Unnatural amino acid modified anti-PD-L1 nanobody

Abstract

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, wherein one or more of positions 8, 23, 26, 70, 71, 77, 85, 87, and 119 of the Nanobody is a non-natural amino acid. The present invention also relates to a double Nanobody construct comprising the Nanobody, nucleic acid molecules, vectors, and host cells encoding the same, and their use for the treatment of diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular, to anti-PD-L1 nanobodies modified with non-natural amino acids, as well as double nanobody constructs comprising the same, nucleic acid molecules, vectors and host cells encoding the same, and their use for treating diseases. Background Art

[0002] PD-L1 (Programmed Death-Ligand 1) plays an important role in the immune system and is often 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 facilitating 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 immune system's response, thereby helping tumors evade 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, which 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, is approved for the treatment of advanced non-small cell lung cancer and urothelial carcinoma. Avelumab (Bavencio), jointly developed by Pfizer and Merck KGaA, is 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 limited therapeutic effects on digestive system tumors such as colorectal cancer. There is an urgent need to develop anti-PD-L1 nanoantibodies with small molecular weight and high affinity.

[0004] Nanobodies ( Nanobodies (VHH) are recombinant single-domain antibodies cloned from the variable region genes of heavy-chain antibodies. They are the smallest fragment known to date that can bind to antigens. In 1993, immunologist Hamers-Casterman first reported a new type of antibody from camelids - heavy-chain antibody (hcIgG) in Nature. Compared with traditional antibody IgG, hcIgG has no light chain and does not have the first constant CH1 domain in the heavy chain. It is composed only of the variable antigen-binding domain (VHH) and heavy chain CH2 and CH3. Nanobodies have the same structure as human immunoglobulin VH: 4 framework regions (FR1-4) and 3 complementary variable regions (CDR1-3). Antigen-antibody specific binding mainly depends on the CDR3 of nanobodies, while the antigen-antibody binding strength depends on CDR1 and CDR2. The average length of the CDR3 of nanobodies is 18 amino acids, which is much longer than the CDR3 length of human or mouse VH. The longer CDR3 also makes the structure of nanobodies more flexible, and after binding to the antigen, they can be fixed in a single structure. The disulfide bonds within the CDR region further enhance the stability of nanobodies. As a prominent representative of miniaturized antibodies, nanobodies are only 1 / 10 the size of full-length antibodies and possess excellent tissue penetration, structural stability, and resistance to high temperatures, pH, and osmotic pressure. Furthermore, VHHs have an amino acid sequence highly homologous to human VHs, making them less immunogenic and easier to humanize.

[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 comparison, the cysteine-based method is more selective, but due to the multiple disulfide bonds in the antibody molecule, the reaction is more complex and it is more difficult to obtain a uniform target product. The traditional method of constructing 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 repeat sequences, resulting in the possibility of a single overall sequence. The conformation of this bispecific nanoantibody often shows an adverse effect on the binding affinity between the antigen and the antibody. This phenomenon may be due to the steric hindrance effect blocking the normal binding between the antigen and the antibody. In contrast to the above methods, the chemical coupling method based on unnatural amino acids can be used as a simple, high-yield and universal method to generate bispecific antibodies or ADC drugs of any design. Summary of the Invention

[0006] The inventors of this 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, as well as bi-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 the CDR1 shown in SEQ ID NO: 2, the CDR2 shown in SEQ ID NO: 3, and the 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.

[0009] As used herein, the expression “the Nanobody at the positions 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 the equivalent positions to 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 achieve the highest percentage identity.

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

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

[0012] In certain embodiments, the Nanobody is a mutant of a wild-type Nanobody whose sequence is 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, or 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-azidopiperidinane (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, comprising the CDR1 shown in SEQ ID NO: 2, the CDR2 shown in SEQ ID NO: 3, and the 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.

[0019] As used herein, the expression “the Nanobody at the positions 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 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 achieve the highest percentage identity.

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

[0021] In certain embodiments, one or more of positions 71, 8, 23, 26, 70, 74, 82, 85, 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, or 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-azidopiperidinane (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 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.

[0028] Bis-nanobody constructs

[0029] In a fifth aspect, the present invention provides a double nanobody construct, which comprises a first VHH that specifically binds to a first antigen and a second VHH that specifically binds to a second antigen, wherein the first antigen is PD-L1 and the second antigen is the same as or different from the first antigen; the first VHH is the nanobody according to the first or second aspect of the 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 substitution of the first VHH at 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 is replaced by pAcF, and the amino acid substitution of the second VHH at 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 is replaced by pAcF.

[0033] In certain embodiments, the first VHH comprises any one of the sequences 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, 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 at the position corresponding to position 77 or 85 of SEQ ID NO: 27 of the first VHH is pAcF, and the amino acid substitution at the position corresponding to position 26 or 71 of SEQ ID NO: 1 of the second VHH 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 by a linker at the site of substitution 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 via 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 linked 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 of the first, second, third or fourth aspect of the invention or the dual Nanobody construct of the fifth aspect of the invention; in the nucleotide sequence, the codon corresponding to the position replaced by the non-natural amino acid is TAG.

