CN111718415A - anti-TIGIT nano antibody and application thereof - Google Patents

anti-TIGIT nano antibody and application thereof Download PDF

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CN111718415A
CN111718415A CN202010632294.5A CN202010632294A CN111718415A CN 111718415 A CN111718415 A CN 111718415A CN 202010632294 A CN202010632294 A CN 202010632294A CN 111718415 A CN111718415 A CN 111718415A
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tigit
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nanobody
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CN111718415B (en
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万亚坤
朱敏
盖军伟
李光辉
乔鹏
沈晓宁
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Shanghai Luoqi Biomedical Technology Co ltd
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Abstract

The invention discloses an anti-TIGIT nano antibody and application thereof. Specifically, the invention provides an anti-TIGIT nano antibody and a sequence thereof. The invention also provides a coding sequence for coding the nano antibody or the VHH chain thereof, a corresponding expression vector, a host cell capable of expressing the nano antibody and a production method of the nano antibody. The nano antibody can block the interaction of TIGIT and CD155 on the cell surface of CT26, and can effectively combine TIGIT protein on the cell surface; the nano antibody can identify human and cynomolgus monkey TIGIT; the blocking activity of the nano antibody is obviously superior to that of a control antibody Tiragolumab; the nano antibody has obvious activation effect on T cells, and the activation effect is obviously superior to that of a control antibody Tiragolumab.

Description

anti-TIGIT nano antibody and application thereof
Technical Field
The invention relates to the technical field of biomedicine or biopharmaceutical, in particular to an anti-TIGIT nano antibody and application thereof.
Background
In recent years, the immune checkpoint protein of TIGIT has become one of the hot spots in the development field of cancer immunotherapy. The full names of TIGIT are T cell immunoglobulin and ITIM domain proteins (T cell immunoglobulin with Ig and ITIMDomains). It is an inhibitory receptor, expressed on the surface of many types of T cells. T cells express a number of different activating and inhibitory receptors on their surface, which often pair-wise to fine-tune T cell activity. TIGIT and the activating receptor CD226 are paired, and their common ligand is PVR (CD 155). TIGIT inhibits the activation of T cells by competing with CD226 and binding PVR, disturbing the activation of CD226 and other mechanisms. TIGIT is highly expressed in tumor infiltrating T cells of multiple cancer types.
The research shows that most tumor NK cells express TIGIT, the effect of the TIGIT in tumor immunosuppression is similar to that of PD-1/PD-L1, and a TIGIT inhibitor and a PD-1/PD-L1 inhibitor can play a synergistic antitumor effect. 18 th 2018, journal of nature-immunology (Nature Immunology) of the International Top-level journal, which published the Ministry of Life sciences and medicine of the university of Chinese science and technology, the Central academy of China's Natural immunity and Chronic disease emphasis laboratory, and the research paper Block of the research center of Fertilizer micro-scale substance science, Massachusetts of Shigngguang, Sun 27757, the subject group, the research results revealed that TIGIT (tungsten inert gas) as an inhibitory receptor can cause the exhaustion of NK cells in the process of tumor development, and that TIGIN can reverse the exhaustion of NK cells and be used for immunotherapy of various tumors. In the world, various pharmaceutical enterprises have been put into the development of TIGIT antibodies, such as tiragolumab of Roche, BMS-986207 of Bethes-Messaging (BMS), OMP-313M32 of OncoMed pharmaceuticals, MTIG7192A of Take, MK-7684 of the Min Shadong (MSD), AB154 of Arcus Biosciences, BGB-A1217 of TIGIT monoclonal antibody of Kyowa, HLX53 of Rehong Han, JS006 of Junchen organism, and mAb-7 of Standivid organism.
At present, patents such as CN109734806A, US20200062859A1, WO2019168382A1, WO2020020281A1, CN110997720A, CN110818795B, WO2019129221A1 and the like report TIGIT antibodies and related applications. Among them, patent WO2019129221a1 discloses a TIGIT construct comprising a single domain antibody (nanobody) specifically recognizing TIGIT, and patent CN110818795B discloses an anti-TIGIT antibody comprising a single domain antibody binding to TIGIT. At present, not less than 12 TIGIT antibodies enter clinic, but no TIGIT antibody is approved to be on the market all over the world, so that the development of new TIGIT antibodies with remarkable differentiation and more suitable for clinical application, in particular TIGIT nano antibodies, is still needed.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the invention provides an anti-TIGIT nano antibody and application thereof, and aims to provide a TIGIT antibody which is remarkably different and more suitable for clinical application.
The technical problem to be solved by the invention is realized by adopting the following technical scheme:
in a first aspect of the invention, there is provided a complementarity determining region CDR of an anti-TIGIT nanobody VHH chain, comprising one or more of the following combinations:
(1) CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 3;
(2) CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11, and CDR12 shown in SEQ ID NO. 12;
(3) CDR1 shown in SEQ ID NO. 19, CDR2 shown in SEQ ID NO. 20, and CDR3 shown in SEQ ID NO. 21.
The CDRs 1, 2 and 3 are separated by framework regions FR1, FR2, FR3 and FR 4.
