Non-natural amino acid modified anti-PD-L1 nano antibody

Abstract

The invention relates to the field of biological medicine, in particular to an unnatural amino acid modified anti-PD-L1 nano antibody, and one or more sites of the 8th site, the 23rd site, the 26th site, the 70th site, the 71th site, the 77th site, the 85th site, the 87th site and the 119th site of the nano antibody are unnatural amino acids. The invention also relates to dual nanoantibody constructs comprising said nanoantibodies, nucleic acid molecules, vectors and host cells encoding them, and their use in the treatment of diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular, to an anti-PD-L1 nanobody modified with a non-natural amino acid, and a double nanobody construct comprising the same, nucleic acid molecules, vectors and host cells encoding the same, and their use for disease treatment. Background Art

[0002] PD-L1 (Programmed Death-Ligand 1) plays an important role in the immune system and is usually expressed by tumor cells or other somatic cells. PD-L1 can bind to the PD-1 receptor on the surface of T cells. This binding can inhibit the activity of T cells, thereby helping immune escape and suppressing immune responses. PD-L1 and its interaction with PD-1 are of great significance in cancer immunotherapy. The role of PD-L1 in tumors is mainly through its binding to the PD-1 (Programmed Cell Death Protein 1) receptor, regulating the response of the immune system, thereby helping tumors escape the body's immune attack. Anti-PD-L1 antibodies promote the immune system's attack on tumors through the immune activity of T cells.

[0003] The currently approved anti-PD-L1 antibody drugs mainly include Atezolizumab (Tecentriq): developed by Genentech, it has been approved for the treatment of various cancers, such as non-small cell lung cancer, breast cancer, bladder cancer, etc. Durvalumab (Imfinzi): developed by AstraZeneca, approved for the treatment of advanced non-small cell lung cancer and urothelial carcinoma. Avelumab (Bavencio): jointly developed by Pfizer and Merck KGaA, approved for the treatment of mucinous adenocarcinoma, urothelial carcinoma, etc. The main defects of existing antibody drugs are as follows: monoclonal drugs have poor tissue penetration ability, and the therapeutic effect on digestive system tumors such as colorectal cancer is limited. It is urgent to develop anti-PD-L1 nanoantibodies with small molecular weight and high affinity.

[0004] Nanobodies ( VHH) is a recombinant single-domain antibody cloned from the variable region gene of a heavy chain antibody. It is the smallest fragment known to bind to an antigen. In 1993, immunologist Hamers-Casterman first reported a new 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 a 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 binding strength of antigen-antibody depends on CDR1 and CDR2. The average length of 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, it can be fixed in a single structure. The disulfide bonds in the CDR region also further enhance the stability of nanobodies. As an outstanding representative of miniaturized antibodies, nanobodies are only 1 / 10 the size of full-length antibodies and have excellent tissue penetration, stable structure, high temperature resistance, pH resistance, and osmotic pressure resistance. In addition, VHH has an amino acid sequence that is highly homologous to human VHs, making it low in immunogenicity and easy to humanize.

[0005] Bispecific / bivalent nanobodies can simultaneously recognize two different antigens or different epitopes of the same antigen, showing better specificity and lower off-target toxicity. The chemical construction methods of traditional bispecific antibodies and ADC drugs mainly utilize the reactivity of lysine or cysteine ​​residues in antibodies. However, due to the presence of multiple reactive surface lysines in antibodies, lysine modification usually produces heterogeneous products. In contrast, the cysteine-based method is more selective, but due to the presence of multiple disulfide bonds in antibody molecules, the reaction is more complex and it is difficult to obtain a uniform and good target product. The traditional construction method of bispecific nanobodies is to design the sequence through genetic engineering methods, and the corresponding bispecific nanobodies 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 the bispecific nanobody 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 the antibody. Compared with the above methods, the chemical coupling method based on unnatural amino acids can be used as a simple, high-yield and general method to generate bispecific antibodies or ADC drugs of any design. Summary of the invention

[0006] The inventors of the present application have developed a method for screening amino acid modification sites of nanobodies, and based on the method provide anti-PD-L1 nanobodies containing non-natural amino acid substitutions, and double nanobody constructs comprising the nanobodies, which have improved stability and affinity compared to unmodified nanobodies. The following invention is thus provided.

[0007] Nanobodies

[0008] In a first aspect, the present invention provides a Nanobody that specifically binds to PD-L1, comprising CDR1 shown in SEQ ID NO:2, CDR2 shown in SEQ ID NO:3, and CDR3 shown in SEQ ID NO:28, and comprising a non-natural amino acid replacement at one or more positions corresponding to positions 77, 8, 23, 26, 70, 71, 85, 87, and 119 of SEQ ID NO:27.

[0009] As used herein, the expression "the Nanobody at the position corresponding to position 77, 8, 23, 26, 70, 71 , 85, 87, 119 of SEQ ID NO: 27" refers to the amino acid residues in the amino acid sequence of the Nanobody being compared that are at equivalent positions to the amino acid residues at position 77, 8, 23, 26, 70, 71 , 85, 87, 119 of SEQ ID NO: 27 when the amino acid sequence of the Nanobody is optimally aligned with SEQ ID NO: 27, i.e. when the amino acid sequence of the Nanobody is aligned with SEQ ID NO: 27 to obtain the highest percentage identity.

[0010] Unless otherwise specified or clearly contradicted by context, the meanings of the remaining similar expressions herein shall be defined in a similar manner to the above.

[0011] In certain embodiments, one or more of positions 77, 8, 23, 26, 70, 71, 85, 87, and 119 of the Nanobody is an unnatural amino acid.

[0012] In certain embodiments, the Nanobody is a mutant of a wild-type Nanobody with a sequence as shown in SEQ ID NO: 27, which comprises a substitution of an unnatural amino acid at one or more positions 77, 8, 23, 26, 70, 71, 85, 87, and 119 compared to the sequence shown in SEQ ID NO: 27.

[0013] 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.

[0014] In certain embodiments, the unnatural amino acid is one or more selected from the group consisting of p-acetylphenylalanine (pAcF), p-azidopiperidine (pAzF), acetylphenylalanine (pAF), and a cyclopropene derivative of lysine (CypK).

[0015] In certain embodiments, the unnatural amino acid is pAcF.

[0016] In certain embodiments, the Nanobody comprises any one sequence selected from SEQ ID NO:41, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:47.

[0017] In a second aspect, the present invention provides a nanobody that specifically binds to PD-L1, wherein the nanobody comprises any one sequence selected from SEQ ID NO:41, SEQ ID NO:101, SEQ ID NO:31, SEQ ID NO:91, SEQ ID NO:33, SEQ ID NO:93, SEQ ID NO:35, SEQ ID NO:95, SEQ ID NO:37, SEQ ID NO:97, SEQ ID NO:39, SEQ ID NO:99, SEQ ID NO:43, SEQ ID NO:103, SEQ ID NO:45, SEQ ID NO:105, SEQ ID NO:47 and SEQ ID NO:107.

[0018] In a third aspect, the present invention provides a Nanobody that specifically binds to PD-L1, which comprises CDR1 shown in SEQ ID NO:2, CDR2 shown in SEQ ID NO:3, and CDR3 shown in SEQ ID NO:4, and which comprises a non-natural amino acid replacement at one or more positions corresponding to positions 71, 8, 23, 26, 70, 74, 82, 85, and 96 of SEQ ID NO:1.

[0019] As used herein, the expression "the Nanobody at the position corresponding to position 71, 8, 23, 26, 70, 74, 82, 85, 96 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 the amino acid residues at position 71, 8, 23, 26, 70, 74, 82, 85, 96 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.

[0020] Unless otherwise specified or clearly contradicted by context, the meanings of the remaining similar expressions herein shall be defined in a similar manner to the above.

[0021] In certain embodiments, one or more of positions 71, 8, 23, 26, 70, 74, 82, 85, and 96 of the Nanobody is an unnatural amino acid.

[0022] In certain embodiments, the Nanobody is a mutant of a wild-type Nanobody whose sequence is shown in SEQ ID NO: 1, which comprises a substitution of an unnatural amino acid at one or more positions 71, 8, 23, 26, 70, 74, 82, 85, and 96 compared to the sequence shown in SEQ ID NO: 1.

[0023] 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.

[0024] In certain embodiments, the unnatural amino acid is one or more selected from the group consisting of p-acetylphenylalanine (pAcF), p-azidopiperidine (pAzF), acetylphenylalanine (pAF), and a cyclopropene derivative of lysine (CypK).

[0025] In certain embodiments, the unnatural amino acid is pAcF.

[0026] In certain embodiments, the Nanobody comprises any one sequence selected from SEQ ID NO: 17, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 and SEQ ID NO: 25.

[0027] In a fourth aspect, the present invention provides a nanobody that specifically binds to PD-L1, wherein the nanobody comprises any one of SEQ ID NO: 17, SEQ ID NO: 79, SEQ ID NO: 9, SEQ ID NO: 71, SEQ ID NO: 11, SEQ ID NO: 73, SEQ ID NO: 13, SEQ ID NO: 75, SEQ ID NO: 15, SEQ ID NO: 77, SEQ ID NO: 19, SEQ ID NO: 81, SEQ ID NO: 21, SEQ ID NO: 83, SEQ ID NO: 23, SEQ ID NO: 85, SEQ ID NO: 25 and SEQ ID NO: 87.

[0028] Bis-nanobody constructs

[0029] In a fifth aspect, the present invention provides a dual nanobody construct, comprising 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 PD-L1, 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, and the first VHH and the second VHH are connected by a linker at the site of the non-natural amino acid replacement.

[0030] In certain embodiments, the second antigen is PD-L1, and the second VHH is the Nanobody described in the third aspect or the fourth aspect of the invention.

[0031] 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.

[0032] In certain embodiments, the amino acid in any one of the positions corresponding to position 77, position 8, position 23, position 26, position 70, position 71, position 85, position 87, and position 119 of SEQ ID NO: 27 of the first VHH is replaced with pAcF, and the amino acid in any one of the positions corresponding to position 71, position 8, position 23, position 26, position 70, position 74, position 82, position 85, and position 96 of SEQ ID NO: 1 of the second VHH is replaced with pAcF.

[0033] In certain embodiments, the first VHH comprises any one of SEQ ID NO:41, SEQ ID NO:101, SEQ ID NO:31, SEQ ID NO:91, SEQ ID NO:33, SEQ ID NO:93, SEQ ID NO:35, SEQ ID NO:95, SEQ ID NO:37, SEQ ID NO:97, SEQ ID NO:39, SEQ ID NO:99, SEQ ID NO:43, SEQ ID NO:103, SEQ ID NO:45, SEQ ID NO:105, SEQ ID NO:47 and SEQ ID NO:107, and / or,

[0034] The second VHH comprises any one sequence selected from SEQ ID NO:17, SEQ ID NO:79, SEQ ID NO:9, SEQ ID NO:71, SEQ ID NO:11, SEQ ID NO:73, SEQ ID NO:13, SEQ ID NO:75, SEQ ID NO:15, SEQ ID NO:77, SEQ ID NO:19, SEQ ID NO:81, SEQ ID NO:21, SEQ ID NO:83, SEQ ID NO:23, SEQ ID NO:85, SEQ ID NO:25 and SEQ ID NO:87.