[0054] In certain embodiments, the isolated nucleic acid molecule comprises a sequence 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, or 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 (e.g., 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 bi-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 culture 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 an 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 invention or the double Nanobody construct of the fifth aspect of the 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, and examples thereof include, but are not limited to, His, Flag, GST, MBP, HA, Myc, GFP, or biotin, and those skilled in the art know how to select an appropriate protein tag (e.g., a purification tag, a detection tag, or a tracing tag) according to the desired purpose.

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

[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 additional biologically active polypeptides.

[0069] In an eleventh aspect, the present invention provides an antibody-drug conjugate comprising the Nanobody of the first, second, third or fourth aspect of the invention, or the bi-Nanobody construct of the fifth aspect of the invention, and a coupling portion; wherein the coupling portion is a cytotoxic drug, and the coupling portion is connected to the Nanobody or bi-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 (PDB), indole benzodiazepines (IGN) or its analogs, antibiotics (e.g., dactinomycin (formerly known as actinomycin), bleomycin, daunorubicin (formerly known as daunorubicin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)), anti-mitotic agents (e.g., microtubule targeting agents), such as diphtheria toxin and related molecules (e.g., diphtheria A chain and active fragments and hybrid molecules thereof), ricin (e.g., ricin A or deglycosylated ricin A chain toxin), cholera toxin, Shiga-like toxins (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, Pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin, saponin, modeccin, gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, pokeweed proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotonin, saponin inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin toxin, antimicrobial / lytic peptides (e.g., CLIP, Magainin 2, melittin, cecropin, and P18), ribonuclease (RNase), DNase I, Staphylococcus enterotoxin-A, and Pseudomonas endotoxin.

[0072] Pharmaceutical composition

[0073] In the twelfth aspect, the present invention provides a pharmaceutical composition comprising 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, the isolated nucleic acid molecule described in the sixth aspect of the invention, the vector described in the seventh aspect of the invention, the host cell described in the eighth aspect of the invention, the antibody-drug conjugate described in the eleventh aspect of the invention, or the conjugate described in the tenth aspect of the 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 Nanobody, bis-Nanobody construct, isolated nucleic acid molecule, vector, host cell, antibody-drug conjugate or conjugate of the invention and the additional pharmaceutically active agent may be provided as separate components or as mixed components. Thus, the Nanobody, bis-Nanobody construct, isolated nucleic acid molecule, vector, host cell, antibody-drug conjugate or conjugate of the invention and the additional pharmaceutically active agent may be administered simultaneously, separately or sequentially.

[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 liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0078] The pharmaceutical compositions of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of a Nanobody, a bis-Nanobody construct, an isolated nucleic acid molecule, a vector, a host cell, an antibody-drug conjugate, or a conjugate as described herein. A "prophylactically effective amount" is an amount sufficient to prevent, arrest, or delay the onset of a disease. A "therapeutically effective amount" is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently.

[0079] Therapeutic applications

[0080] In a 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, second, third or fourth aspects 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 medicament 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, diabody construct, isolated nucleic acid molecule, vector, host cell, antibody-drug conjugate, conjugate or pharmaceutical composition is used alone or in combination with another pharmaceutically active agent (e.g., an anti-tumor drug).

[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 injection solutions, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use.

[0087] A preferred dosage form is an injection. Such an injection can be a sterile injectable solution. For example, a sterile injectable solution can be prepared by the following method: incorporating the necessary dose of the nanobody or bi-nanobody construct of the invention into an appropriate solvent, and optionally, simultaneously incorporating other desired ingredients (including but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonicity agents, preservatives, diluents, or any combination thereof), followed by filtration sterilization. In addition, the sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powder can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0088] The nanobodies of the invention, the bis-nanobody constructs, the isolated nucleic acid molecules, the vectors, the host cells, the antibody-drug conjugates, the conjugates or the pharmaceutical compositions of the invention can be administered by any suitable method known in the art, including but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powders, ointments or drops), or nasal routes. However, for many therapeutic uses, the preferred route / mode of administration is parenteral administration (e.g., intravenous or bolus, subcutaneous, intraperitoneal, intramuscular). It will be understood by the skilled person that the route and / or mode of administration will vary depending on the intended purpose. In certain embodiments, the nanobodies of the invention, the bis-nanobody constructs, the isolated nucleic acid molecules, the vectors, the host cells, the antibody-drug conjugates or the conjugates or the pharmaceutical compositions of the invention are administered by intravenous or bolus injection.

[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, 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.

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

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

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

[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, diabody construct or 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 has a disease associated with PD-L1. In such embodiments, the method may further comprise a step of comparing the amount of 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 is increased relative to a negative reference value, it indicates that the subject has a disease associated with PD-L1.

[0102] In certain embodiments, the PD-L1-related disease is characterized by elevated PD-L1 expression and / or excessive PD-L1 activity. In certain embodiments, the PD-L1-related disease is a solid tumor. In certain embodiments, the PD-L1-related disease is selected from the group consisting of 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, and the like.

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

[0105] In the fifteenth aspect, there is provided the use of the nanobody of the first, second, third or fourth aspect of the invention, the double nanobody construct of the fifth aspect of the invention, or the conjugate of 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 or diabody construct used to prepare the detection reagent is detectably labeled.

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

[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 includes the number of atoms, atom names, atom types, atomic coordinates, amino acid names, polypeptide chain length, and the number of polypeptide chains, for example, in the form of a protein PDB file;

[0112] (2) using the three-dimensional structural 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 six-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 the unnatural amino acid pAcF) to obtain the average Cartesian ddG of the six-frame Nanobody mutants, 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., the 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 a 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 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 biochemical experiments 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, immunoblotting (Western Blot), and Tm value detection.