In a second aspect of the invention, there is provided an anti-TIGIT nanobody VHH chain comprising a framework region FR comprising FRl, FR2, FR3 and FR4 and a complementarity determining region CDR of the first aspect of the invention, the framework region FR comprising one or more of the following combinations:
(a) FR1 shown by SEQ ID NO. 4, FR2 shown by SEQ ID NO. 5, FR3 shown by SEQ ID NO. 6, and FR4 shown by SEQ ID NO. 7;
(b) FR1 shown by SEQ ID NO. 13, FR2 shown by SEQ ID NO. 14, FR3 shown by SEQ ID NO. 15, and FR4 shown by SEQ ID NO. 16;
(c) FR1 shown by SEQ ID NO. 22, FR2 shown by SEQ ID NO. 23, FR3 shown by SEQ ID NO. 24, and FR4 shown by SEQ ID NO. 25.
Preferably, the VHH chain of the anti-TIGIT nano antibody is selected from one or more of SEQ ID NO 8, SEQ ID NO 17, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 29 and SEQ ID NO 30.
In a third aspect of the present invention, there is provided an anti-TIGIT nanobody that is a nanobody against a TIGIT epitope and has an anti-TIGIT nanobody VHH chain according to the second aspect of the present invention.
Preferably, the anti-TIGIT nanobody comprises a monomer, a bivalent antibody, and/or a multivalent antibody.
Preferably, the anti-TIGIT nanobody comprises one or more VHH chains having amino acid sequences shown as SEQ ID NO 8, SEQ ID NO 17, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30.
Preferably, the anti-TIGIT nanobody comprises any two VHH chains of the amino acid sequences shown as SEQ ID NO. 28, SEQ ID NO. 29 and SEQ ID NO. 30, and the VHH chains are connected through a linker.
Preferably, the linker is selected from the following sequences: (GaSb) x- (GmSn) y, wherein a, b, m, n, x, y is 0 or 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 (preferably, a is 4 and b is 1, m is 3 and n is 1).
Preferably, the linker is selected from the group consisting of: GGGGSGGGS are provided.
Preferably, the anti-TIGIT nanobody is capable of recognizing two different TIGIT epitopes.
Preferably, the amino acid sequence of the anti-TIGIT nano antibody is shown in SEQ ID NO. 34 or SEQ ID NO. 36
The fourth aspect of the invention provides an Fc fusion protein of an anti-TIGIT nano antibody, wherein the structure of the fusion protein from the N end to the C end is shown as formula Ia or Ib:
A-L-B(Ia)；
B-L-A(Ib)；
wherein the content of the first and second substances,
a is the anti-TIGIT nanobody of claim 4;
b is an Fc fragment of IgG; and
l is a non-or flexible linker.
Preferably, the flexible linker is a peptide linker.
Preferably, the peptide linker has 1-50 amino acids, preferably 1-20 amino acids.
Preferably, the Fc fragment of IgG comprises an Fc fragment of human IgG.
Preferably, the Fc fragment of IgG is selected from the group consisting of: an Fc fragment of IgG1, IgG2, IgG3, IgG4, or a combination thereof.
Preferably, the amino acid sequence of the Fc fragment is shown in SEQ ID NO 38.
Preferably, the fusion protein is a nanobody Fc fusion protein aiming at TIGIT epitope.
Preferably, the anti-TIGIT nanobody comprises a monomer, a bivalent antibody, and/or a multivalent antibody.
In a fifth aspect of the invention, there is provided a polynucleotide encoding a protein selected from the group consisting of: the complementarity determining region CDR of the anti-TIGIT nanobody VHH chain according to the first aspect of the present invention, or the anti-TIGIT nanobody VHH chain according to the second aspect of the present invention, or the anti-TIGIT nanobody according to the third aspect of the present invention, or the anti-TIGIT nanobody Fc fusion protein according to the fourth aspect of the present invention.
Preferably, the polynucleotide sequences are in combination and comprise one or more of SEQ ID NO 9, SEQ ID NO 18, SEQ ID NO 27, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33.
Preferably, the polynucleotide has a nucleotide sequence as shown in SEQ ID NO. 35 or SEQ ID NO. 36.
Preferably, the polynucleotide comprises RNA, DNA or cDNA.
In a sixth aspect of the invention, there is provided an expression vector comprising a polynucleotide according to the fifth aspect of the invention.
Preferably, the expression vector is selected from the group consisting of: DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.
Preferably, the expression vector comprises a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.
In a seventh aspect of the invention, there is provided a host cell comprising an expression vector according to the sixth aspect of the invention, or having a polynucleotide according to the fifth aspect of the invention integrated into its genome.
Preferably, the host cell comprises a prokaryotic cell or a eukaryotic cell.
Preferably, the host cell is selected from the group consisting of: escherichia coli, yeast cells, mammalian cells, bacteriophage, or combinations thereof
In an eighth aspect of the present invention, there is provided a method for producing anti-TIGIT nanobody and Fc fusion protein thereof, comprising the steps of:
(a) culturing the host cell according to the seventh aspect of the invention under conditions suitable for production of the anti-TIGIT nanobody according to the third aspect of the invention, thereby obtaining a culture comprising the anti-TIGIT nanobody according to the third aspect of the invention; or culturing a host cell according to the seventh aspect of the invention under conditions suitable for production of an anti-TIGIT nanobody Fc fusion protein according to the fourth aspect of the invention, thereby obtaining a culture comprising an anti-TIGIT nanobody Fc fusion protein according to the fourth aspect of the invention; and
(b) isolating and/or recovering the anti-TIGIT nanobody or the anti-TIGIT nanobody Fc fusion protein from the culture; and
(c) optionally, purifying and/or modifying the anti-TIGIT nanobody or the anti-TIGIT nanobody Fc fusion protein obtained in step (b).