[0035] In certain embodiments, the amino acid substitution of the first VHH at the position corresponding to position 77 or 85 of SEQ ID NO: 27 is pAcF, and the amino acid substitution of the second VHH at the position corresponding to position 26 or 71 of SEQ ID NO: 1 is pAcF.

[0036] In certain embodiments, the first VHH comprises any one of SEQ ID NO:41, SEQ ID NO:101, SEQ ID NO:43 and SEQ ID NO:103, and / or the second VHH comprises any one of SEQ ID NO:13, SEQ ID NO:75, SEQ ID NO:17 and SEQ ID NO:79.

[0037] In certain embodiments, the first VHH and the second VHH are connected via a linker at the site replaced with pAcF.

[0038] In certain embodiments, the first VHH and the second VHH are linked by Bicyclononyne and Tetrazine.

[0039] In certain embodiments, the pAcF in the first VHH and the pAcF in the second VHH are linked by Bicyclononyne and Tetrazine.

[0040] In certain embodiments, the carbonyl group of pAcF in the first VHH and the amino group of pAcF in the second VHH are connected to the amino group of Bicyclononyne or Tetrazine via the following route 1:

[0041]

[0042] In certain embodiments, the cyclooctane group of the Bicyclononyne and the azide group of the Tetrazine are connected via the following route 2:

[0043]

[0044] In certain embodiments, (1) the first VHH comprises the sequence shown in SEQ ID NO:41, and the second VHH comprises the sequence shown in SEQ ID NO:17;

[0045] (2) the first VHH comprises the sequence shown in SEQ ID NO: 41, and the second VHH comprises the sequence shown in SEQ ID NO: 13;

[0046] (3) the first VHH comprises the sequence shown in SEQ ID NO: 43, and the second VHH comprises the sequence shown in SEQ ID NO: 13;

[0047] (4) the first VHH comprises the sequence shown in SEQ ID NO: 43, and the second VHH comprises the sequence shown in SEQ ID NO: 17;

[0048] (5) the first VHH comprises the sequence shown in SEQ ID NO: 101, and the second VHH comprises the sequence shown in SEQ ID NO: 79;

[0049] (6) the first VHH comprises the sequence shown in SEQ ID NO: 101, and the second VHH comprises the sequence shown in SEQ ID NO: 75;

[0050] (7) the first VHH comprises the sequence shown in SEQ ID NO: 103, and the second VHH comprises the sequence shown in SEQ ID NO: 75; or

[0051] (8) The first VHH comprises the sequence shown in SEQ ID NO: 103, and the second VHH comprises the sequence shown in SEQ ID NO: 79.

[0052] Preparation of nanobodies or diabody constructs

[0053] In a sixth aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the nanobody described in the first, second, third or fourth aspect of the invention or the double nanobody construct described in the fifth aspect of the invention; in the nucleotide sequence, the codon corresponding to the position replaced by a non-natural amino acid is TAG.

[0054] In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid selected from the group consisting of SEQ ID NO:32, SEQ ID NO:92, SEQ ID NO:34, SEQ ID NO:94, SEQ ID NO:36, SEQ ID NO:96, SEQ ID NO:38, SEQ ID NO:98, SEQ ID NO:40, SEQ ID NO:100, SEQ ID NO:42, SEQ ID NO:102, SEQ ID NO:44, SEQ ID NO:104, SEQ ID NO:46, SEQ ID NO:106, SEQ ID NO:48, SEQ ID NO:108, SEQ ID NO:10, SEQ ID NO:72, SEQ ID NO:12, SEQ ID NO:74, SEQ ID NO:14, SEQ ID NO:76, SEQ ID NO:16, SEQ ID NO:78, SEQ ID NO:18, SEQ ID NO:80, SEQ ID NO:20, SEQ ID NO:82, SEQ ID NO:22, SEQ ID NO:84, SEQ ID NO: Any one of SEQ ID NO:24, SEQ ID NO:86, SEQ ID NO:26 and SEQ ID NO:88.

[0055] In a seventh aspect, the present invention provides a vector (eg, a cloning vector or an expression vector) comprising the isolated nucleic acid molecule of the sixth aspect of the present invention. In certain embodiments, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, and the like.

[0056] In an eighth 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 (such as mammalian cells, such as mouse cells, human cells, etc.).

[0057] In a ninth aspect, a method for preparing the nanobody of the first, second, third or fourth aspect of the invention or the double nanobody construct of the fifth aspect of the invention is provided, comprising:

[0058] - co-transfecting a host cell with an isolated nucleic acid molecule or vector of the present invention and a vector encoding an amber codon suppressor tRNA and an aminoacyl-tRNA synthetase specific for an unnatural amino acid;

[0059] - cultivating the host cell in a medium containing the unnatural amino acid.

[0060] In certain embodiments, the unnatural amino acid is pAcF.

[0061] In certain embodiments, the aminoacyl-tRNA synthetase specific for the unnatural amino acid is a pAcF-specific aminoacyl-tRNA synthetase (eg, Mj-TyrRS).

[0062] Conjugate

[0063] In a tenth aspect, the present invention provides a conjugate comprising the Nanobody of the first, second, third or fourth aspect of the present invention or the double Nanobody construct of the fifth aspect of the present invention and a coupling portion.

[0064] In certain embodiments, the Nanobody or diabody construct of the invention is conjugated to said coupling moiety, optionally via a linker.

[0065] In certain embodiments, the coupling moiety is selected from a protein tag. Such protein tags are well known in the art, examples of which 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 a suitable protein tag (e.g., a purification tag, a detection tag, or a tracing tag) according to the desired purpose.

[0066] 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 material 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 labels. In certain embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, detectable labels as described above can be connected to the nanobody or bi-nanobody constructs of the invention by linkers of varying lengths to reduce potential steric hindrance.

[0067] In certain embodiments, the conjugated moiety is selected from a therapeutic agent, such as an anti-tumor drug.

[0068] In certain embodiments, the conjugation moiety is selected from another biologically active polypeptide.

[0069] In the eleventh aspect, the present invention provides an antibody-drug conjugate, which comprises the nanobody described in the first, second, third or fourth aspects of the present invention, or the double nanobody construct described in the fifth aspect of the present 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.

[0070] 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.

[0071] 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 of the class (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 its active fragments and hybrid molecules), 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, Phytolacca americana 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), ribonucleases (RNases), DNase I, Staphylococcus enterotoxin-A, and Pseudomonas endotoxin.

[0072] Pharmaceutical composition

[0073] In the twelfth aspect, the present invention provides a pharmaceutical composition, which contains the nanobody described in the first, second, third or fourth aspect of the present invention, the double nanobody construct described in the fifth aspect of the present invention, the isolated nucleic acid molecule described in the sixth aspect of the present invention, the vector described in the seventh aspect of the present invention, the host cell described in the eighth aspect of the present invention, the antibody-drug conjugate described in the eleventh aspect of the present invention, or the conjugate described in the tenth aspect of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0074] In certain embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent.

[0075] In certain embodiments, the additional pharmaceutically active agent is an anti-tumor drug.

[0076] In certain embodiments, in the pharmaceutical composition, the Nanobodies, bis-Nanobody constructs, isolated nucleic acid molecules, vectors, host cells, antibody-drug conjugates, or conjugates of the invention and the additional pharmaceutically active agent can be provided as separate components or as mixed components. Thus, the Nanobodies, bis-Nanobody constructs, isolated nucleic acid molecules, vectors, host cells, antibody-drug conjugates, or conjugates of the invention and the additional pharmaceutically active agent can be administered simultaneously, separately or sequentially.

[0077] 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 liquids are 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 buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0078] The pharmaceutical composition of the invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of a Nanobody, a bi-Nanobody construct, an isolated nucleic acid molecule, a vector, a host cell, an antibody-drug conjugate, or a conjugate of the invention. A "prophylactically effective amount" refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease. A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent a 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 simultaneously, etc.

[0079] Therapeutic applications

[0080] In the thirteenth aspect, the present invention provides a method for preventing and / or treating a disease associated with PD-L1 in a subject, comprising administering to a subject in need thereof the nanobody of the first aspect, the second aspect, the third aspect or the fourth aspect of the present invention, the double nanobody construct of the fifth aspect of the present invention, the isolated nucleic acid molecule of the sixth aspect of the present invention, the vector of the seventh aspect of the present invention, the host cell of the eighth aspect of the present invention, the antibody-drug conjugate of the eleventh aspect of the present invention, the conjugate of the tenth aspect of the present invention, or the pharmaceutical composition of the twelfth aspect of the present invention. The present invention also relates to the use of the nanobody, double nanobody construct, isolated nucleic acid molecule, vector, host cell, antibody-drug conjugate, conjugate or pharmaceutical composition for the preparation of a drug for preventing and / or treating a disease associated with PD-L1 in a subject.

[0081] In certain embodiments, the PD-L1-associated disease is characterized by elevated PD-L1 expression and / or excessive PD-L1 activity.

[0082] In certain embodiments, the disease associated with PD-L1 is a solid tumor.

[0083] In certain embodiments, the disease associated with PD-L1 is selected from: non-small cell lung cancer, small cell lung cancer, bladder cancer, triple-negative breast cancer, hepatocellular carcinoma, renal cell carcinoma, esophageal cancer, or Merkel cell carcinoma.

[0084] In certain embodiments, the subject is a mammal, such as a human.

[0085] In certain embodiments, the nanobody, bi-nanobody 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 (eg, an anti-tumor drug).

[0086] 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 injections, 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.

[0087] A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by the following method: incorporating the necessary dose of the nanobody or double nanobody construct of the present invention in an appropriate solvent, and optionally, incorporating other desired ingredients (including but not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration sterilization. In addition, sterile injection solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powders 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.

[0088] Nanobodies, bi-nanobody constructs, isolated nucleic acid molecules, vectors, host cells, antibody-drug conjugates, conjugates or pharmaceutical compositions of the invention can be administered by any suitable method known in the art, including but not limited to, oral, oral, 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). The skilled person will appreciate that the route and / or mode of administration will vary depending on the intended purpose. In certain embodiments, nanobodies, bi-nanobody constructs, isolated nucleic acid molecules, vectors, host cells, antibody-drug conjugates or conjugates or pharmaceutical compositions of the invention are administered by intravenous or bolus.

[0089] Detection Application

[0090] In a fourteenth aspect, the present invention provides a method for detecting the presence or content of PD-L1 in a sample, which comprises using the nanobody described in the first aspect, the second aspect, the third aspect or the fourth aspect of the present invention, the double nanobody construct described in the fifth aspect of the present invention, or the conjugate described in the tenth aspect of the present invention.

[0091] In certain embodiments, the method is an immunological assay, such as an immunoblot, an enzyme immunoassay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

[0092] In certain embodiments, the conjugate for use in the methods comprises a Nanobody, a diabody construct of the invention and a detectable label.

[0093] In certain embodiments, the Nanobody or diabody construct used in the methods is detectably labeled.