[0128] In certain embodiments, the biochemical experiment comprises an experiment to detect 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 level of the Nanobody mutant, including but not limited to: enzyme-linked immunosorbent assay (ELISA), immunoblotting (Western Blot), immunoprecipitation, immunohistochemistry, and mass spectrometry.

[0130] Definition of terms

[0131] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, procedures in cell culture, molecular biology, biochemistry, nucleic acid chemistry, immunology, and the like used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0132] When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[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) are to 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 typically 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 T cell activity, thereby promoting immune escape and suppressing immune responses. 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 monomeric variable antibody domain (e.g., a single heavy chain variable region), typically derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or a shark antibody). Typically, a nanobody consists of four framework regions and three complementarity determining regions, with a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A nanobody may be truncated at the N-terminus or C-terminus so that it comprises only a portion of FR1 and / or FR4, or lacks one or two of those framework regions, as long as it substantially maintains antigen binding and specificity. Nanobodies are also referred to as single-domain antibodies (sdAbs), and the two are used interchangeably.

[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, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given Nanobody, a person skilled in the art will easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003).

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

[0138] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its targeted antigen. The strength or affinity of a specific binding interaction can be expressed as the equilibrium dissociation constant (KD) of the interaction. In the present invention, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0139] The specific binding properties between two molecules can be measured using methods well known in the art. One method involves measuring the speed of formation and dissociation of antigen binding sites / antigen complexes. Both "association rate constant" (ka or kon) and "dissociation rate constant" (kdis or koff) can be calculated by concentration and the actual rate of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis / kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). KD, kon and kdis values ​​can be measured by any effective method. In certain embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR). In addition, the dissociation constant can also be measured using bioluminescence interferometry or Kinexa.

[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 and RNA, and its length is not particularly limited. For nucleic acids used to construct recombinant constructs, it is preferably DNA because DNA is more stable and easier to handle than RNA.

[0141] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[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. A host cell may include a single cell or a cell population.

[0143] As used herein, the term "identity" refers to the match between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match). Typically, two sequences are compared when they are aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0144] The twenty conventional amino acids referred to herein are compiled according to conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[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, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like.

[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 related to 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 partial or complete), 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 double nanobody constructs by coupling the nanobodies substituted with non-natural amino acids obtained by screening. The double nanobody constructs have better specificity and lower off-target toxicity and are of important clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0150] Figures 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] Figures 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] Figures 5A-5B : Calculation results of the hydrogen bond numbers 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, Figure 9A is p-acetylphenylalanine (pAcF), Figure 9B Tetrazine (TET) is the linker. Figure 9C The linker is Bicyclononyne (BCN).

[0158] Figures 10A-10B : Electrophoresis diagram of PV2 and PV3 mutant proteins, among which, Figure 10A PV2 monomer, 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; Figure 10BIt is a PV3 monomer. 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, PV3-G119.

[0159] Figures 11A-11B : Tm values ​​of nanoantibodies in PV2 and PV3 groups (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, Figure 11A It is the PV2 group, Figure 11B It is the PV3 group.

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

[0161] Figure 13 : Schematic diagram of the construction principle of the double nanobody construct.

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

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

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

[0165] Figure 17 : Flowchart of animal experiments.

[0166] Figures 18A-18D : Animal experiment results, among which, Figure 18A is the change of tumor volume over time; Figure 18B The weight of mice changes over time; Figure 18C is the weight of tumor tissue collected after the mice in each group were sacrificed; Figure 18D Figures are 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 will now be described in the following non-limiting examples.

[0181] Those skilled in the art will appreciate that the examples are provided to illustrate the present invention by way of example and are not intended to limit the scope of the invention. The experimental methods in the examples are conventional methods unless otherwise specified. Where specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional products.

[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 using Alphafold2 online modeling, 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 in the figure, using the nanobody PV2 as an example, the nanobody sequence was first subjected to structure prediction using Alphafold2 and CDR region prediction using an online tool. Subsequently, MD simulations were performed on the wild-type nanobody PV2 and the PD-L1 antigen. Six equilibrium conformations of PV2 were selected, with an amino acid in PV2 mutated to the unnatural amino acid pAcF. Rosetta Cartesian ddG was used to calculate the six conformations, and the average Cartesian ddG values ​​of the six mutants were obtained and ranked from low to high. The top-ranked mutants were selected and analyzed for parameters such as RMSD, RMSF, hydrogen bonds, and salt bridges, and experimentally validated. Simultaneously, the equilibrium structures of the wild-type nanobody PV2 and the PD-L1 antigen obtained from MD simulations were docked using ZDOCK. A single amino acid in PV2 was mutated to the unnatural amino acid pAcF. Rosetta Flex ddG was used to calculate the affinity maturation of the PV2-PD-L1 complex, and the Flex ddG values ​​were obtained and ranked from low to high. Combining the results of Cartesian ddG, Flex ddG and CDR region predictions, 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. 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 using Alphafold2, with the best of the five optimal structures generated by Alphafold2 being selected. The antigen structures were obtained from the RCSB crystallographic database, with crystal water molecules and irrelevant residues removed from the PDB. The CDR regions of the nanobodies were predicted using 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 in Rosetta was used to calculate the changes in nanobody stability before and after the unnatural amino acid pAcF substitution mutation. In the Cartesian_ddg protocol, the protein-ligand complex model with the lowest score after energy minimization using the relax command was used as input to 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. Rosetta CartesianddG was used to calculate the six-frame conformational equilibrium of the nanobody MD simulation. The average Cartesian ddG score for the six mutants was obtained and ranked from low to high. Finally, several mutants with the highest Cartesian ddG rankings for the two nanobodies were selected. The screening results are shown in Table 2.

