via Bicyclononyne and Tetrazine.

3. The bi-nanobody construct of claim 2, wherein: (1) the sequence of the first VHH is shown in SEQ ID NO: 8, and the sequence of the second VHH is shown in SEQ ID NO: 12; or (2) The sequence of the first VHH is shown in SEQ ID NO: 16, and the sequence of the second VHH is shown in SEQ ID NO:

20.

4. An isolated nucleic acid molecule, which is a nucleotide sequence encoding the Nanobody according to claim 1; in said nucleotide sequence, the codon corresponding to the position substituted with the unnatural amino acid is TAG.

5. The isolated nucleic acid molecule of claim 4, wherein the sequence is shown in SEQ ID NO: 9 or SEQ ID NO:

17.

6. A vector comprising the isolated nucleic acid molecule of claim 4 or 5. The vector according to claim 6 , which is a cloning vector or an expression vector.

8. A host cell comprising the isolated nucleic acid molecule of claim 4 or 5, or the vector of claim 6 or 7.

9. A method for preparing the Nanobody of claim 1, comprising: - co-transfecting a host cell with the isolated nucleic acid molecule of claim 4 or 5 or the vector of claim 6 or 7 and a vector encoding an amber codon suppressor tRNA and an aminoacyl-tRNA synthetase specific for an unnatural amino acid; - cultivating the host cell in a culture medium containing the unnatural amino acid.

10. The method according to claim 9, wherein The unnatural amino acid is pAcF.

11. The method according to claim 10, wherein The aminoacyl-tRNA synthetase specific for unnatural amino acids is pAcF-specific aminoacyl-tRNA synthetase.

12. A conjugate consisting of the Nanobody of claim 1 or the bis-Nanobody construct of claim 2 or 3, and a coupling moiety; in, The coupling moiety is selected from a protein tag or a detectable label.

13. The conjugate according to claim 12, wherein The protein tag is a purification tag.

14. The conjugate according to claim 12, wherein The detectable label is an enzyme, a radionuclide, a fluorescent dye, a luminescent substance or biotin.

15. The conjugate according to claim 14, wherein The enzyme is horseradish peroxidase.

16. The conjugate according to claim 15, wherein The luminescent substance is a chemiluminescent substance.

17. A pharmaceutical composition comprising the Nanobody of claim 1, the diabody construct of claim 2 or 3, the isolated nucleic acid molecule of claim 4 or 5, the vector of claim 6 or 7, the host cell of claim 8, or the conjugate of any one of claims 12 to 16; and a pharmaceutically acceptable carrier and / or excipient.

18. The pharmaceutical composition of claim 17, further comprising an additional pharmaceutically active agent.

19. The pharmaceutical composition according to claim 18, wherein The additional pharmaceutically active agent is an anti-tumor drug, an anti-inflammatory drug or an immunosuppressant.

20. Use of the Nanobody of claim 1, the diabody construct of claim 2 or 3, the isolated nucleic acid molecule of claim 4 or 5, the vector of claim 6 or 7, the host cell of claim 8, the conjugate of any one of claims 12 to 16, or the pharmaceutical composition of any one of claims 17 to 19 for the preparation of a medicament for preventing and / or treating graft-versus-host disease in a subject.

21. The use according to claim 20, wherein The subject is a mammal.

22. The use according to claim 20, wherein The subject is a human.

23. The use according to claim 20, wherein The Nanobodies, diabody constructs, isolated nucleic acid molecules, vectors, host cells, conjugates or pharmaceutical compositions are used alone or in combination with another pharmaceutically active agent.

24. The use according to claim 23, wherein The additional pharmaceutically active agent is an anti-tumor drug, an anti-inflammatory drug or an immunosuppressant.

25. A method for detecting the presence of CCL5 in a sample or its level for non-diagnostic purposes, comprising the use of the Nanobody of claim 1, the diabody construct of claim 2 or 3, or the conjugate of any one of claims 12-16.

26. The method of claim 25, wherein The method is an immunological assay.

27. The method of claim 26, wherein The immunological detection is selected from immunoblotting, enzyme immunoassay, chemiluminescent immunoassay, fluorescent immunoassay or radioimmunoassay.

28. The method of claim 27, wherein The enzyme immunoassay is ELISA.

29. Use of the Nanobody according to claim 1, the bis-Nanobody construct according to claim 2 or 3, or the conjugate according to any one of claims 12 to 16 in the preparation of a detection reagent for detecting the presence or level of CCL5 in a sample, or in the preparation of a detection reagent for diagnosing whether a subject has a disease associated with CCL5.

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