Preferably, the anti-TIGIT nanobody comprises one or more VHH chains having amino acid sequences shown as SEQ ID NO 8, SEQ ID NO 17, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30.
Preferably, the amino acid sequence of the anti-TIGIT nano antibody is shown in SEQ ID NO. 34 or SEQ ID NO. 36.
In a ninth aspect of the invention, there is provided an immunoconjugate comprising:
(a) an anti-TIGIT nanobody VHH chain according to the second aspect of the invention, or an anti-TIGIT nanobody according to the third aspect of the invention, or an anti-TIGIT nanobody Fc fusion protein according to the fourth aspect of the invention; and
(b) a coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle/nanorod, a nanomagnet, a viral coat protein, or a VLP, or a combination thereof.
Preferably, the radionuclide comprises:
(i) a diagnostic isotope selected from the group consisting of: tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or combinations thereof; and/or
(ii) A therapeutic isotope selected from the group consisting of: lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or a combination thereof.
Preferably, the coupling moiety is a drug or toxin.
Preferably, the drug is a cytotoxic drug.
Preferably, the cytotoxic drug is selected from the group consisting of: an anti-tubulin drug, a DNA minor groove binding agent, a DNA replication inhibitor, an alkylating agent, an antibiotic, a folate antagonist, an anti-metabolite drug, a chemotherapeutic sensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.
Preferably, examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs include, for example, auristatins (auristatins), camptothecins (camptothecins), duocarmycins/duocarmycins (duocarmycins), etoposides (etoposides), maytansinoids (maytansinoids) and maytansinoids (e.g., DM1 and DM4), taxanes (taxanes), benzodiazepines (benzodiazepines), or benzodiazepine-containing drugs (e.g., pyrrolo [1,4] benzodiazepines (pbps), indoline benzodiazepines (olipids) and benzodiazepines (pbazolidines)), or combinations thereof.
Preferably, the toxin is selected from the group consisting of: auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), aureomycin, maytansinoid, ricin A-chain, combretastatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthrax toxin dione, actinomycin, diphtheria toxin, Pseudomonas Exotoxin (PE) A, PE40, abrin a chain, modeccin a chain, alpha-sarcina, gelonin, mitogelonin (mitogellin), restrictocin (rettstricon), phenomycin, enomycin, curcin (curcin), crotin, calicheamicin, soapwort (Sapaonaria officinalis) inhibitor, glucocorticoid, or a combination thereof.
Preferably, the conjugated moiety is a detectable label.
Preferably, the coupling moiety is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl-hydrolase-like protein (BPHL)), or nanoparticles of any form.
Preferably, the immunoconjugate comprises: a multivalent (e.g. bivalent) anti-TIGIT nanobody VHH chain according to the second aspect of the invention, an anti-TIGIT nanobody according to the third aspect of the invention, or an anti-TIGIT nanobody Fc fusion protein according to the fourth aspect of the invention.
Preferably, the multivalent refers to the anti-TIGIT nanobody VHH chain according to the second aspect of the present invention, the anti-TIGIT nanobody according to the third aspect of the present invention, or the anti-TIGIT nanobody Fc fusion protein according to the fourth aspect of the present invention, comprising multiple repeats in the amino acid sequence of the immunoconjugate.
In a tenth aspect of the invention, there is provided a use of the anti-TIGIT nanobody VHH chain of the second aspect of the invention, or the anti-TIGIT nanobody of the third aspect of the invention, or the anti-TIGIT nanobody Fc fusion protein of the fourth aspect of the invention for the preparation of a medicament for the diagnosis or treatment of a tumor.
Preferably, the tumor includes, but is not limited to: hematologic tumors, solid tumors, non-small cell lung cancer, colorectal cancer, melanoma, breast cancer, esophageal cancer, gastric tumors.
In an eleventh aspect of the present invention, there is provided a pharmaceutical composition comprising:
(i) a complementarity determining region CDR region of an anti-TIGIT nanobody VHH chain according to the first aspect of the invention, an anti-TIGIT nanobody VHH chain according to the second aspect of the invention, or an anti-TIGIT nanobody according to the third aspect of the invention, or an anti-TIGIT nanobody Fc fusion protein according to the fourth aspect of the invention, or an immunoconjugate according to the ninth aspect of the invention; and
(ii) a pharmaceutically acceptable carrier.
Preferably, the conjugating moiety of the immunoconjugate is a drug, toxin, and/or therapeutic isotope.
Preferably, the pharmaceutical composition is in the form of injection.
Preferably, the pharmaceutical composition is used for preparing a medicament for diagnosing or treating tumors.
Preferably, the tumor includes, but is not limited to: hematologic tumors, solid tumors, non-small cell lung cancer, colorectal cancer, melanoma, breast cancer, esophageal cancer, gastric tumors.
In a twelfth aspect of the invention, there is provided a use of one or more of the complementarity determining regions CDRs of an anti-TIGIT nanobody VHH chain according to the first aspect of the invention, or an anti-TIGIT nanobody VHH chain according to the second aspect of the invention, or an anti-TIGIT nanobody according to the third aspect of the invention, or an anti-TIGIT nanobody Fc fusion protein according to the fourth aspect of the invention:
(a) a drug for diagnosing or treating tumors;
(b) used for detecting human TIGIT molecules.