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

[0191]

[0192] Based on the Rosetta Cartesian ddG results, the top six sites with the highest average Cartesian ddG values ​​in PV2 and PV3 were selected. The top mutants all had similar scores, around -9 kcal / mol. Therefore, further MD analysis and experiments will be needed to confirm that these sites are optimal mutation sites and verify the accuracy of the Cartesian ddG calculations.

[0193] (2) MD simulation

[0194] like Figure 1 As shown in Figure 2, the first MD simulations were performed at 300 K for the wild-type and different mutant systems of the nanobody, as well as the antigen system. PROPKA3 was used to predict the protonation state of the protein at the experimental pH. The MD simulations were run in Gromacs 2023 using the CHARMM36 protein force field. The harmonic bias potential force constant was 1000 kJ mol. -1 nm 2 . Sodium ions and chloride ions were added to the solution to maintain the neutrality of the system. 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, and then a 200 ps simulation was run 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 performed, and the equilibrium conformation was collected at intervals of 10 ps.

[0195] Throughout the simulation, a velocity-regulated thermostat with a time constant of 0.1 ps was used (see Giovanni Bussi, (2007). Canonical sampling through velocity rescaling. THE JOURNAL OF CHEMICAL PHYSICS, 126.) to maintain 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. The pressure time constant and isothermal compressibility were set to 2 ps and 4.5 × 10, respectively. -5 bar -1 The integration time step of the equation of motion is 2 fs throughout the simulation. The cutoff value for non-bonded interactions is The particle mesh Ewald algorithm is used to calculate long-range electrostatic interactions.

[0196] The 50ns structure in the MD simulation is selected and the mutation results in PV2 and PV3 are marked. The result diagram is shown as follows: Figures 2A-2B As shown in the figure, 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 two groups of nanoantibodies were analyzed after 10ns of MD simulation. Figures 3A-3B shown.

[0198] The black lines in the figure correspond to the RMSD fluctuations of the respective wild-types. Using this as a benchmark, T116pAcF exhibits significantly greater fluctuations than the wild-type, while the remaining mutants, S71pAcF, S26pAcF, S8pAcF, S85pAcF, and Q82pAcF, all display lower and more stable RMSD curves than the wild-type. This suggests that T116F is less stable than the wild-type, while the remaining mutants exhibit better stability, with S26pAcF and S85pAcF showing the best performance. In PV3, N77pAcF, S8pAcF, S26pAcF, and Q82pAcF exhibit significantly greater fluctuations than the wild-type, while S71pAcF exhibits a significantly more stable RMSD than the wild-type. The remaining mutants, K87pAcF and S85pAcF, exhibit RMSD curves similar to those of the wild-type. It can be speculated that the stability of N77pAcF, S8pAcF, S26pAcF and Q82pAcF is worse than that of the wild type, the stability of S71pAcF is relatively good, and the stability of the other mutants is similar to that of the wild type.

[0199] In addition, the RMSF of the two groups of nanoantibodies were analyzed after 10ns of MD simulation equilibrium. Figures 4A-4BAs shown in the figure, the black lines correspond to the RMSF fluctuations of the respective wild-type mutants. Using this as a standard, in PV2, the Cα of T116pAcF exhibits significantly greater fluctuations than the wild-type, while the remaining mutants (S71pAcF, S26pAcF, S8pAcF, S85pAcF, and Q82pAcF) all display RMSF curves similar to the wild-type. This suggests that T116F is less stable than the wild-type, while the remaining mutants all have similar stability to the wild-type. In PV3, the Cα of S8pAcF, S26pAcF, Q82pAcF, N77pAcF, and K87pAcF exhibits significantly greater fluctuations than the wild-type, while the remaining mutants, S85pAcF and S71pAcF, display 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: Figures 5A-5B shown.

[0201] Compared to the wild type, the number of stable hydrogen bonds decreased in all mutants in the PV2 group. However, the decreases in S71pAcF and S85pAcF were smaller, but they still had the highest number of hydrogen bonds among all mutants. S26pAcF and T116pAcF showed the largest decrease, with T116pAcF showing the greatest reduction, suggesting that T116pAcF may have poorer stability. In the PV3 group, the number of stable hydrogen bonds in S71pAcF increased compared to the wild type. The number of stable hydrogen bonds in all remaining mutants decreased, while S26pAcF showed a smaller decrease, similar to that of the wild type. N77pAcF and S8pAcF showed the largest decrease. It is speculated that S71pAcF and S26pAcF have improved or maintained the same stability as the wild type, while the remaining mutants, K87pAcF, S85pAcF, and Q82pAcF, have decreased in stability, with N77pAcF and S8pAcF potentially showing poorer stability.