[0094] In certain embodiments, the nanobody or bi-nanobody construct used in the method does not carry a detectable label. Therefore, the method may also include the use of other reagents (such as a second antibody) with a detectable label to detect the nanobody or bi-nanobody construct of the invention.

[0095] In certain embodiments, the method comprises the following steps:

[0096] (1) contacting the sample with a Nanobody, a bis-Nanobody construct or a conjugate of the invention;

[0097] (2) detecting the formation of a complex between the Nanobody, the bis-Nanobody construct or the conjugate and the antigen or detecting the amount of the complex.

[0098] In certain embodiments, the formation of the complex indicates the presence of the antigen or a cell expressing the antigen.

[0099] In certain embodiments, the antigen is PD-L1.

[0100] In certain embodiments, the methods can be used for diagnostic purposes, or for non-diagnostic purposes (eg, the sample is a cell sample rather than a sample from a patient).

[0101] In certain embodiments, the method is used to diagnose whether a subject suffers from a disease associated with PD-L1. In such embodiments, the method may further include a step of comparing the amount of the PD-L1 in a sample from the subject with a reference value. The reference value may be the level of PD-L1 in a sample from a subject (e.g., a healthy control) known not to have a disease associated with PD-L1 (also referred to as a "negative reference value"). For example, if the amount of PD-L1 in a sample from the subject increases relative to a negative reference value, it indicates that the subject suffers from a disease associated with PD-L1.

[0102] In certain embodiments, the disease associated with PD-L1 is characterized by elevated PD-L1 expression and / or excessive PD-L1 activity. In certain embodiments, the disease associated with PD-L1 is a solid tumor. In certain embodiments, the disease associated with PD-L1 is selected from: non-small cell lung cancer, small cell lung cancer, bladder cancer, triple-negative breast cancer, hepatocellular carcinoma, renal cell carcinoma, esophageal cancer, or Merkel cell carcinoma.

[0103] 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, etc.

[0104] In certain embodiments, the PD-L1 is human PD-L1.

[0105] In the fifteenth aspect, there is provided the use of the nanobody described in the first, second, third or fourth aspect of the invention, the double nanobody construct described in the fifth aspect of the invention, or the conjugate described in the tenth aspect of the invention in the preparation of a detection reagent, wherein the detection reagent is used to detect the presence or level of PD-L1 in a sample, or to diagnose whether a subject has a disease related to PD-L1.

[0106] In certain embodiments, the conjugate used to prepare a detection reagent comprises a Nanobody, a diabody construct of the invention and a detectable label.

[0107] In certain embodiments, the Nanobody, diabody construct used to prepare the detection reagent is detectably labeled.

[0108] In certain embodiments, the nanobodies and bi-nanobody constructs used to prepare detection reagents do not carry a detectable label. In such embodiments, the detection reagents may further comprise other reagents (such as a second antibody) capable of detecting the nanobodies and bi-nanobody constructs of the invention.

[0109] Method for screening amino acid modification sites of nanobodies

[0110] In a sixteenth aspect, the present invention provides a method for screening amino acid modification sites of Nanobodies, comprising the following steps:

[0111] (1) obtaining three-dimensional structural data of the target Nanobody, the target antigen and the antigen-antibody complex of the target Nanobody; preferably, the three-dimensional structural data comprises the number of atoms, atom names, atom types, atomic coordinates, amino acid names, polypeptide chain lengths, and the number of polypeptide chains, for example, in the form of a protein PDB file;

[0112] (2) using the three-dimensional structure data of the Nanobody obtained in step (1), performing MD simulation on the Nanobody and the antigen-antibody complex to obtain 6-frame conformations of the Nanobody after MD simulation equilibrium (for example, presented in the form of a protein PDB file), and 1-frame conformation of the antigen-antibody complex MD simulation (for example, presented in the form of a protein PDB file);

[0113] (3) using Rosetta Cartesian ddG to calculate the 6-frame conformation of the Nanobody obtained in step (2), and at the same time mutating the amino acid at any site in the Nanobody (for example, replacing it with an unnatural amino acid pAcF), to obtain the average Cartesian ddG of the 6-frame Nanobody mutant, selecting mutants with negative results, and ranking them from low to high according to the size of the negative value, and selecting the Nanobody mutants ranked in the top 30%-50%;

[0114] (4) performing RMSD value, RMSF value, number of hydrogen bonds, and number of salt bridges detection on the Nanobody mutants screened in step (3), ranking the detection results of the RMSD value and RMSF value from high to low according to stability, and ranking the detection results of the number of hydrogen bonds and the number of salt bridges from high to low, and selecting the Nanobody mutants whose RMSD value, RMSF value, number of hydrogen bonds, and number of salt bridges are all ranked in the top 30%-50%;

[0115] (5) using Rosetta Flex ddG to calculate the three-dimensional structural data of the antigen-antibody complex obtained in step (1) and the one-frame conformation of the antigen-antibody complex obtained in step (2), mutating the amino acid at any site in the nanobody (for example, replacing it with an unnatural amino acid pAcF) to obtain the Flex ddG of the mutated antigen-antibody complex, and combining the Rosetta Cartesian ddG result of the nanobody mutant obtained in step (3) to select the nanobody mutant for which both the Rosetta Cartesian ddG and Rosetta Flex ddG results are negative, or selecting the nanobody mutant for which the Rosetta Cartesian ddG result is negative and the Rosetta Flex ddG result is within 1 kcal / mol;

[0116] (6) simultaneously selecting the Nanobody mutant obtained in step (4) and the Nanobody mutant obtained in step (5) to obtain the final screened Nanobody mutant, wherein the mutation site of the Nanobody mutant is the screened amino acid modification site.

[0117] In certain embodiments, the three-dimensional structural data of the target antigen, target nanobody, or the antigen-antibody complex of the target antigen and the target nanobody are derived from a protein database known in the art (e.g., RCSB crystal database), or obtained by computer modeling (e.g., Alphafold2 calculation). In certain embodiments, the three-dimensional structure of the antigen-antibody complex of the target antigen and the target nanobody is generated by rigid docking with ZDOCK.

[0118] In certain embodiments, the amino acid modification is an amino acid substitution, such as substitution with an unnatural amino acid. In certain embodiments, the unnatural amino acid is pAcF.

[0119] In certain embodiments, the amino acid modification site is located in a non-CDR region.

[0120] In certain embodiments, in step (3), before using Rosetta Cartesian ddG calculation, the calculation parameters of the unnatural amino acid are established in Rosetta software.

[0121] In certain embodiments, in step (5), the computational parameters for the unnatural amino acid are established in Rosetta software prior to calculation using Rosetta Cartesian ddG and Rosetta FlexddG.

[0122] In certain embodiments, the operational parameters for the unnatural amino acid include a parameter file, a library of rotators, and an unfolding energy.

[0123] In certain embodiments, the parameter file for the unnatural amino acid is obtained using the molfile2params_polymer.py script.

[0124] In certain embodiments, in Rosetta Flex ddG calculations, the talaris 2014 scoring function is used.

[0125] In certain embodiments, steps (1) and (2) are performed before steps (3) to (6). In certain embodiments, steps (3) and (4) are performed before step (5) or simultaneously with step (5). In certain embodiments, step (6) is performed after steps (1) to (5).

[0126] In certain embodiments, the method further comprises a biochemical experiment to verify the nanobody mutant obtained in step (6).

[0127] In certain embodiments, the biochemical experiment comprises an experiment to detect the stability of the Nanobody mutant, including but not limited to: enzyme-linked immunosorbent assay (ELISA), differential scanning fluorimetry, Western Blot, and Tm value detection.

[0128] In certain embodiments, the biochemical experiment comprises an experiment for detecting the affinity of the nanobody mutant to the antigen, including but not limited to: enzyme-linked immunosorbent assay (ELISA), immunoblotting (Western Blot), KD value detection, surface plasmon resonance (SPR) technology, and fluorescence resonance energy transfer (FRET) technology.

[0129] In certain embodiments, the biochemical experiment comprises an experiment to detect the expression amount of the Nanobody mutant, including but not limited to: enzyme-linked immunosorbent assay (ELISA), Western Blot, immunoprecipitation, immunohistochemistry, and mass spectrometry.

[0130] Definition of terms

[0131] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular biology, biochemistry, nucleic acid chemistry, immunology and other operating steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0132] When the terms "for example," "such as," "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."

[0133] 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) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0134] As used herein, the term "PD-L1 (Programmed Death-Ligand 1)" refers to programmed cell death-ligand 1, which is usually expressed by tumor cells or other somatic cells. PD-L1 can bind to the PD-1 receptor on the surface of T cells, and this binding can inhibit the activity of T cells, thereby helping immune escape and inhibiting immune response. The sequence of PD-L1 is well known to those skilled in the art (see, for example, NCBI Gene ID: 29126).

[0135] 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 monomer variable antibody domain (e.g., a single heavy chain variable region), usually derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or a shark antibody). Typically, a nanobody consists of 4 framework regions and 3 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 contains only part 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 (single-domainantibody, sdAb), and the two are used interchangeably.

[0136] 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, named CDR1, CDR2 and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as 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).

[0137] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0138] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it is directed. The strength or affinity of a specific binding interaction can be represented by 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.

[0139] The specific binding properties between two molecules can be measured using methods 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, surface plasmon resonance (SPR) can be used to measure the dissociation constant in Biacore. In addition, bioluminescence interferometry or Kinexa can be used to measure the dissociation constant.

[0140] As used herein, the term "nucleic acid" can be any polymer comprising deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA, RNA, and its length is not subject to any particular restrictions. For nucleic acids used to construct recombinant constructs, it is preferred that the nucleic acid is DNA, because DNA is more stable and easy to handle relative to RNA.

[0141] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are 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 (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. 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, papillomaviruses (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. In addition, the vector may also contain a replication initiation site.

[0142] 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, etc. Host cells may include a single cell or a cell population.

[0143] As used herein, the term "identity" is used to refer to the matching of sequences 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 a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by 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.

[0144] The compilation of the twenty conventional amino acids involved in this article follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR 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. And 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.

[0145] 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, e.g., 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, 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.

[0146] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or disorder or symptom (e.g., a disease associated with PD-L1) in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to the expected survival (if not receiving treatment).

[0147] Advantageous Effects of the Invention

[0148] The present invention provides a method for screening non-natural amino acid modification sites of nanobodies, which obtains mutants of nanobodies with improved affinity and stability by analyzing the changes in affinity and stability of nanobodies substituted with non-natural amino acids, and obtains a double nanobody construct by coupling the nanobodies substituted with non-natural amino acids obtained by screening, which has better specificity and lower off-target toxicity and has important clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0149] Figure 1: Flowchart for screening non-natural amino acid mutation sites in nanobodies.

[0150] Figure 2A-2B : The mutation sites of PV2 and PV3 for MD simulation, among which, Figure 2A It is the PV2 group, Figure 2B It is the PV3 group.

[0151] Figure 3A-3B : RMSD results of PV2 and PV3 during MD simulation, where: Figure 3A It is the PV2 group, Figure 3B It is the PV3 group.