Preferably, the use is diagnostic and/or non-diagnostic, and/or therapeutic and/or non-therapeutic.
In a thirteenth aspect of the present invention, there is provided a recombinant protein having:
(i) a VHH chain of an anti-TIGIT nanobody according to the second aspect of the invention, or an anti-TIGIT nanobody according to the third aspect of the invention, or an anti-TIGIT nanobody Fc fusion protein according to the fourth aspect of the invention; and
(ii) optionally a tag sequence to facilitate expression and/or purification.
Preferably, the tag sequence comprises an Fc tag, an HA tag and a 6His tag.
Preferably, the recombinant protein specifically binds to TIGIT protein.
In a fourteenth aspect of the invention, there is provided the use of a VHH chain of an anti-TIGIT nanobody according to the second aspect of the invention, or an anti-TIGIT nanobody according to the third aspect of the invention, or an anti-TIGIT nanobody Fc fusion protein according to the fourth aspect of the invention, or an immunoconjugate according to the ninth aspect of the invention, for the preparation of a medicament, a reagent, a detection plate or a kit;
wherein the reagent, assay plate or kit is for: detecting TIGIT protein in the sample;
wherein the medicament is for the diagnosis or treatment of a tumor.
Preferably, the tumor includes, but is not limited to: hematologic tumors, solid tumors, non-small cell lung cancer, colorectal cancer, melanoma, breast cancer, esophageal cancer, gastric tumors.
Preferably, the detection comprises flow detection and cell immunofluorescence detection.
In a fifteenth aspect of the invention, a kit is provided, said kit comprising a VHH chain of an anti-TIGIT nanobody according to the second aspect of the invention, or an anti-TIGIT nanobody according to the third aspect of the invention, or an anti-TIGIT nanobody Fc fusion protein according to the fourth aspect of the invention, or an immunoconjugate according to the ninth aspect of the invention.
The invention has the beneficial effects that:
(a) the invention can block the interaction of TIGIT and CD155 on the cell surface of CT 26;
(b) the invention can effectively combine TIGIT protein on the cell surface, and the combination activity of the invention is similar to that of a control antibody Tiragolumab;
(c) the method can identify the TIGIT of the human and the cynomolgus monkey and cannot identify the TIGIT of the mouse;
(d) the blocking activity of the invention is obviously superior to that of a control antibody Tiragolumab;
(e) the invention has obvious activation effect on T cells, and the activation effect is obviously better than that of a control antibody Tiragolumab.
Drawings
FIG. 1 shows the result of SDS-PAGE detection of hTIGIT (ECD) -Fc protein. The results show that:
the purity of the hTIGIT (ECD) -Fc protein is more than 90 percent.
FIG. 2 shows the results of library capacity detection of phage display TIGIT nanobody library. The results show that: the library size constructed was 1.9x109 CFU.
FIG. 3 shows the result of detecting the insertion rate of antibody gene fragments in the phage display TIGIT nano antibody library. The results show that: the insertion rate of the constructed library fragment was 95.8%.
FIG. 4 shows the enrichment of positive colonies of TIGIT nanobodies screened by phage display technology. Wherein "+" represents enrichment of positive clone phage, "-" represents enrichment of negative clone phage, and ratio is the calculated result of "+/-". The results show that: after three rounds of screening, positive antibodies were enriched 176-fold.
Fig. 5 and 6 are the results of flow cytometry screening for nanobodies capable of blocking TIGIT interaction with CD 155. The results show that: of the 10 candidate antibodies, Nb49, Nb66, and Nb100 antibodies were able to block TIGIT interaction with CT26 cell surface CD 155.
FIG. 7 shows the results of flow cytometry to detect TIGIT binding ability of candidate antibodies to cell surfaces. The results show that: nb49, Nb66 and Nb100 can effectively bind to TIGIT protein on the cell surface, and the binding activity of the antibody is similar to that of a control antibody Tiragolumab.
FIG. 8 shows the results of flow cytometry to detect the epitope difference of antigen recognition between the candidate antibody and the control antibody Tiragolumab. The results show that: nb49 recognizes a different epitope than the control antibody Tiragolumab; nb66 and Nb100 recognize the same or similar epitope as the control antibody Tiragolumab.
FIG. 9 shows the results of flow cytometry for the blocking activity of candidate humanized antibodies. The results show that: blocking activity after humanization of the three Nb49, Nb66, and Nb100 antibodies was comparable to that of the pre-humanized antibody.
FIG. 10 shows the results of ELISA detection of candidate antibodies cross-binding to TIGIT of different species. The results show that: the three antibodies can recognize human and cynomolgus monkey TIGIT, but can not recognize mouse TIGIT.
FIG. 11 is the results of flow cytometry to detect the blocking activity of the bi-epitopic antibody against TIGIT/CD 155. The results show that: compared with single antibodies and control antibodies, the blocking activity of the double-epitope antibodies Nb49-Nb66 and Nb49-Nb100 is obviously improved.
FIG. 12 is a functional assay of T cell activation by bi-epitopic bivalent antibodies. The results show that: the double-epitope antibodies Nb49-Nb66 and Nb49-Nb100 both show significant activation on T cells, and the activation is significantly stronger than TIGIT nano antibody and a control antibody Tiragolumab alone.