[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 four stable salt bridges. S71pAcF, S26pAcF, S8pAcF, S85pAcF, and Q82pAcF each have one additional stable salt bridge compared to the wild type, primarily 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 a significant effect on stability. The PV3 wild type has four stable salt bridges. S8pAcF and S85pAcF have two additional stable salt bridges compared to the wild type, primarily the Glu47-Arg39 and Glu45-Lys113 salt bridges. N77pAcF and K87pAcF each have one additional stable salt bridge compared to the wild type, primarily replacing the wild type's Glu89-Lys87 with the 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 during 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. However, 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 requires the construction of three non-natural amino acid files, namely parameters, rotator libraries, and unfolding energies. First, the molfile2params_polymer.py script is used to obtain the parameter file of the non-natural amino acid, and the new non-natural amino acid side chain is incorporated into Rosetta for design. Then, a rotator library related to the skeleton is created for the non-natural amino acid, and the unfolding energy is calculated for the non-natural amino acid (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 maturation after non-natural amino acid mutations are 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 was the average of the generalized additive model obtained from 35 iterations of the protocol.

[0207] In Rosetta, we established the computational parameters for p-acetylphenylalanine (pAcF), performed Rosetta Cartesian ddG calculations on the 6-frame MD equilibrium conformation of the nanobody, and performed Rosetta Flex ddG calculations on the 1-frame conformation of the antigen-nanobody complex obtained after docking. We plotted the energies of Rosetta Cartesian ddG and Flex ddG into a scatter plot, deleted the mutation sites in the CDR region, and found sites with relatively negative energies for both Cartesian ddG and Flex ddG, or sites with relatively negative energies for Cartesian ddG and Flex ddG within 1 kcal / mol. The above results are as follows: Figure 7 shown.

[0208] In the figure, the CDR region sites of each of the two groups of nanobodies are marked with green X scatter points. A total of five possible optimal mutations were found in the PV2 group: N74pAcF, S26pAcF, S8pAcF, S71pAcF, and Q82pAcF. Although the Flex ddG of S85pAcF exceeds 1 kcal / mol, its Cartesian ddG prediction is good, so it was still selected for subsequent experiments. The above sites are marked with pink scatter points. In addition, the energies of both Cartesian ddG and Flex ddG are found to be relatively positive for comparison in subsequent experiments. A total of three possible inferior mutations, I70pAcF, C96pAcF, and C23pAcF, were selected and marked with orange scatter points. A total of nine mutants were selected for subsequent experiments. Screening of the PV3 group was similar, resulting in the identification of five potential optimal mutations: S71pAcF, S26pAcF, S8pAcF, N77pAcF, and K87pAcF. Furthermore, S85pAcF, whose Flex ddG exceeded 1 kcal / mol but whose Cartesian ddG prediction was good, was also selected for further experiments. In addition, three potential inferior mutations were selected: I70pAcF, C23pAcF, and G119pAcF. A total of nine mutants were selected for further experiments.

[0209] Example 2: Construction of Nanobody Expression Plasmids with Unnatural Amino Acid Insertion - Construction of Single-Point Mutation Nanobodies and Mutant Nanobody Expression Plasmids 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 stored in the laboratory and inserted with the PV2 wild-type antibody gene (sequence shown in SEQ ID NO: 8) as a template, a TAG codon mutation was introduced into the target gene sequence through primer design. 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). 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. 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 PCR program. Set the extension time to 2 kb / min. The annealing temperature is generally set to 58°C, but can also be adjusted according to the Tm value of the primers. The PCR program settings are shown in Table 5.

[0219] Table 5: PCR reaction program

[0220]

[0221] (2) The PCR products were verified to be correct by nucleic acid gel electrophoresis, and then the PCR products were 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 each mutant nanobody. 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℃ 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. Mix gently and let it stand for 30 minutes. After standing, place the competent cells in a 42℃ water bath for heat shock for 90 seconds, then quickly transfer to ice and let it stand for 4 minutes. Finally, add 1 mL LB medium to mix the competent cells, place them at 37℃ for 1 hour, centrifuge at 6000 rpm for 2 minutes and discard 800 μL supernatant. After resuspending, spread it on a plate containing Amp resistance and culture it at 37℃ overnight. The next day, pick the monoclonal colony from the plate and culture it in LB medium containing Amp resistance. When the OD600 of the bacterial solution reaches 0.6, add 30% glycerol and store it at -80℃. At the same time, send the sample 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 compare it with 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) was inserted into the strain (shown), and the transformed strain was 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). The mutant nanobodies containing the unnatural amino acid pAcF were expressed using PV3-K87 (SEQ ID NO: 43), PV3-K87 (SEQ ID NO: 45), and PV3-G119 (SEQ ID NO: 47), 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 PV2 and PV3 wild type and mutants 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 Figure 9A ) solution, and at the same time, only 200 μL IPTG (isopropyl-β-d-thiogalactoside) solution was added to the negative control group, and cultured at 16°C and 160 rpm for 16 h.

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

[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 seconds of disruption and 3 seconds of rest, and the working time was 30 minutes.

[0235] (5) After ultrasonic disruption, centrifuge the bacterial suspension at 4°C and 10,000 rpm for 15 min. Aspirate the supernatant with a syringe and filter it through a 0.45 μm PES filter for later use.

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

[0239] 3) Replace the input liquid from deionized water with 80 mM imidazole solution and flow it into the prepacked column at a flow rate of 4 mL / min for 8-10 column volumes to equilibrate the prepacked column.