[0152] Figures 4A-4B : RMSF results of PV2 and PV3 during MD simulation, where: Figure 4A It is the PV2 group, Figure 4B It is the PV3 group.

[0153] Figure 5A-5B : Calculation results of the number of hydrogen bonds of PV2 and PV3 during MD simulation, where Figure 5A It is the PV2 group, Figure 5B It is the PV3 group.

[0154] Figure 6 : The probability and number of stable salt bridges in the wild type and mutants of PV2 and PV3 in the balanced 1000-frame trajectory.

[0155] Figure 7 :Rosetta Cartesian ddG calculation results of PV2 and PV3 nanoantibodies.

[0156] Figure 8 :Schematic diagram of the plasmid pultra-CNFRS structure.

[0157] Figures 9A-9C : Chemical formula, in which, Fig. 9A is p-acetylphenylalanine (pAcF), Fig. 9B The linker is Tetrazine (TET), Fig. 9C It is a linking agent Bicyclononyne (BCN).

[0158] Figures 10A-10B : Electrophoresis diagram of PV2 and PV3 mutant proteins, among which, Fig. 10A It is a PV2 monomer. The bands from left to right are: M: marker; 1-10: PV2-WT, PV2-S8, PV2-C23, PV2-S26, PV2-I70, PV2-S71, PV2-N74, PV2-Q82, PV2-S85, PV2-C96; Fig. 10BIt is a PV3 monomer, and the bands from left to right are M: marker; 1-10: PV3-WT, PV3-S8, PV3-C23, PV3-S26, PV3-I70, PV3-S71, PV3-N77, PV3-S85, PV3-K87, and PV3-G119.

[0159] Figures 11A-11B : Tm values ​​of nanoantibodies in each group of PV2 and PV3 (data in the figure are n=5; where there was a significant difference compared with the respective WT groups, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001), where, Fig.11A It is the PV2 group, Fig. 11B It is the PV3 group.

[0160] Figures 12A-12D : PV2, PV3 nanobody affinity test results, among which Fig. 12A ELISA results of each mutant in the PV2 group; Fig. 12B These are the ELISA results of each mutant in the PV3 group; Fig. 12C This is the affinity data of each mutant in the PV2 group; Fig.12D Affinity data for each mutant in the PV3 group.

[0161] Fig.13 : Schematic diagram of the construction principle of the dual nanobody construct.

[0162] Figures 14A-14D :Size exclusion chromatograms of four groups of double antibody construction groups, among which, Fig.14A PV2-S26+PV3-N77; Fig. 14B PV2-S71+PV3-N77; Fig. 14C PV2-S26+PV3-S85; Fig.14D It is PV2-S71+PV3-S85.

[0163] Figures 15A-15E : Evaluation of PV2-S71+PV3-N77 dual antibodies, among which, Fig.15A The mass spectrometry verification results of PV2-S71+PV3-N77; Fig. 15B This is the SDS-PAGE run result of PV2-S71+PV3-N77; Fig. 15C It is the thermal stability determination of PV2-S71+PV3-N77; Fig.15D This is the PV2-S71+PV3-N77 ELISA result; Fig.15E Affinity data for PV2-S71+PV3-N77.

[0164] Figures 16A-16B:Macrophage phagocytosis detection under laser confocal scanning microscope, among which, Fig.16A For characteristic visual field, Fig. 16B The phagocytosis rate results of each group (the data in the figure are n=3; there was a significant difference compared with the PBS group, *: p<0.05).

[0165] Fig.17 : Flowchart of animal experiments.

[0166] Figures 18A-18D : Animal experiment results, among which, Fig.18A is the change of tumor volume over time; Fig.18B The weight of mice changes over time; Fig.18C is the weight of tumor tissue collected after mice in each group were sacrificed; Fig.18D Figure 3 shows the in vitro tumor tissues of mice in each group.

[0167] Sequence information

[0168] The information of the sequences involved in the present invention is described in the following table:

[0169] Table 1: Sequence description

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179] Wherein, X represents pAcF. DETAILED DESCRIPTION

[0180] The invention is now described in the following non-limiting examples.

[0181] Those skilled in the art will appreciate that the embodiments describe the present invention by way of example and are not intended to limit the scope of protection claimed in the present application. The experimental methods in the embodiments are conventional methods unless otherwise specified. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0182] Example 1: Screening of non-natural amino acid insertion sites

[0183] The 3D structures of nanoantibodies PV2 and PV3 (anti-PD-L1 nanoantibodies, wherein the PV2 VHH sequence is shown in SEQ ID NO: 1 and the PV3 VHH sequence is shown in SEQ ID NO: 27) were obtained by online modeling using Alphafold2, and the CDR regions in PV2 and PV3 nanoantibodies were predicted using the CDR region online prediction website (http: / / aligncdr.labshare.cn / ). This patent uses computational methods such as Alphafold2, molecular dynamics (MD) simulation, molecular docking, and Rosetta to accurately predict the stability of nanoantibodies and changes in the affinity of nanoantibodies after the introduction of non-natural amino acid mutations.

[0184] The calculation process is as follows Figure 1 As shown, taking the nanobody PV2 as an example, the nanobody sequence is first predicted by Alphafold2 for structure prediction and CDR region prediction by online tools. After that, the wild-type nanobody PV2 and the antigen PD-L1 are simulated by MD respectively. The 6-frame conformation after PV2 equilibrium is selected, and a certain amino acid in PV2 is mutated to the non-natural amino acid pAcF. The 6-frame conformation is 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 are selected for parameter analysis such as RMSD, RMSF, hydrogen bond, and salt bridge, and experimental verification is performed. At the same time, the equilibrium structure obtained by MD simulation of the wild-type nanobody PV2 and the antigen PD-L1 is docked with ZDOCK, and a certain amino acid in PV2 is mutated to the non-natural amino acid pAcF. Rosetta Flex ddG is used to calculate the affinity maturation of the PV2-PD-L1 complex, and the Flex ddG value is obtained, which is ranked from low to high. Combining the results of Cartesian ddG, Flex ddG and CDR region prediction, mutants that were not in the CDR region and had high and low Cartesian ddG and FlexddG rankings were selected for experimental verification, and finally suitable nanobody PV2 and PV3 mutants were selected, and the sequences are shown in Table 1.

[0185] The calculation process is as follows:

[0186] (1) Computing system settings

[0187] The structures of the nanobodies were calculated by Alphafold2, and the best structures among the five optimal structures generated by Alphafold2 were selected. The antigen structures were taken from the RCSB crystal database, and the crystal water molecules and irrelevant residues in the PDB were deleted. The CDR regions of the nanobodies were predicted by the online tool AbRSA (http: / / aligncdr.labshare.cn / aligncdr / abrsa.php) and the Chothia and Kabat methods in Novo Pro (https: / / www.novopro.cn / tools / ).

[0188] Antibody-antigen docking was performed using ZDOCK for rigid docking (see Pierce et al., (2014). ZDOCK server: interactive docking prediction of protein-protein complexes and symmetric multimers. Bioinformatics, 30(12), 1771-1773.), and the best structure among the 10 optimal structures generated by ZDOCK was selected.

[0189] The Cartesian_ddG protocol was used in Rosetta to calculate the changes in the stability of the nanobody before and after the substitution mutation of the non-natural amino acid pAcF. In the Cartesian_ddg protocol, the protein-ligand complex model with the lowest score after energy minimization using the relax command was used as the input of the protocol. The final ddG score was obtained by averaging the results of 50 iterations of the Cartesian_ddg protocol, using the talaris 2015 scoring function. The six-frame conformations after the equilibrium of the nanobody MD simulation were calculated using Rosetta CartesianddG to obtain the average Cartesian ddG of the six-frame mutants, which were ranked from low to high, and finally several mutants with the top Cartesian ddG rankings in the two nanobodies were screened out. The screening results are shown in Table 2.

[0190] Table 2: Rosetta Cartesian ddG ranking of mutation sites

[0191]

[0192] According to the results of Rosetta Cartesian ddG, the top six sites with the highest average Cartesian ddG in PV2 and PV3 were screened. The scores of the top mutants were close, all around -9 kcal / mol, so MD analysis and experiments were needed to confirm whether these sites were the best mutation sites to verify the accuracy of Cartesian ddG calculation.

[0193] (2) MD simulation

[0194] like Figure 1 As shown, the first batch of MD simulations were performed at 300 K for the wild-type and different mutant systems of nanobodies, as well as the antigen system. PROPKA3 was used to predict the distribution of protein protonation states at the experimental pH. The MD simulations were run by Gromacs 2023 and the CHARMM36 protein force field was used for MD simulations. The force constant of the harmonic bias potential was 1000 kJ mol -1 nm 2 . Sodium ions and chloride ions were added to the solution to keep the system neutral. In the initial stage, the system was minimized for 10,000 steps using the steepestdescent algorithm. In the pre-equilibrium stage, a 200 ps simulation was run using the NVT ensemble, followed by a 200 ps simulation using the NPT ensemble to gradually heat the system to 300 K at 1 atm. In the equilibrium stage, the NPT ensemble was continued to be used for 100 ns simulations at 1 atm and 300 K. After obtaining the equilibrium system, a 10 ns production simulation was then performed, and the equilibrium conformations were collected at 10 ps intervals.

[0195] Throughout the simulation, a velocity-regulated thermostat with a time constant equal to 0.1 ps was used (see Giovanni Bussi, (2007). Canonical sampling through velocity rescaling. THE JOURNAL OF CHEMICAL PHYSICS, 126.) to keep the temperature constant. To maintain the pressure, a Berendsen pressure coupler was used in the pre-equilibrium run and a Parrinello-Rahman pressure coupler was used in the equilibrium and production simulations, with the pressure time constant and isothermal compressibility set to 2 ps and 4.5×10 -5 bar -1 The equations of motion were integrated with a time step of 2 fs throughout the simulation. The cutoff for non-bonded interactions is The particle mesh Ewald algorithm is used to calculate long-range electrostatic interactions.

[0196] Select the 50ns structure in the MD simulation and mark the mutation results in PV2 and PV3. The result diagram is shown in the figure below. Figure 2A-2B As shown, the CDR region of the nanobody is marked in black, the selected mutation sites are all far away from the CDR region, and the amino acid side chains are all unfolded outward.

[0197] The RMSD of the skeletons of the two groups of nanoantibodies were analyzed after 10 ns of MD simulation equilibrium. Figure 3A-3B shown.

[0198] The black lines in the figure correspond to the RMSD fluctuations of the wild type. Based on this standard, T116pAcF obviously shows a larger fluctuation than the wild type, while the remaining mutants S71pAcF, S26pAcF, S8pAcF, S85pAcF, and Q82pAcF all show a lower and more stable RMSD curve than the wild type. It can be speculated that the stability of T116F is worse than that of the wild type, while the remaining mutants have better stability, among which S26pAcF and S85pAcF are better. In PV3, N77pAcF, S8pAcF, S26pAcF, and Q82pAcF obviously show larger fluctuations than the wild type, S71pAcF obviously shows a more stable RMSD than the wild type, and the remaining mutants K87pAcF and S85pAcF show RMSD curves similar to the wild type. It can be speculated that the stability of N77pAcF, S8pAcF, S26pAcF and Q82pAcF is poorer than that of the wild type, the stability of S71pAcF is relatively good, and the stability of the remaining mutants is similar to that of the wild type.