Detailed Description
The inventor successfully obtains a plurality of anti-TIGIT nano antibodies through extensive and intensive research and a large amount of screening. Specifically, the invention utilizes human TIGIT extracellular segment antigen protein to immunize camels to obtain a high-quality immune nano antibody gene library. Then coupling TIGIT protein molecules on an enzyme label plate, displaying the correct spatial structure of the TIGIT protein, and screening an immune nano antibody gene library (a camel heavy chain antibody phage display gene library) by using the antigen in the form through a phage display technology, thereby obtaining the TIGIT specific nano antibody gene. In addition, relevant experiment results show that the anti-TIGIT nano antibody obtained by the invention can be effectively combined with TIGIT on the cell surface, and simultaneously can block the interaction with the ligand of the cell surface. The present invention has been completed based on this finding.
Term(s) for
As used herein, the terms "antibody of the invention", "anti-TIGIT nanobody of the invention", "anti-TIGIT nanobody", "TIGIT nanobody" have the same meaning and are used interchangeably and all refer to an antibody that specifically recognizes and binds to TIGIT, including human TIGIT. Preferably, the amino acid sequence of the VHH chain of the nanobody of the present invention is selected from one or more of SEQ ID NO 8, SEQ ID NO 17, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30.
As used herein, the term "antibody" or "immunoglobulin" is an heterotetrameric glycan protein of about 150000 daltons with the same structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable region (VH) followed by a plurality of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Particular amino acid residues form the interface between the variable regions of the light and heavy chains.
As used herein, the terms "single domain antibody", "VHH", "nanobody", "single domain antibody, sdAb, or nanobody" have the same meaning and are used interchangeably to refer to the cloning of the variable region of an antibody heavy chain, creating a single domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with full function. Typically, single domain antibodies (VHHs) consisting of only one heavy chain variable region are constructed by first obtaining an antibody that naturally lacks the light and heavy chain constant region 1(CH1) and then cloning the variable region of the antibody heavy chain.
As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, in a substantially b-folded configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a b-folded structure. The CDRs in each chain are held close together by the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIH Publ. No.91-3242, Vol I, 647-669 (1991)). The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.
As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines (cytokines), radionuclides, enzymes, and other diagnostic or therapeutic molecules are conjugated to the antibodies or fragments thereof of the present invention to form conjugates. The invention also comprises a cell surface marker or antigen combined with the anti-TIGIT nano antibody or the fragment thereof.
As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably.
As used herein, the term "variable region" is used interchangeably with "Complementarity Determining Region (CDR).
In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises three complementarity determining regions CDR1, CDR2, and CDR 3.
In a preferred embodiment of the invention, the heavy chain of the antibody comprises the above-described heavy chain variable region and heavy chain constant region.
In the present invention, the terms "antibody of the invention", "protein of the invention", or "polypeptide of the invention" are used interchangeably and all refer to a polypeptide that specifically binds to TIGIT, e.g., a protein or polypeptide having a heavy chain variable region. They may or may not contain the initial methionine.
The invention also provides other proteins or fusion expression products having an antibody of the invention. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain comprising a variable region, provided that the variable region is identical or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of an antibody of the invention.
In general, the antigen binding properties of an antibody can be described by 3 specific regions in the heavy chain variable region, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, and the β -sheets formed by the FRs between them are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type.
The variable regions of the heavy chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding to antigen. Thus, the invention includes those molecules having an antibody heavy chain variable region with CDRs whose homology to the CDRs identified herein is greater than 90% (preferably greater than 95%, most preferably greater than 98%).
The invention includes not only intact antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.
As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as an antibody of the invention. A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g. a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.
The antibody of the present invention refers to a polypeptide having TIGIT binding activity comprising the CDR regions described above. The term also includes variants of the polypeptides comprising the CDR regions described above that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.
Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes under high or low stringency conditions with DNA encoding an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.
The invention also provides other polypeptides, such as fusion proteins comprising antibodies or fragments thereof. In addition to almost full-length polypeptides, the invention also encompasses fragments of the antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids of the antibody of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.
In the present invention, "conservative variant of the antibody of the present invention" means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are substituted by amino acids having similar or similar properties as compared with the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variant polypeptides are preferably generated by amino acid substitutions according to Table 1.
Initial residue(s) Representative substitutions Preferred substitutions
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
The invention also provides polynucleotide molecules encoding the above antibodies or fragments or fusion proteins thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.
Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.
The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.
The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more. Also, the polynucleotides that hybridize to the mature polypeptide encode polypeptides having the same biological functions and activities as the mature polypeptide.
The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be obtained by a PCR amplification method, a recombinant method, or an artificial synthesis method. One possibility is to use synthetic methods to synthesize the sequence of interest, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. Alternatively, the coding sequence for the heavy chain and an expression tag (e.g., 6His) can be fused together to form a fusion protein.
Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules in an isolated form.
At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; CHO, COS7, 293 cells, etc.
Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, such as E.coli, competent cells capable of DNA uptake can be harvested after the exponential growth phase and treated by the CaCl2 method using procedures well known in the art. Another method is to use MgCl 2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.
The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.
The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.
The antibodies of the invention may be used alone or in combination or conjugated with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties or combinations of any of the above.
Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.
Therapeutic agents that may be conjugated or conjugated to the antibodies of the invention include, but are not limited to: 1. a radionuclide; 2. biological toxicity; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; 5. a viral particle; 6. a liposome; 7. nano magnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), and the like.