[0240] 4) Load the supernatant prepared in the previous step and select an infusion pump for injection at a 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, 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% and set the volume to 50 mL for gradient elution at a flow rate of 3 mL / min. Start collecting when the elution peak appears and continue collecting until the UV280 line flattens. Then, flush the line and column with deionized water until the line conductivity is essentially zero.

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

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

[0246] The 9 mutant nanobodies in the PV2 and PV3 groups were all successfully expressed as mutants introduced with pAcF. 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 PV2 group is about 13 kDa, and the molecular weight of the nanoantibodies in each PV3 group is about 14 kDa. The electrophoresis results are compared with the markers and 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] Differential scanning fluorescence was used to detect the amount of fluorescent dye bound to the structurally altered Nanobodies during heating, thereby evaluating the thermal stability of the Nanobodies. Detection was performed using a LightCycler 480 II fluorescent quantitative PCR instrument, and the reaction system was shown in Table 8.

[0249] Table 8: Differential Scanning Fluorescence Reaction System

[0250]

[0251] Each group of experiments was set up in parallel, and the system was protected from light throughout the preparation. After preparation, the samples were divided into 96-well plates and centrifuged at low speed to mix the system and remove bubbles. Then, the samples were 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-test. After the self-test 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 465 nm, and the emission wavelength is 580 nm. Set the Analysis Mode to Melting Curves, the Acquisition Mode to Continuous, the Ramp Rate to 0.01°C / s, and the Acquistions to 50 per °C.

[0254] (3) After the experiment, the data were exported and analyzed using the LightCycler Thermal Shift Analysis software (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 important to ensure that the stability of the altered protein conformation is not significantly reduced. Differential scanning fluorimetry was used to determine the Tm values ​​of the mutants and wild-type nanobodies in the PV2 and PV3 groups. This result can be used as a thermal stability criterion to screen for optimal 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] Figure 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; Figure 11B The thermal stability of each mutant in the PV3 group varies greatly, with more obvious differences within the group. The Tm value of the PV3 wild type is 51.96°C. Among them, the mutant with the worst stability is PV3-K87, with a Tm value of 44.42°C. The groups with significantly improved Tm values ​​include 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 binding to the antigen after the introduction of pAcF, the affinity of each mutant and the subsequently constructed anti-PD-L1 double nanobody construct was tested by indirect ELISA.

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

[0261] (ab280943, abcam) was coated in a 96-well plate and tested by ELISA. 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 of PBST solution to each well, and wash three times, each time for 5 minutes.

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

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

[0266] (5) Prepare gradient concentrations of nanoantibodies. The highest concentration of the nanoantibody monomer group is 100 μg / mL, and the highest concentration of the bivalent nanoantibody group is 400 μg / mL. Perform 4-fold gradient dilutions, with a total of 10 gradient concentrations. Set up a zero concentration control group (PBS) and incubate in a 37°C incubator for 2 h.

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

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

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

[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 a microplate reader.

[0272] The final results of each group were as follows Figures 12A-12D As shown, Figure 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. Figure 12C 、 12D Listed in.

[0273] The affinity of the PV2 wild type is 15.14 nM. The affinity of most mutants in the PV2 group was improved after the introduction of pAcF. Among them, the affinity of the PV2-S71 and PV2-S26 groups was significantly improved, reaching 1.702 nM and 1.571 nM, 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 37.41 nM. The affinity of the PV3 wild type was 51.01 nM. Among the PV3 mutants, the PV3-K87, PV3-C23, PV3-S85, and PV3-N77 groups had the most significant increase in affinity. Among them, the group with the highest affinity increase was PV3-K87, with an affinity of 13.67 nM, a 3.8-fold increase compared to the wild type.

[0274] In summary, the computer prediction-assisted screening method narrows the scope of experimental screening. Most of the predicted mutation sites have improved thermal stability and affinity compared with 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 adaptability to the development of other non-natural amino acid insertion antibodies based on nanobodies.

[0275] Example 6: Construction and evaluation of anti-PD-L1 bi-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-nanoantibody constructs was screened out. Taking into account the Tm value and affinity data of each mutant, the mutation sites in PV2 and PV3 that were most suitable for the construction of dual-nanoantibody constructs were selected. Finally, the PV2 group selected PV2-S26 (Tm value 3rd, affinity 1st), PV2-S71 (Tm value 2nd, affinity 2nd); the PV3 group selected PV3-S85 (Tm value 4th, affinity 3rd), PV3-N77 (Tm value 1st, affinity 4th). The mutants corresponding to the above different nanoantibodies were combined in pairs, and a total of four groups of dual-nanoantibody constructs were constructed. The construction principle of the dual-nanoantibody construct is as follows. Figure 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 into 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. One group was added with a linker, Bicyclononyne (BCN) (e.g. Figure 9C ), and the other group added the linker Tetrazine (TET) (such as Figure 9B ), the molar ratio of the linker added to the nanobody was 50:1, and the reaction was allowed to proceed at 30°C for 24h.

[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 nanobody-linker complexes after the reaction were measured using Nanodrop and the nanobody concentration was adjusted to 2 mg / mL. The two different complexes were mixed in equal volumes, and the mixed solution was placed at 30°C for reaction for 24 h.