[0199] In addition, the RMSF of the skeletons of the two groups of nanoantibodies were analyzed after 10 ns of MD simulation equilibrium. Figures 4A-4BAs shown. The black line in the figure corresponds to the RMSF fluctuation of the respective wild type. Taking this as a standard, in PV2, the Cα of T116pAcF obviously fluctuates more than the wild type, while the remaining mutants (S71pAcF, S26pAcF, S8pAcF, S85pAcF, Q82pAcF) all show RMSF curves similar to the wild type. It can be speculated that the stability of T116F is poorer than that of the wild type, while the remaining mutants are similar in stability to the wild type. In PV3, the Cα of S8pAcF, S26pAcF, Q82pAcF, N77pAcF, and K87pAcF obviously fluctuates more than the wild type, while the remaining mutants S85pAcF and S71pAcF all show RMSF curves similar to the wild type. It can be speculated that the stability of S8pAcF, S26pAcF, Q82pAcF, N77pAcF, and K87pAcF is poorer than that of the wild type, while the stability of the remaining mutants is similar to that of the wild type.

[0200] Hydrogen bonds are an important factor affecting protein conformational stability. The calculation of the number of hydrogen bonds during MD simulation can reflect the stability of each mutant. The calculation results of the number of hydrogen bonds in each group are as follows: Figure 5A-5B shown.

[0201] Compared with the wild type, the number of relatively stable hydrogen bonds of all mutants in the PV2 group decreased, but the decrease in S71pAcF and S85pAcF was smaller, and they still had a large number of hydrogen bonds among all mutants. The decrease in S26pAcF and T116pAcF was larger, especially the decrease in the number of hydrogen bonds of T116pAcF was the largest, which can be inferred that the stability of T116pAcF may be poor. Compared with the wild type, the number of relatively stable hydrogen bonds of S71pAcF in the PV3 group increased, and the number of relatively stable hydrogen bonds of all other mutants decreased, while the decrease in S26pAcF was smaller, which was similar to the number of hydrogen bonds of the wild type. The decrease in N77pAcF and S8pAcF was larger. It can be inferred that the stability of S71pAcF and S26pAcF is improved or equal to that of the wild type, and the stability of the remaining K87pAcF, S85pAcF, and Q82pAcF is reduced, and the stability of N77pAcF and S8pAcF may be poor.

[0202] In addition to hydrogen bonds, the number of salt bridges that may be formed between amino acid sites within the protein molecule, the connection sites, and the probability of occurrence are calculated. The calculation results are as follows: Figure 6 shown.

[0203] The PV2 wild type has 4 stable salt bridges, and S71pAcF, S26pAcF, S8pAcF, S85pAcF, and Q82pAcF all have one more stable salt bridge than the wild type, mainly adding the Asp52-Lys65 salt bridge. The remaining T116pAcF has the same number of salt bridges as the wild type. Therefore, it can be seen that T116pAcF may not have much effect on improving stability. The PV3 wild type has 4 stable salt bridges, and S8pAcF and S85pAcF have two more stable salt bridges than the wild type, mainly adding the two groups of Glu47-Arg39 and Glu45-Lys113 salt bridges. N77pAcF and K87pAcF both have one more stable salt bridge than the wild type, mainly replacing the wild type's Glu89-Lys87 with the two groups of Glu47-Arg39 and Glu45-Lys113 salt bridges. The remaining S71pAcF, S26pAcF, and Q82pAcF have the same number of salt bridges as the wild type, which may not have much effect on improving stability.

[0204] Based on the calculation results of RMSD, RMSF, hydrogen bonds and salt bridges in the above MD simulation process, except for PV2-T116, the mutants in the PV2 and PV3 groups all achieved better calculation results than the wild type in at least one calculation result, while PV2-T116 showed poor data in RMSD, RMSF, hydrogen bonds and salt bridge data, so T116pAcF was not subjected to subsequent experimental verification.

[0205] (3)Rosetta calculation

[0206] Ordinary Rosetta calculations only contain calculation parameters related to natural amino acids. In order to use non-natural amino acid related functions in Rosetta, this patent needs to build three non-natural amino acid files, namely parameters, rotator libraries and unfolding energy. First, the molfile2params_polymer.py script is used to obtain the parameter file of non-natural amino acids, and the new non-natural amino acid side chains are incorporated into Rosetta for design. Then, a rotator library related to the skeleton is created for non-natural amino acids, and unfolding energy is calculated for non-natural amino acids (see, Uversky et al., (2012). Incorporation of Noncanonical Amino Acids into Rosetta and Use in Computational Protein-Peptide Interface Design. Plos One, 7 (3).). The changes in affinity maturity after non-natural amino acid mutations were calculated using the Flex_ddg protocol in Rosetta. In the Flex_ddg protocol, the talaris 2014 scoring function was used (see Leaver-Fay et al., (2013). Scientific Benchmarks for Guiding Macromolecular Energy Function Improvement. In Methods in Protein Design (pp. 109-143); O'Meara et al., (2015). Combined Covalent-Electrostatic Model of Hydrogen Bonding Improves Structure Prediction with Rosetta. Journal of Chemical Theory and Computation, 11 (2), 609-622.). For the Flex_ddg protocol, the final ddG prediction value is the average of the generalized additive model obtained from 35 iterations of the protocol.

[0207] In Rosetta, the calculation parameters for pAcF of acetylphenylalanine were established, and the Rosetta Cartesian ddG calculation was performed on the 6-frame MD equilibrium conformation of the nanobody, and the Rosetta Flex ddG calculation was performed on the 1-frame conformation of the antigen-nanobody complex obtained after docking. The energy of Rosetta Cartesian ddG and Flex ddG was plotted as a scatter plot, and the mutation sites in the CDR region were deleted to find the energy of both Cartesian ddG and Flex ddG, or the energy of Cartesian ddG was relatively negative and Flex ddG was within 1 kcal / mol. The above results are as follows Figure 7 shown.

[0208] In the figure, the CDR region sites of the two groups of nanoantibodies are marked with green X scatter points. A total of 5 possible optimal mutations were found in the PV2 group, including N74pAcF, S26pAcF, S8pAcF, S71pAcF, and Q82pAcF. Although the Flex ddG of S85pAcF exceeds 1kcal / mol, its Cartesian ddG prediction is better, so it is still selected for subsequent experiments. The above sites are marked with pink scatter points. In addition, the energy of both Cartesian ddG and Flex ddG is found to be relatively positive for comparison in subsequent experiments. A total of 3 possible poor mutations of I70pAcF, C96pAcF and C23pAcF are selected, marked with orange scatter points. A total of 9 mutants were selected for subsequent experiments. The PV3 group was screened in the same way, and a total of 5 possible optimal mutations were found, including S71pAcF, S26pAcF, S8pAcF, N77pAcF, and K87pAcF. In addition, the Flex ddG of S85pAcF exceeded 1 kcal / mol, but its Cartesian ddG prediction was good, and it was also selected for subsequent experiments. In addition, 3 possible inferior mutations of I70pAcF, C23pAcF and G119pAcF were selected. A total of 9 mutants were selected for subsequent experiments.

[0209] Example 2: Construction of Nanobody Expression Plasmid with Unnatural Amino Acid Insertion - Construction of Single-point Mutation Nanobody and Mutant Nanobody Expression Plasmid by CPEC (Circular Polymerase Extension Cloning) Method

[0210] All feasible mutation sites in PV2 and PV3 nanobodies were scored and ranked, and several mutation sites at the top and bottom of the scoring results were selected for mutant expression to verify the calculation results.

[0211] Taking the PV2 mutant as an example, the specific steps are as follows:

[0212] (1) Using the pET-32a(+) plasmid inserted with the PV2 wild-type antibody gene (sequence as shown in SEQ ID NO: 8) stored in the laboratory as a template, the TAG codon mutation was introduced into the target gene sequence by primer design, and the upstream and downstream primers of the mutation site were designed using the PrimeX online point mutation primer design website (https: / / www.bioinformatics.org / primerx / index.htm), and the upstream and downstream primers were designed roughly symmetrically with the mutation site on the plasmid. The primer sequences are shown in Table 3 (the underlined part is the TAG codon mutation region). The target protein was amplified by PCR, and the PCR system is shown in Table 4.

[0213] Table 3: PCR primer sequences for CPEC experimental design

[0214]

[0215]

[0216] Table 4: PCR reaction system

[0217]

[0218] After configuring the reaction system according to Table 4, set the reaction PCR program, set the extension time according to 2kb / min, and the annealing temperature is generally set to 58°C, which can also be adjusted according to the Tm value of the primer. The PCR program settings are shown in Table 5.

[0219] Table 5: PCR reaction program

[0220]

[0221] (2) The PCR product was verified to be correct by nucleic acid gel electrophoresis, and then the PCR product was recovered using a DNA gel rapid purification kit.

[0222] (3) The two PCR products obtained after purification were cyclized by the CPEC method to construct expression plasmids with mutant sequences for expressing various mutant nanobodies. The CPEC reaction system and reaction procedure are shown in Tables 6 and 7.

[0223] Table 6: CPEC cyclization reaction system

[0224]

[0225] Table 7: CPEC Response Procedure

[0226]

[0227] (4) Take 100 μL Trans5α competent cells (Beijing Quanshijin Biotechnology) stored at -80°C and place them on ice to melt. In a sterile clean bench, take 10 μL CPEC cyclization product and slowly add it to the competent cells. After gentle mixing, let it stand for 30 minutes. After standing, place the competent cells in a 42°C water bath for heat shock for 90 seconds, then quickly transfer them to ice and let them stand for 4 minutes. Finally, add 1 mL LB medium to mix the competent cells, place them at 37°C for 1 hour, centrifuge at 6000 rpm for 2 minutes and discard 800 μL of supernatant. After resuspending, spread them on a plate containing Amp resistance and culture them at 37°C overnight. The next day, pick out the monoclonal colonies on the plate and culture them in LB medium containing Amp resistance. When the OD600 of the bacterial solution reaches 0.6, add 30% glycerol and store it at -80°C. At the same time, send the samples to the company for sequencing to confirm the successful introduction of the TAG codon single point mutation.

[0228] (5) Extract the point mutation expression plasmid stored in Trans5α and separate it from the pULTRA-TSDV plasmid (in the plasmid pultra-CNFRS (as Figure 8 The mutant of the archaeal tyrosyl-tRNA synthetase (see SEQ ID NO: 12 in CN202211225132.5 for the sequence) was inserted into the strain and transformed into the TransB strain (Beijing Quanshijin Biotechnology). After the transformed strain was sequenced correctly, 30% glycerol was added to preserve the strain for subsequent expression.