TIGIT (T cell immunoglobulin and ITIM structural domain protein)
The full names of TIGIT are T cell immunoglobulin and ITIM domain proteins (T cell immunoglobulin with Ig and ITIM domains). It is an inhibitory receptor, expressed on the surface of many types of T cells. T cells express a number of different activating and inhibitory receptors on their surface, which often pair-wise to fine-tune T cell activity. TIGIT and the activating receptor CD226 are paired, and their common ligand is PVR (CD 155). TIGIT inhibits the activation of T cells by competing with CD226 and binding PVR, disturbing the activation of CD226 and other mechanisms. TIGIT is highly expressed in tumor infiltrating T cells of multiple cancer types.
VHH chain combinations
The invention also comprises a combination of the nano antibody VHH chains. Wherein the combination comprises at least one set of the following CDR regions:
(1) CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, CDR3 shown in SEQ ID NO. 3;
(2) CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11, CDR3 shown in SEQ ID NO. 12;
(3) CDR1 shown in SEQ ID NO. 19, CDR2 shown in SEQ ID NO. 20, CDR3 shown in SEQ ID NO. 21;
pharmaceutical composition
The invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the above antibody or an active fragment thereof or a fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intraperitoneal, intravenous, or topical administration.
The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described antibody (or conjugate thereof) of the present invention and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.
In the case of pharmaceutical compositions, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 10 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
anti-TIGIT nano antibody
In the present invention, the anti-TIGIT nanobody includes a monomer, a bivalent body (bivalent antibody), a tetravalent body (tetravalent antibody), and/or a multivalent body (multivalent antibody).
In a preferred embodiment of the invention, the anti-TIGIT nanobody comprises one or more VHH chains having the amino acid sequence shown as SEQ ID NO 8, 17, 26, 28, 29, 30.
Typically, the anti-TIGIT nano antibody comprises any two VHH chains of amino acid sequences shown as SEQ ID NO. 28, SEQ ID NO. 29 and SEQ ID NO. 30, and the VHH chains are connected through a linker.
In a preferred embodiment of the invention, the linker is selected from the following sequences: (GaSb) x- (GmSn) y, wherein a, b, m, n, x, y is 0 or 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 (preferably, a is 4 and b is 1, m is 3 and n is 1).
In a preferred embodiment of the present invention, the linker is selected from the group consisting of: GGGGSGGGS are provided.
In a preferred embodiment of the invention, the anti-TIGIT nanobody is capable of recognizing two different TIGIT epitopes.
In a preferred embodiment of the invention, the amino acid sequence of the anti-TIGIT nano antibody is shown as SEQ ID NO. 34 or SEQ ID NO. 36.
Labeled antibodies
In a preferred embodiment of the invention, the antibody is detectably labeled. More preferably, the marker is selected from the group consisting of: isotopes, colloidal gold labels, coloured labels or fluorescent labels.
The colloidal gold labeling can be performed by methods known to those skilled in the art. In a preferred embodiment of the present invention, the antibody to TIGIT is labeled with colloidal gold to obtain a colloidal gold-labeled antibody.
The anti-TIGIT nano antibody can effectively bind to TIGIT protein on the surface of cells.
Detection method
The invention also relates to a method for detecting the TIGIT protein. The method comprises the following steps: obtaining a cell and/or tissue sample; dissolving the sample in a medium; detecting the level of TIGIT protein in the solubilized sample.
The sample used in the detection method of the present invention is not particularly limited, and a typical example is a cell-containing sample present in a cell preservation solution.
Reagent kit
The present invention also provides a kit comprising an antibody (or fragment thereof) or assay plate of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like.
The invention also provides a detection kit for detecting the TIGIT level, which comprises an antibody for identifying the TIGIT protein, a lysis medium for dissolving a sample, general reagents and buffers required for detection, such as various buffers, detection markers, detection substrates and the like. The test kit may be an in vitro diagnostic device.
Applications of
As described above, the antibody of the present invention has wide biological and clinical applications, and its applications are in various fields such as diagnosis and treatment of diseases associated with TIGIT, research in basic medicine, and biological research. One preferred application is for clinical diagnosis and treatment for TIGIT.
The main advantages of the invention include:
(a) the antibody of the invention can block the interaction of TIGIT and CD155 on the cell surface of CT 26.
(b) The antibody can effectively bind TIGIT protein on the surface of cells, and has similar binding activity to a control antibody Tiragolumab.
(c) The antibody can identify human and cynomolgus monkey TIGIT, but cannot identify mouse TIGIT.
(d) The blocking activity of the antibody of the invention is obviously superior to that of a control antibody Tiragolumab.
(e) The antibody of the invention has obvious activation effect on T cells, and the activation effect is obviously better than that of a control antibody Tiragolumab.
The following specific examples further illustrate the invention. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out according to conventional conditions, for example as described in Sambrook and Russell et al, Molecular Cloning: A laboratory Manual (third edition) (2001) CSHL Press, or according to the conditions as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.