[0280] (4) After 24 hours, the reaction solution was concentrated to 600-1000 μL using an ultrafiltration tube (10,000 MWCO) and the unconjugated nanobody monomers and small molecule linkers in the solution system were removed by size exclusion chromatography. The size exclusion purification steps are as follows:

[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 tubing until the baseline was flat.

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

[0284] 4) Fill the sample volume to 1 mL with PBS solution and inject it into the sample loop from the sample loop injection port. Select the sample loop injection mode at the same time, with an injection flow rate of 0.4 mL / min and an injection volume of 2 mL.

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

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

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

[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 bispecific antibody constructs were separated by size exclusion chromatography from the uncoupled free monoclonal antibody, and the coupling efficiency differences between the groups were compared by the ratio of the bispecific antibody peak area to the monoclonal antibody peak area in the chromatogram. The results of the four groups of size exclusion peaks are shown in FIG. Figures 14A-14D The peak areas and proportions are given in Table 9.

[0290] Table 9: Size exclusion chromatography peak area ratio 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 Jinweizhi Biotechnology Co., Ltd. and 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, Figure 15A , B. The successful construction of PV2-S71+PV3-N77 was verified by SDS-PAGE gel electrophoresis and MALDI-TOF mass spectrometry. Figure 15C , D, E The thermal stability and affinity of the two double-nanoantibody constructs, PV2-S71+PV3-N77 and fusion-expressed PV2-PV3, were detected by differential scanning fluorimetry and indirect ELISA, respectively, to compare the differences in the properties of the double-nanoantibody constructs obtained by different construction methods.

[0293] like Figure 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. The 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 higher thermal stability as a whole.

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

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

[0296] Anti-PD-L1 antibodies have 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 immunity. By co-culturing tumor cells HT29 and induced differentiated THP-1 cells, the cells are labeled with fluorescent staining, and the biological activity of anti-PD-L1 antibodies 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 to a final concentration of 80 ng / mL. THP-1 cells were cultured in the PMA-containing medium for 24 hours. After 24 hours, observation under an inverted microscope revealed that the cells had differentiated from suspension cells to adherent cells. After confirming that most cells were adherent, the PMA-containing 1640 medium was removed with a Pasteur pipette. After rinsing twice with PBS, the cells were transferred to PMA-free 1640 complete medium and allowed to rest for 24 hours to obtain induced differentiated human macrophages.

[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 ratio 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 treatment 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. 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 Macrophages were added into each well in a volume of 1 mL and cultured at 37°C for 2 h 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 trypsinized and resuspended in serum-free 1640 medium to adjust the cell density to 1 × 10 6 Cells were centrifuged at 4 °C for 10 min. After centrifugation at 1000 rpm for 5 min, the supernatant was discarded and the cells were washed twice with PBS. The cells were resuspended in 1640 complete medium and allowed to rest for 10 min. Then, each antibody was added at 10 μg / mL and incubated in a 37°C incubator for 1 h.

[0304] After the incubation, HT29 cells were plated at 2×10 5 Add 500 μL of Hoechst 33342 working solution to each well of a 12-well plate containing macrophages and incubate at 37°C for 3 hours. Wash twice with PBS. Remove Hoechst 33342 dye stored at -20°C and dilute 100-fold with PBS to the working concentration. Add 500 μL of 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. Carefully remove the cell slide from each well and transfer it to a laser confocal scanning microscope for imaging and examination.

[0305] The local image results in the microscope are as follows Figures 16A-16B As shown, three replicates were performed in each group, and over 100 images were taken for each experimental group. 100 blue fluorescently labeled macrophages were randomly selected from these samples. The number of macrophages exhibiting red-blue fusion and phagocytosis was observed and recorded, and the corresponding phagocytic rate for each group was calculated. The results showed that among the antibody drug groups, the phagocytic efficiency of the PV2-S71+PV3-N77 group and the Durvalumab 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 bi-nanobody constructs

[0307] To explore the effects of different groups of PD-L1-targeting antibody drugs on the growth of subcutaneous tumors of mouse colon cancer, hPD-L1-MC38 cells with humanized PD-L1 gene were injected into the right flank 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 human PD-L1-targeting antibodies.

[0308] Twenty-four female C57BL / 6 Smoc-Pdcd1em1 (hPDCD1) / Smoc mice (7 weeks old) expressing humanized PD-1 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 of acclimatization. Mice had free access to water and food under a standard 12-h dark:12-h light cycle.

[0309] like Figure 17 As shown, after the mice were acclimated for one week, a subcutaneous tumor model was established. 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.) and trypsinized. The cell pellet was collected by centrifugation, washed twice with PBS solution, and counted. 1×10 6 hPD-L1-MC38 cells were resuspended in sterile PBS solution containing 25% matrigel, 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. About 10 days after inoculation, the tumor volume reached 100 mm3 (tumor volume = length × width 2 × 0.5). The mice were randomly divided into four groups (6 per group): saline group, fusion protein dual antibody group (PV2-PV3), durvalumab group, and unnatural amino acid constructed dual antibody group (PV2-S71 + PV3-N77). The four groups of mice were administered with intraperitoneal injection every three days at a concentration of 10 mg / kg. The body 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 endpoint is a humanitarian endpoint. Euthanasia was performed when tumor volume exceeded this threshold or when extensive ulceration occurred within the tumor area. After three days of rest following the final dose, all remaining mice were euthanized. Fresh blood was collected and allowed to stand overnight at 4°C. Serum was then separated by centrifugation at 3000 rpm for 10 minutes at 4°C.