[0229] Example 3: Expression and purification of nanobodies with non-natural amino acid insertion

[0230] The nine mutants of PV2 (PV2-S8 (SEQ ID NO: 9), PV2-C23 (SEQ ID NO: 11), PV2-S26 (SEQ ID NO: 13), PV2-I70 (SEQ ID NO: 15), PV2-S71 (SEQ ID NO: 17), PV2-N74 (SEQ ID NO: 19), PV2-Q82 (SEQ ID NO: 21), PV2-S85 (SEQ ID NO: 23), PV2-C96 (SEQ ID NO: 25)) and the nine mutants of PV3 (PV3-S8 (SEQ ID NO: 31), PV3-C23 (SEQ ID NO: 33), PV3-S26 (SEQ ID NO: 35), PV3-I70 (SEQ ID NO: 37), PV3-S71 (SEQ ID NO: 39), PV3-N77 (SEQ ID NO: 41), PV3-S85 (SEQ ID NO: 42), NO:43), PV3-K87 (SEQ ID NO:45), PV3-G119 (SEQ ID NO:47)) were used to express mutant nanobodies with the introduction of the unnatural amino acid pAcF, and the successful introduction of pAcF into the mutants was verified by SDS-PAGE gel electrophoresis. The specific steps are as follows:

[0231] (1) Take out the Trans5α expression strain containing wild-type and mutant PV2 and PV3 constructed in Example 2 (Quanshijin Company).

[0232] (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 Fig. 9A ) solution, and the negative control group was added with only 200 μL IPTG (isopropyl-β-d-thiogalactoside) solution, and cultured at 16°C and 160 rpm for 16 h.

[0233] (3) Collect the cells by centrifugation at 4°C and 10,000 rpm for 10 min, discard the supernatant, and wash the cell sludge twice with PBS solution.

[0234] (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 s on, 3 s off, and a working time of 30 min.

[0235] (5) The bacterial suspension after ultrasonic disruption was centrifuged at 4°C and 10,000 rpm for 15 min. The supernatant was aspirated with a syringe and filtered through a 0.45 μm PES filter for later use.

[0236] (6) Purify the nanoantibody using AKTA prime plus protein purifier. The specific steps are as follows:

[0237] 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.

[0238] 2) Install the HIS TrapTM prepacked column on the instrument and flush the column at a flow rate of 4 mL / min until the instrument shows that the UV280 line and the conductivity line are flat and close to zero. Approximately 50 mL of deionized water is required.

[0239] 3) The input liquid was replaced from deionized water to 80 mM imidazole solution, and flowed into the pre-packed column at a flow rate of 4 mL / min for 8-10 column volumes to equilibrate the pre-packed column.

[0240] 4) Load the supernatant prepared in the previous step, select the infusion pump for injection, and select the injection flow rate of 1 mL / min.

[0241] 5) After loading, flush the column with 80 mM imidazole solution at a flow rate of 1 mL / min for 10 min, and then increase the flow rate to 4 mL / min until the UV280 line flattens.

[0242] 6) Set pump A to 80 mM imidazole solution and pump B to 500 mM imidazole solution, set pump B from 0 to 100%, set the volume to 50 mL for gradient elution, and the flow rate to 3 mL / min. Start collecting when the elution peak appears, and stop collecting until the UV280 line flattens out. Use deionized water to flush the pipeline and chromatography column until the pipeline conductivity is essentially zero.

[0243] 7) Rinse the column and tubing with deionized water until the UV280 line and the conductivity line are flat and close to zero.

[0244] 8) Rinse the system with 20% ethanol solution, flush 50 mL to make the pipeline and chromatography column full of 20% ethanol solution, remove the HIS pre-packed column, store it at 4°C, and turn off the instrument.

[0245] 9) Analyze the purified fractions by SDS-PAGE gel electrophoresis.

[0246] The 9 mutant nanobodies in the PV2 and PV3 groups were successfully expressed as mutants with pAcF introduced. The electrophoresis results of wild-type and mutant expressed proteins are shown in Figure 2. Figures 10A-10B As shown, the molecular weight of the nanoantibodies in each group of PV2 is about 13 kDa, and the molecular weight of the nanoantibodies in each group of PV3 is about 14 kDa. By comparing the electrophoresis results with the marker, it can be seen that the molecular weight of each mutant is consistent with the theoretical molecular weight.

[0247] Example 4: Verification of thermal stability of mutant nanobodies by differential scanning fluorimetry

[0248] The thermal stability of the nanobody was evaluated by using a differential scanning fluorescence method to detect the amount of the fluorescent dye combined with the nanobody with a changed structure during the heating process. A LightCycler 480II fluorescent quantitative PCR instrument was used for detection, and the reaction system was shown in Table 8.

[0249] Table 8: Differential scanning fluorescence reaction system

[0250]

[0251] Each group of the experiment was set up with 5 parallels. The system was protected from light during the whole process. After the configuration, the samples were divided into 96-well plates, centrifuged at low speed to mix the system and remove bubbles, and then tested in a fluorescent quantitative PCR instrument. The detection steps are as follows:

[0252] (1) Start the instrument and wait for the instrument to complete the self-check. After the self-check is completed, place the 96-well plate in the sample rack.

[0253] (2) Enter the LightCycler 480 program interface and set the experimental parameters. The experimental temperature gradient is 25-95°C, the dye excitation wavelength is 465nm, and the emission wavelength is 580nm. The Analysis Mode is set to Melting Curves, the Acquisition Mode is set to continuous, the Ramp Rate is set to 0.01°C / s, and the Acquistions are set to 50per°C.

[0254] (3) After the experiment, the data was exported and analyzed using the LightCycler Thermal Shift Analysissoftware (Roche Applied Science) plug-in to calculate the Tm value of each mutant.

[0255] Thermal stability is an important indicator of antibody properties. During the introduction of mutations, it is necessary to ensure that the stability of the altered protein conformation does not decrease too much. The Tm values ​​of each mutant and wild-type nanobody in the PV2 and PV3 groups were obtained by differential scanning fluorimetry. This result can be used as a measure of thermal stability to screen the best mutation sites.

[0256] After the qPCR instrument was completed, the LightCycler Thermal Shift Analysis software plug-in was used for analysis and calculation, and the Tm values ​​of the two groups of nanoantibodies were finally obtained as follows: Figures 11A-11B shown.

[0257] Fig.11A The results showed that the Tm value of the PV2 wild type was 47.89°C, and the Tm values ​​of the mutants were all within 5°C of the wild type. The mutant with the worst stability was PV2-C96, with a Tm value of 42.17°C. The mutants with relatively high Tm values ​​were PV2-S26, PV2-S71, and PV2-Q82, with corresponding Tm values ​​of 52.24°C, 52.90°C, and 53.1°C, respectively. These three sites can enter the next step of PV2 group optimal site screening; Fig. 11B The thermal stability of each mutant in the PV3 group varied greatly, with more obvious differences within the group. The Tm value of the PV3 wild type was 51.96°C. The mutant with the worst stability was PV3-K87, with a Tm value of 44.42°C. The groups with significantly improved Tm values ​​included PV3-S71 and PV3-N77, with corresponding Tm values ​​of 58.66°C and 59.99°C, respectively. These two sites are suitable for further screening.

[0258] Example 5: ELISA test of mutant nanobody affinity

[0259] In order to evaluate the changes in the affinity of each mutant for antigen binding after the introduction of pAcF, the affinity of each mutant and the subsequently constructed anti-PD-L1 dual-nanobody construct was tested by indirect ELISA.

[0260] PD-L1 antigen (recombinant human PD-L1 protein (Active)

[0261] (ab280943), abcam) were coated in a 96-well plate for ELISA test. The test steps are as follows:

[0262] (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.

[0263] (2) Discard the coating solution, add 200 μL PBST solution to each well, and wash three times, 5 min each time.

[0264] (3) Add 100 μL of freshly prepared blocking solution to each well and transfer to a 37°C incubator for 1 h.

[0265] (4) Discard the blocking solution and add 200 μL of PBST solution to each well. Wash three times, 5 min each time.

[0266] (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 divalent nanoantibody group being 400 μg / mL. Perform 4-fold gradient dilutions, with a total of 10 gradient concentrations, and set up a zero concentration control group (PBS). Incubate in a 37°C incubator for 2 h.

[0267] (6) Discard the liquid in the wells, add 200 μL PBST solution to each well, and wash 5 times, 5 min each time.

[0268] (7) Dilute HRP-labeled mouse anti-HIS monoclonal antibody at 1:10,000, add 100 μL to each well, and incubate in a 37°C incubator for 1 h.

[0269] (8) Discard the liquid in the wells, add 200 μL PBST solution to each well, and wash 5 times, 5 min each time.

[0270] (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.

[0271] (10) Add 100 μL of stop solution to each well and measure the OD450 value of each well using an ELISA reader.

[0272] The final results of each group are as follows Figures 12A-12D As shown, Fig. 12A , 12B The horizontal axis is the logarithmic molar concentration of the nanoantibody, and the vertical axis is the OD450 absorbance. The affinity values ​​of each group are fitted. Fig. 12C , 12D Listed in.

[0273] The affinity of the PV2 wild type is 15.14nM. The affinity of most mutants in the PV2 group was improved after the introduction of pAcF. Among them, the affinity of the PV2-S71 group and the PV2-S26 group was greatly improved, 1.702nM and 1.571nM respectively, which was about 9 times higher than the wild type. The PV2-C96 group had the most obvious decrease in affinity, with an affinity of 37.41nM. The affinity of the PV3 wild type was 51.01nM. Among the PV3 group mutants, the PV3-K87, PV3-C23, PV3-S85, and PV3-N77 groups had a more obvious increase in affinity. Among them, the group with the highest affinity increase was PV3-K87, with an affinity result of 13.67nM, which was 3.8 times higher than the wild type.

[0274] In summary, the computer prediction-assisted screening method narrows the scope of experimental screening. Most of the mutation sites predicted to be better have improved thermal stability and affinity compared to the wild type, proving the feasibility of the non-natural amino acid insertion site screening scheme for nanobodies developed in this patent. Both antibodies have significant effects, proving its universality and can be adapted to the development of other non-natural amino acid insertion antibodies based on nanobodies.

[0275] Example 6: Construction and evaluation of anti-PD-L1 dual nanobody constructs based on unnatural amino acids

[0276] Through computer prediction and Tm value and affinity testing of each mutant, the most suitable mutant combination for the construction of dual-nano antibody constructs was screened out. Taking into account the Tm value and affinity data of each mutant, the mutation sites in PV2 and PV3 that are most suitable for the construction of dual-nano antibody constructs were selected. Finally, the PV2 group selected PV2-S26 (3rd in Tm value, 1st in affinity) and PV2-S71 (2nd in Tm value, 2nd in affinity); the PV3 group selected PV3-S85 (4th in Tm value, 3rd in affinity) and PV3-N77 (1st in Tm value, 4th in affinity). The mutants corresponding to the above different nano antibodies were combined in pairs, and a total of four groups of dual-nano antibody constructs were constructed. The construction principle of the dual-nano antibody construct is as follows. Fig.13 As shown, the construction process is as follows:

[0277] (1) Taking the construction of the PV2-S71 + PVE-N77 group as an example, the nanobody monomer purified in Example 3 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 to a weakly acidic buffer environment.