Example 1: human TIGIT extracellular segment protein expression and camel immune library construction
Transient expression of human TIGIT extracellular segment protein by using mammalian cell HEK 293F: mixing the pFUSE-IgG recombinant plasmid with cloned human TIGIT extracellular segment gene with a transfection reagent PEI, and transfecting to HEK293F cells; 37 ℃ and 6% CO2Culturing for 5 days in a shaking incubator; subsequently, cell supernatants were collected and bound to Protein A beads for 1h at room temperature; after washing the beads with phosphate buffer pH 7.0, the protein was eluted with 0.1M glycine pH3.0 solution; the eluted proteins were then ultrafiltered into PBS solution, and samples were taken for SDS-PAGE detection after yield determination. The detection result is shown in figure 1, the purity of the hTIGIT (ECD) -Fc protein expressed and purified is more than 90 percent, and the hTIGIT-Fc protein can be used for camel immunization and antibody screening.
The purified hTIGIT (ECD) -Fc protein is used for immunizing 1 Xinjiang bactrian camel, total RNA is separated from camel peripheral blood after 7 times of immunization, VHH gene is amplified through reverse transcription and PCR, the VHH gene is cloned to a phage vector pMECS, and the phage display library is constructed by transforming the phage vector pMECS to TG1 host cells. The library volume constructed was determined to be 1.9X109 CFU (FIG. 2) and the library insertion rate was 95.8% (FIG. 3).
Then, the phage display technology is used for library screening, and the phage enrichment containing antibody genes is obtained 176 times through 3 rounds of 'adsorption-washing-enrichment' screening processes (FIG. 4). Randomly selecting 600 phage clones from the enriched phage clones to carry out PE-ELISA identification, carrying out sequencing identification on all obtained positive clones, taking all antibodies with different sequences as candidate objects, and expressing and purifying various antibodies by using escherichia coli for subsequent identification.
Example 2: flow cytometry screening blocking type TIGIT nano antibody
Cloning the nanometer antibody genes with different strain sequences to a pFUSE-IgG carrier, then mixing the recombinant plasmid with a transfection reagent PEI 1:3, and transfecting to HEK293F cells; 37 ℃ and 6% CO2Culturing for 5 days in a shaking incubator; subsequently, cell supernatants were collected and bound to Protein A beads for 1h at room temperature; after washing the beads with phosphate buffer pH 7.0, the protein was eluted with 0.1M, pH3.0 glycine solution; then, the product is processedEluted proteins were ultrafiltered into PBS solution for candidate functional activity studies.
The cultured CHOZEN/TIGIT stably transfected cells are distributed into 96-well plates, 3E5 cells are distributed in each well, the supernatant is removed by centrifugation at 3000rpm for 3min, and diluted antibodies (Nb5, Nb10, Nb14, Nb16, Nb49, Nb66, Nb72, Nb86, Nb96 and Nb100) and CD155-Biotin protein are added for incubation for 20min, wherein the antibody concentration gradient is 2ug/mL, 1ug/mL and 0.5 ug/mL. The supernatant was discarded by centrifugation, and the diluted SA-PE antibody was added and incubated at 4 ℃ for 20 min. The supernatant was centrifuged again, 200uL of PBS was added to each well to resuspend the cells, and the PE signal of the sample was detected by flow cytometry. The results are shown in fig. 5 and 6: candidate antibodies Nb49, Nb66 and Nb100 were able to block TIGIT interaction with CT26 cell surface CD 155.
Example 3: detection of binding activity of TIGIT (tungsten inert gas) nano antibody and cell surface antigen
And (3) performing antibody binding function verification by using stably transformed cells CHOZN/TIGIT with high expression TIGIT: after digesting the cultured CHOZN/TIGIT cells with pancreatin and neutralizing with complete medium, washing the cells once with PBS, and then collecting the cells; evenly subpackaging into 96-well plates, adding diluted antibodies (the dilution concentration of each group of antibodies is respectively 40ug/mL, 20ug/mL, 10ug/mL, 5ug/mL, 2.5ug/mL, 1.25ug/mL, 0.625ug/mL, 0.313ug/mL, 0.156ug/mL, 0.078ug/mL, 0.039ug/mL and 0.019ug/mL), and incubating at 4 ℃ for 20 min; after centrifugation, the cells were washed once with PBS, added with Goatanti human IgG-FITC (diluted 1: 200), and incubated at 4 ℃ for 20 min; after washing the cells once with PBS, centrifuging at 3000rpm and 4 ℃ for 4min, discarding the supernatant, adding 200uL PBS/well, resuspending the cells, transferring to a flow tube, and flow-detecting the FITC signal of each sample. As shown in fig. 7, the three nanobodies (Nb49, Nb66, Nb100) were able to effectively bind to TIGIT protein on the cell surface, and had similar binding activity to the control antibody Tiragolumab. The sequence numbers of the three strains of nano antibodies are shown in Table 2.