[0310] The results are as follows Figures 18A-18D As shown, Figure 18A It 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 had 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. Figure 18B It can be seen that there was no significant change in the weight of the four groups of mice during the experiment, that is, the therapeutic antibodies PV2-S71+PV3-N77 had no obvious toxic side effects and had no significant impact on the normal growth and health of the mice. Figures 18C-18D The subcutaneous tumor tissues were taken after the mice were killed on the 18th day. One mouse in the normal saline group was killed early during the experiment due to the excessive size of the tumor.

[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 variations may be made to the details based on all the teachings disclosed, and that such modifications are within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A nanobody that specifically binds to PD-L1, wherein: The sequence of the nanobody is shown in SEQ ID NO: 41 or SEQ ID NO: 101, wherein the amino acid at position X is pAcF.

2. A double nanobody construct, which consists of a first VHH that specifically binds to a first antigen, a second VHH that specifically binds to a second antigen, and a linker connecting the first VHH and the second VHH; in, The first antigen is PD-L1, and the first VHH is the nanobody of claim 1; The second antigen is PD-L1, and the sequence of the second VHH is shown in SEQ ID NO: 17 or SEQ ID NO: 79, wherein the amino acid at position X is pAcF; The pAcF of the first VHH and the pAcF of the second VHH are connected via Bicyclononyne and Tetrazine.

3. The bi-nanobody construct of claim 2, wherein: (1) the sequence of the first VHH is shown in SEQ ID NO: 41, and the sequence of the second VHH is shown in SEQ ID NO: 17; or (2) The sequence of the first VHH is shown in SEQ ID NO: 101, and the sequence of the second VHH is shown in SEQ ID NO:

79.

4. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the Nanobody of claim 1 or the dual Nanobody construct of claim 2 or 3; wherein the codon corresponding to the position substituted with the unnatural amino acid is TAG.

5. The isolated nucleic acid molecule of claim 4, comprising any one sequence selected from the group consisting of SEQ ID NO:42, SEQ ID NO:102, SEQ ID NO:18, and SEQ ID NO:

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

8. A host cell comprising the nucleic acid molecule according to claim 4 or 5 or the vector according to claim 6 or 7, wherein The host cell is a non-plant cell.

9. A method for preparing the Nanobody of claim 1 or the bis-Nanobody construct of claim 2 or 3, comprising: - co-transfecting a host cell with the isolated nucleic acid molecule of claim 4 or 5 or the vector of claim 6 or 7 and a vector encoding an amber codon suppressor tRNA and an aminoacyl-tRNA synthetase specific for an unnatural amino acid; - cultivating the host cell in a culture medium containing the unnatural amino acid.

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

11. The method of claim 9, wherein The aminoacyl-tRNA synthetase specific for unnatural amino acids is pAcF-specific aminoacyl-tRNA synthetase.

12. A conjugate comprising the Nanobody of claim 1 or the bis-Nanobody construct of claim 2 or 3, and a coupling moiety; the coupling moiety being selected from a purification tag, horseradish peroxidase, a radionuclide, a fluorescent dye or a chemiluminescent substance.

13. A pharmaceutical composition comprising the Nanobody of claim 1, the bis-Nanobody construct of claim 2 or 3, the isolated nucleic acid molecule of claim 4 or 5, the vector of claim 6 or 7, the host cell of claim 8, or the conjugate of claim 12; and a pharmaceutically acceptable carrier and / or excipient.

14. The pharmaceutical composition of claim 13, further comprising an additional pharmaceutically active agent.

15. The pharmaceutical composition according to claim 14, wherein The additional pharmaceutically active agent is an anti-tumor drug.

16. Use of the Nanobody of claim 1, the bis-Nanobody construct of claim 2 or 3, the isolated nucleic acid molecule of claim 4 or 5, the vector of claim 6 or 7, the host cell of claim 8, the conjugate of claim 12, or the pharmaceutical composition of any one of claims 13 to 15 for the preparation of a medicament for preventing and / or treating a disease selected from the group consisting of 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 in a subject.

17. The use according to claim 16, wherein The subject is a mammal.

18. The use according to claim 16, wherein The subject is a human.

19. The use according to claim 16, wherein The Nanobodies, diabody constructs, isolated nucleic acid molecules, vectors, host cells, conjugates or pharmaceutical compositions are used alone or in combination with another pharmaceutically active agent.

20. The use according to claim 19, wherein The additional pharmaceutically active agent is an anti-tumor drug.

21. A method for detecting the presence or level of PD-L1 in a sample for non-diagnostic purposes, comprising using the Nanobody of claim 1, the bis-Nanobody construct of claim 2 or 3, or the conjugate of claim 12.

22. The method of claim 21, wherein The method is an immunological assay.

23. The method of claim 22, wherein The immunological detection is selected from immunoblotting, enzyme immunoassay, chemiluminescent immunoassay, fluorescent immunoassay or radioimmunoassay.

24. The method of claim 23, wherein The enzyme immunoassay is ELISA.

25. Use of the Nanobody according to claim 1, the bis-Nanobody construct according to claim 2 or 3, or the conjugate according to claim 12 in the preparation of a detection reagent 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, wherein 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.

Images