[0278] (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. The linker Bicyclononyne (BCN) (e.g. Fig. 9C ), and another group added the linker Tetrazine (TET) (such as Fig. 9B ), the molar ratio of linker addition to nanobody was 50:1, and the reaction was allowed to proceed at 30°C for 24h.

[0279] (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 nanoantibody-linker complexes after the reaction were measured using Nanodrop and the nanoantibody 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.

[0280] (4) After 24 hours, the reaction solution was concentrated to 600-1000 μL using an ultrafiltration tube (10000 MWCO), and the uncoupled nanobody monomers and small molecule linkers in the solution system were removed by size exclusion chromatography. The size exclusion purification steps are as follows:

[0281] 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.

[0282] 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 pipeline until the baseline was flat.

[0283] 3) Replace the solution with PBS solution, and let 1 to 2 column volumes of solution flow through the pipeline until the UV280 line is flat. At the same time, use a 1 mL syringe to suck up the PBS solution and inject it into the sample loop injection port. Repeat the cleaning five times.

[0284] 4) Use PBS solution to make up the volume of the sample to be loaded to 1 mL, inject it into the sample loop from the sample loop inject port, and select the sample loop injection at the same time, the injection flow rate is 0.4 mL / min, and the injection volume is set to 2 mL.

[0285] 5) When the instrument shows that the UV line is rising, start collecting samples until the first peak ends and the peak corresponding to the second monomer does not rise significantly.

[0286] 6) Replace the solution with deionized water and clean the pipeline with 1 to 2 column volumes.

[0287] 7) Replace the solution with 20% ethanol, adjust the flow rate to 0.25 mL / min, flow 30 mL of solution to protect the tubing and column, remove the column and store at 4°C.

[0288] 8) The collected fractions were analyzed by SDS-PAGE gel electrophoresis, and the double nanobody construct was concentrated into one tube using an ultrafiltration tube.

[0289] 9) The coupled bi-nanobody constructs were separated by size exclusion chromatography to separate the coupled bi-antibodies and the uncoupled free monoclonal antibodies, and the coupling efficiency differences between the groups were compared by the ratio of the bi-antibody peak area to the monoclonal antibody peak area in the chromatographic peak graph. The results of the four groups of size exclusion peak graphs are shown in Figures 14A-14D As shown, the peak areas and proportions are given in Table 9.

[0290] Table 9: Size exclusion chromatographic peak area ratios of each group

[0291]

[0292] The PV2-S71+PV3-N77 group had the best connection efficiency and the highest yield, so it was used for subsequent evaluation. The PV2WT-(GGGGS)3-PV3WT double nanobody construct based on the flexible peptide connection fusion protein was used for control verification. The designed sequence was synthesized by Genewise Biotechnology Co., Ltd. The pET-32a(+) plasmid carrying the sequence was transferred into the TransB strain for expression. The specific steps are shown in Example 3. Figures 15A-15E As shown, Fig.15A , B. The successful construction of PV2-S71+PV3-N77 was verified by SDS-PAGE gel electrophoresis and MALDI-TOF mass spectrometry. Fig. 15C , D, E The thermal stability and affinity of the two double nanobody constructs, PV2-S71+PV3-N77 and fusion expression PV2-PV3, were detected by differential scanning fluorescence and indirect ELISA, respectively, to compare the differences in the properties of the double nanobody constructs obtained by different construction methods.

[0293] like Fig. 15C As shown, the Tm value of the PV2-S71+PV3-N77 group was measured to be 56.75°C, and the Tm value of the PV2-PV fusion protein group was 50.466°C. The thermal stability of the dual antibodies constructed based on non-natural amino acids was higher than that of the fusion protein group. Analysis showed that after the introduction of pAcF at the mutation site, the thermal stability of the mutant monomers in the PV2-S71 and PV3-N77 groups was improved compared with the wild-type monomers. Therefore, the dual nanoantibody construct constructed based on the mutants also has a higher thermal stability as a whole.

[0294] like Fig.15EAs shown, the affinity results showed that the affinity of the PV2-S71+PV3-N77 group was 6.13 nM, which was much higher than the 61.1 nM of the PV2-PV3 fusion protein and significantly improved relative to the affinity of the wild-type monomer, proving the feasibility and necessity of the construction of the dual nanobody construct.

[0295] Example 7: Cell phagocytosis assay to evaluate the biological activity of anti-PD-L1 dual nanobody constructs

[0296] The anti-PD-L1 antibody has the ability to block the binding of PD-1 of macrophages to PD-L1 of tumor cells, thereby promoting the phagocytic ability of macrophages and reducing the ability of tumor cells to escape immune system. By co-culturing tumor cells HT29 and induced differentiated THP-1 cells, the cells are labeled with fluorescent staining, and the biological activity of the anti-PD-L1 antibody is evaluated by calculating the cell phagocytic efficiency. The cell lines THP-1 cells (human monocytic leukemia cells) and HT29 cells (human colon cancer cells) used in this patent are both from the ATCC cell bank.

[0297] (1) THP-1 cell differentiation induction

[0298] THP-1 cells can be induced to differentiate into macrophages in vitro by adding the stimulatory factor PMA. The main steps are as follows:

[0299] THP-1 cells were sampled and counted under an inverted microscope and the cell density was adjusted to 2×10 5 PMA was added to the cell culture medium, and the final concentration of the inducer PMA in the culture medium was 80 ng / mL. THP-1 cells were cultured in the medium containing PMA for 24 hours. After 24 hours, the cells were observed under an inverted microscope to differentiate from suspension cells into adherent cells. After confirming that most of the cells were attached to the wall under the microscope, the 1640 medium containing PMA was removed with a Pasteur pipette, and after rinsing twice with PBS solution, the cells were transferred to 1640 complete medium without PMA. After resting for 24 hours, induced differentiated human macrophages were obtained.

[0300] (2) Laser confocal scanning microscopy detection of HT29 cells and macrophages co-culture

[0301] HT29 cells and macrophages were fluorescently labeled with DiD dye and Hoechst 33342 dye, respectively. After co-culture, the number and proportion of blue and blue-red fused fluorescent cells were observed under a laser confocal scanning microscope to determine the changes in the phagocytic rate of macrophages after the antibody action in each experimental group. The specific steps are as follows:

[0302] The human macrophages obtained after trypsin digestion and differentiation were transferred to a 12-well plate, and each well of the 12-well plate was pre-plated with a cell slide of appropriate size. Each well was diluted to 5×10 4 / mL, 1mL of macrophages were added to each well and cultured at 37℃ for 2h until the cells attached to the wall. The experiment was divided into five groups: PBS negative control group, PV2 monoclonal antibody control group, fusion protein PV2-PV3 control group, PV2-S71+PV3-N77 experimental group, and Durvalumab positive control group.

[0303] DiD mother solution (2 mM) stored at -20°C was diluted to a working concentration (2 μM) at a ratio of 1:1000 with serum-free 1640 medium. HT29 cells were digested with trypsin and resuspended in serum-free 1640 medium to adjust the cell density to 1×10 6 / mL, add DiD working solution to the cell suspension at a ratio of 5μL / mL, and gently blow it evenly with a pipette. Transfer the cell suspension to a 37℃ environment and incubate for 5min, then transfer it to a 4℃ environment and incubate for 10min. Centrifuge at 1000rpm for 5min, discard the supernatant, and resuspend and wash twice with PBS solution. Resuspend the cells in 1640 complete culture medium and let it rest for 10min, then add each group of antibodies at a ratio of 10μg / mL, and transfer to a 37℃ incubator and incubate for 1h.

[0304] After the incubation, HT29 cells were plated at 2×10 5 The ratio of 100 μL per well was added to a 12-well plate containing macrophages and co-cultured at 37°C for 3 hours. Wash twice with PBS solution, take out the Hoechst 33342 dye stored at -20°C, dilute it 100 times with PBS to the working concentration, add 500 μL Hoechst 33342 working solution to each well of the 12-well plate, and stain at 37°C for 20 minutes. After staining, aspirate the dye and wash twice with PBS solution, carefully remove the cell slides in each well, and transfer them to a laser confocal scanning microscope for shooting and detection.

[0305] The local image results in the microscope are as follows Figures 16A-16B As shown, three replicates were performed in each group, and more than 100 photos were taken for each experimental group. 100 blue fluorescently labeled macrophages were randomly selected, and the number of macrophages with red and blue fusion phagocytosis in the 100 macrophages was observed and recorded, and the phagocytic rate of each group was calculated. The calculation results showed that the phagocytic efficiency of the PV2-S71+PV3-N77 group and the Durvalumab group in the antibody drug group showed significant differences (p<0.05, n=3), while the PV2-PV3 and PV2 groups did not show significant differences from the PBS group.

[0306] Example 8: Anti-tumor activity of anti-PD-L1 dual nanobody constructs

[0307] In order to explore the effects of different groups of antibody drugs targeting PD-L1 on the growth of subcutaneous tumors of mouse colon cancer, hPD-L1-MC38 cells with humanized PD-L1 gene were injected into the right side of hPD-1-C57BL / 6Smoc-Pdcd1em1 (hPDCD1) / Smoc mice with humanized PD-1 gene to construct a subcutaneous tumor model that can verify the efficacy of antibodies targeting human PD-L1.

[0308] Twenty-four female PD-1 humanized C57BL / 6Smoc-Pdcd1em1(hPDCD1) / Smoc mice (7 weeks old) were purchased from Shanghai Model Organisms Technology Co., Ltd. and housed in a specific pathogen-free laboratory animal facility at a temperature of 22±3°C and a relative humidity of 55±10% for one week. The mice had free access to water and food under a standard 12-hour dark:12-hour light cycle.

[0309] like Fig.17 As shown, after the mice were adapted for one week, the mouse subcutaneous tumor model was established. The hPD-L1-MC38 (humanized gene-modified hPD-L1-MC38 (PD-L1 gene humanized mouse colon cancer cells) with good growth status were obtained from Beijing Kangyuan Bochuang Biotechnology Co., Ltd.) cells were digested with trypsin and centrifuged to collect the cell pellet, washed twice with PBS solution and counted, 1×10 6 hPD-L1-MC38 cells were resuspended in a sterile PBS solution containing 25% matrix gel, and 100 μL of the cell suspension was subcutaneously injected into the right side of hPD-1-C57BL6 mice. The tumor growth of the mice was then continuously observed, and the long and short axes of the tumor were measured with a vernier caliper. The tumor volume reached 100 mm3 (tumor volume = length × width 2 × 0.5) about 10 days after inoculation. The mice were randomly divided into 4 groups (6 mice in each group): saline group, fusion protein dual antibody group (PV2-PV3), Durvalumab group, and non-natural amino acid constructed dual antibody group (PV2-S71 + PV3-N77). The four groups of mice were administered intraperitoneally every three days at a concentration of 10 mg / kg. The weight and tumor volume of the mice were recorded every two days. A total of six administrations were performed during the experiment, including 2000 mm 3 This is a humanitarian endpoint. When the volume exceeds this value or ulceration occurs in a large area of ​​the tumor, the mice are euthanized. After three days of rest after the last administration, all the remaining mice are euthanized. Fresh blood is collected, left to stand at 4°C overnight, and then centrifuged at 3000 rpm for 10 minutes at 4°C to separate the serum.

[0310] The results are as follows Figures 18A-18D As shown, Fig.18A The figure is a schematic diagram of the change of tumor volume over time. The final results show that on the 18th day, there was no significant difference in tumor volume between the fusion protein dual antibody treatment group (PV2-PV3) and the normal saline control group (p=0.1775, n=6), while the tumor volume of the positive control group (Durvalumab) and the non-natural amino acid constructed dual antibody group (PV2-S71+PV3-N77) was significantly different from that of the PBS group (p<0.05, p<0.01, n=6). During the entire treatment cycle, the average tumor volume of the PV2-S71+PV3-N77 group was always significantly lower than that of the PV2-PV3 group. The tumor volume reached its maximum on the 14th day and then showed a downward trend, while the PV2-PV3 group showed no obvious downward trend. The experimental results show that the constructed PV2-S71+PV3-N77 bispecific anti-PD-L1 nanoantibody has better anti-tumor activity. Fig.18B It can be seen that there was no significant change in body weight of the four groups of mice during the experiment, that is, the therapeutic antibody PV2-S71+PV3-N77 had no obvious toxic side effects and had no significant effect on the normal growth and health of mice. Figures 18C-18D The subcutaneous tumor tissues obtained after the mice were killed on the 18th day were photographed. One mouse in the normal saline group was killed early during the experiment due to the large tumor volume.

[0311] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A Nanobody that specifically binds to PD-L1, comprising CDR1 shown in SEQ ID NO: 2, CDR2 shown in SEQ ID NO: 3, and CDR3 shown in SEQ ID NO: 28, and comprising a non-natural amino acid substitution at one or more positions corresponding to positions 77, 8, 23, 26, 70, 71, 85, 87, and 119 of SEQ ID NO: 27; Preferably, the Nanobody is a mutant of a wild-type Nanobody whose sequence is shown in SEQ ID NO: 27, and the Nanobody comprises a substitution of an unnatural amino acid at one or more positions 77, 8, 23, 26, 70, 71, 85, 87, 119 compared to the sequence shown in SEQ ID NO: 27; Preferably, 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; Preferably, the unnatural amino acid is pAcF.

2. A nanobody that specifically binds to PD-L1, wherein: The Nanobody comprises any one sequence selected from SEQ ID NO:41, SEQ ID NO:101, SEQ ID NO:31, SEQ ID NO:91, SEQ ID NO:33, SEQ ID NO:93, SEQ ID NO:35, SEQ ID NO:95, SEQ ID NO:37, SEQ ID NO:97, SEQ ID NO:39, SEQ ID NO:99, SEQ ID NO:43, SEQ ID NO:103, SEQ ID NO:45, SEQ ID NO:105, SEQ ID NO:47 and SEQ ID NO:

107.

3. A Nanobody that specifically binds to PD-L1, comprising CDR1 shown in SEQ ID NO: 2, CDR2 shown in SEQ ID NO: 3, and CDR3 shown in SEQ ID NO: 4, and comprising a non-natural amino acid substitution at one or more positions corresponding to positions 71, 8, 23, 26, 70, 74, 82, 85, and 96 of SEQ ID NO: 1; Preferably, the Nanobody is a mutant of a wild-type Nanobody having a sequence as shown in SEQ ID NO: 1, and the Nanobody comprises a substitution of an unnatural amino acid at one or more of positions 71, 8, 23, 26, 70, 74, 82, 85, and 96 compared to the sequence shown in SEQ ID NO: 1; Preferably, 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; Preferably, the unnatural amino acid is pAcF.

4. A nanobody that specifically binds to PD-L1, wherein: The Nanobody comprises any one sequence selected from SEQ ID NO: 17, SEQ ID NO: 79, SEQ ID NO: 9, SEQ ID NO: 71, SEQ ID NO: 11, SEQ ID NO: 73, SEQ ID NO: 13, SEQ ID NO: 75, SEQ ID NO: 15, SEQ ID NO: 77, SEQ ID NO: 19, SEQ ID NO: 81, SEQ ID NO: 21, SEQ ID NO: 83, SEQ ID NO: 23, SEQ ID NO: 85, SEQ ID NO: 25 and SEQ ID NO:

87.

5. A double nanobody construct comprising 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 PD-L1, and the second antigen is the same as or different from the first antigen; the first VHH is the Nanobody of claim 1 or 2, and the first VHH and the second VHH are connected by a linker at the site of the non-natural amino acid replacement; Preferably, the second antigen is PD-L1, and the second VHH is the Nanobody of claim 3 or 4; Preferably, the amino acid in any one of the positions corresponding to positions 77, 8, 23, 26, 70, 71, 85, 87 and 119 of SEQ ID NO: 27 of the first VHH is substituted with pAcF, and the amino acid in any one of the positions corresponding to positions 71, 8, 23, 26, 70, 74, 82, 85 and 96 of SEQ ID NO: 1 of the second VHH is substituted with pAcF; Preferably, the amino acid substitution of the first VHH in the position corresponding to position 77 or 85 of SEQ ID NO: 27 is pAcF, and the amino acid substitution of the second VHH in the position corresponding to position 26 or 71 of SEQ ID NO: 1 is pAcF; Preferably, the first VHH and the second VHH are connected via a linker at the site replaced with pAcF; Preferably, the first VHH and the second VHH are connected by Bicyclononyne and Tetrazine; Preferably, the first VHH comprises any one of SEQ ID NO: 41, SEQ ID NO: 101, SEQ ID NO: 43 and SEQ ID NO: 103, and / or the second VHH comprises any one of SEQ ID NO: 13, SEQ ID NO: 75, SEQ ID NO: 17 and SEQ ID NO: 79; Preferably, (1) the first VHH comprises the sequence shown in SEQ ID NO:41, and the second VHH comprises the sequence shown in SEQ ID NO:17; (2) the first VHH comprises the sequence shown in SEQ ID NO:41, and the second VHH comprises the sequence shown in SEQ ID NO:13; (3) the first VHH comprises the sequence shown in SEQ ID NO: 43, and the second VHH comprises the sequence shown in SEQ ID NO: 13; (4) the first VHH comprises the sequence shown in SEQ ID NO: 43, and the second VHH comprises the sequence shown in SEQ ID NO: 17; (5) the first VHH comprises the sequence shown in SEQ ID NO: 101, and the second VHH comprises the sequence shown in SEQ ID NO: 79; (6) the first VHH comprises the sequence shown in SEQ ID NO: 101, and the second VHH comprises the sequence shown in SEQ ID NO: 75; (7) the first VHH comprises the sequence shown in SEQ ID NO: 103, and the second VHH comprises the sequence shown in SEQ ID NO: 75; or (8) The first VHH comprises the sequence shown in SEQ ID NO:103, and the second VHH comprises the sequence shown in SEQ ID NO:

79.

6. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the Nanobody according to any one of claims 1 to 4, or the double Nanobody construct according to claim 5; in the nucleotide sequence, the codon corresponding to the position replaced by the non-natural amino acid is TAG; Preferably, the isolated nucleic acid molecule comprises a nucleic acid molecule selected from the group consisting of SEQ ID NO:32, SEQ ID NO:92, SEQ ID NO:34, SEQ ID NO:94, SEQ ID NO:36, SEQ ID NO:96, SEQ ID NO:38, SEQ ID NO:98, SEQ ID NO:40, SEQ ID NO:100, SEQ ID NO:42, SEQ ID NO:102, SEQ ID NO:44, SEQ ID NO:104, SEQ ID NO:46, SEQ ID NO:106, SEQ ID NO:48, SEQ ID NO:108, SEQ ID NO:10, SEQ ID NO:72, SEQ ID NO:12, SEQ ID NO:74, SEQ ID NO:14, SEQ ID NO:76, SEQ ID NO:16, SEQ ID NO:78, SEQ ID NO:18, SEQ ID NO:80, SEQ ID NO:20, SEQ ID NO:82, SEQ ID NO:22, SEQ ID NO:84, SEQ ID NO: Any one of SEQ ID NO:24, SEQ ID NO:86, SEQ ID NO:26 and SEQ ID NO:

88.

7. A vector comprising the nucleic acid molecule according to claim 6; preferably, the vector is a cloning vector or an expression vector.

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

9. A method for preparing the Nanobody of any one of claims 1 to 4 or the double Nanobody construct of claim 5, comprising: - co-transfecting a host cell with 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 medium containing an unnatural amino acid; Preferably, the non-natural amino acid is pAcF; preferably, the aminoacyl-tRNA synthetase specific for the non-natural amino acid is a pAcF-specific aminoacyl-tRNA synthetase (such as Mj-TyrRS).

10. An antibody-drug conjugate comprising a nanobody as described in any one of claims 1 to 4, or a double nanobody construct as described in claim 5, 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.

11. A conjugate comprising the Nanobody of any one of claims 1 to 4, or the double Nanobody construct of claim 5, and a coupling moiety; Preferably, the coupling moiety is selected from a protein tag, such as a purification tag; a detectable label, such as an enzyme (e.g. horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g. chemiluminescent substance) or biotin; a therapeutic agent, such as an anti-tumor drug; or, another biologically active polypeptide.

12. A pharmaceutical composition comprising the Nanobody according to any one of claims 1 to 4, the double Nanobody construct according to claim 3, the isolated nucleic acid molecule according to claim 6, the vector according to claim 7, the host cell according to claim 8, the antibody-drug conjugate according to claim 10, or the conjugate according to claim 11; and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises another pharmaceutically active agent, such as an anti-tumor drug.

13. Use of the Nanobody of any one of claims 1 to 4, the bi-Nanobody construct of claim 5, the isolated nucleic acid molecule of claim 6, the vector of claim 7, the host cell of claim 8, the antibody-drug conjugate of claim 10, the conjugate of claim 11, or the pharmaceutical composition of claim 12 for the preparation of a drug for preventing and / or treating a disease associated with PD-L1 in a subject; Preferably, the disease associated with PD-L1 is a solid tumor; Preferably, the disease associated with PD-L1 is selected from: non-small cell lung cancer, small cell lung cancer, bladder cancer, triple-negative breast cancer, hepatocellular carcinoma, renal cell carcinoma, esophageal cancer, or Merkel cell carcinoma; Preferably, the subject is a mammal, such as a human; Preferably, the Nanobody, diabody construct, isolated nucleic acid molecule, vector, host cell, conjugate or pharmaceutical composition is used alone or in combination with another pharmaceutically active agent (eg, an anti-tumor drug).

14. A method for detecting the presence or level of PD-L1 in a sample, comprising using the Nanobody of any one of claims 1 to 4, the double Nanobody construct of claim 5, or the conjugate of claim 11; Preferably, the method is an immunological assay, such as immunoblotting, enzyme immunoassay (eg ELISA), chemiluminescent immunoassay, fluorescent immunoassay or radioimmunoassay.

15. Use of the Nanobody of any one of claims 1 to 4, the double Nanobody construct of claim 5, or the conjugate of claim 11 in the preparation of a method for detecting the presence or level of PD-L1 in a sample, or in the preparation of a detection reagent for diagnosing whether a subject has a disease associated with PD-L1.

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