TABLE 2 TIGIT NanoAbody sequence numbering
Antibody numbering Nb49 Nb66 Nb100
CDR1 amino acid sequence SEQ ID NO：1 SEQ ID NO：10 SEQ ID NO：19
CDR2 amino acid sequence SEQ ID NO：2 SEQ ID NO：11 SEQ ID NO：20
CDR3 amino acid sequence SEQ ID NO：3 SEQ ID NO：12 SEQ ID NO：21
FR1 amino acid sequence SEQ ID NO：4 SEQ ID NO：13 SEQ ID NO：22
FR2 amino acid sequence SEQ ID NO：5 SEQ ID NO：14 SEQ ID NO：23
FR3 amino acid sequence SEQ ID NO：6 SEQ ID NO：15 SEQ ID NO：24
FR4 amino acid sequence SEQ ID NO：7 SEQ ID NO：16 SEQ ID NO：25
Full length amino acid sequence SEQ ID NO：8 SEQ ID NO：17 SEQ ID NO：26
full-Length base sequence SEQ ID NO：9 SEQ ID NO：18 SEQ ID NO：27
Example 4: antigen recognition epitope assay of candidate antibodies
Detecting antigen recognition epitope differences of the candidate antibody and the control antibody by using flow cytometry: the cultured CHOZN/TIGIT cells were dispensed into 96-well plates, 3E5 cells per well, centrifuged, and the supernatant discarded. 50uL4ug/mL of control antibody Tiragolumab and 50uL 40ug/mL of candidate nanobodies Nb49, Nb66 and Nb100 (expressed by E.coli) are mixed and added to the cells, and incubated at 4 ℃ for 20 min. Subsequently, the cells were washed with PBS, and then added with the goat anti human IgG-FITC (diluted at 1: 200), and incubated at 4 ℃ for 20 min; after washing the cells once with PBS, centrifuging at 3000rpm and 4 ℃ for 4min, discarding the supernatant, adding 200uL PBS/well, resuspending the cells, transferring to a flow tube, and flow-detecting the FITC signal of each sample. As shown in fig. 8, Nb49 failed to block the binding of the control antibody to TIGIT on the cell surface, indicating that the antibody recognizes a different epitope than the control antibody Tiragolumab; and Nb66 and Nb100 can block the combination of the control antibody and TIGIT on the cell surface, which shows that the two antibodies and the control antibody Tiragolumab recognize the same or similar epitope.
Example 5: species-specific detection of candidate antibodies
And detecting whether the candidate antibody can generate cross reaction with other TIGIT species by ELISA. Adding 1ug/mL human TIGIT, mouse TIGIT and cynomolgus monkey TIGIT antigen protein into an enzyme label plate to be coated overnight, and coating at 4 ℃ for 100 uL/hole; after washing 5 times with PBST, 300uL of 1% BSA was added to each well and blocked for 2 hours at room temperature; after washing 5 times with PBST, 100uL of 10ug/mL biotin-labeled Nb49, Nb66, and Nb100 were added, respectively, and incubated at 37 ℃ for 1 hour; further washed 5 times with PBST, added 100uL diluted SA-HRP (1:2000 dilution) and incubated at 37 ℃ for 1 hour; washing with PBST for 5 times, adding color developing solution, and measuring absorption value with enzyme labeling instrument at 450nm wavelength. The results are shown in FIG. 9: the three antibodies can recognize human and cynomolgus monkey TIGIT, but can not recognize mouse TIGIT.
Example 6: antibody humanization design and Activity detection
The amino acid sequences of the three strains of nano antibodies are placed in a structure database to search homologous structures, the structures with higher sequence isogenity are taken, the structures are compared, finally, proteins including 3dwt are selected according to the resolution of the crystal structure and the constructed evolutionary tree, multi-template homologous modeling of the target nano antibody sequence is carried out, and then the structure with the lowest molpdf is selected according to the high-low ordering of a scoring function; the solvent accessibility of the residues was then calculated for the constructed optimal structure using the ProtSA server, and the constructed optimal structure and DP-47 were aligned to replace the corresponding solvent exposed residues. Finally, a humanized TIGIT nano antibody is determined, and the sequence of the humanized antibody corresponds to the following table 3:
TABLE 3 humanized TIGIT Nanobody sequence numbering
Antibody numbering Amino acid sequence numbering Base sequence number
HuNb49 SEQ ID NO.28 SEQ ID NO.31
HuNb66 SEQ ID NO.29 SEQ ID NO.32
HuNb100 SEQ ID NO.30 SEQ ID NO.33
After the amino acid sequence of the humanized antibody is optimized according to human codons, the base sequence of the humanized antibody is synthesized to a pFUSE vector, and then the recombinant plasmid is transfected into HEK293F cells, wherein the transfection method is shown in example 1.
The blocking activity of the antibody expressed and purified above was detected by flow cytometry, which was performed in the same manner as in example 2, and the results are shown in FIG. 9: the blocking activity after humanization of the three-strain antibody was comparable to that of the antibody before humanization, and thus it was considered that antibody humanization was successful.
Example 7: construction of bi-epitopic bivalent antibodies
In order to further improve the functional activity of candidate antibodies, the construction of bi-epitopic antibodies was attempted. The serial design of HuNb49/HuNb66 and HuNb49/HuNb100 is respectively carried out, and the structural formula is as follows:
HuNb49-GGGGSGGGS-HuNb66-Fc (amino acid sequence is shown as SEQ ID NO:34, base sequence is shown as SEQ ID NO: 35)
HuNb49-GGGGSGGGS-HuNb100-Fc (amino acid sequence is shown as SEQ ID NO:36, base sequence is shown as SEQ ID NO: 37)
The amino acid sequence of the antibody was arranged according to the above structure, and then it was codon-optimized according to human codon preference, and then the optimized base sequence was synthesized onto the pcdna3.1 vector. The constructed plasmid was transformed into HEK293F cells, and the transfection and culture methods were the same as example 1. The candidate antibody is then purified from the supernatant for subsequent activity detection.
Example 8: detection of blocking Activity of Biepitope bivalent antibody
The blocking activity of the bi-epitope antibody was detected by flow cytometry: the cultured CHOZEN/TIGIT stably transfected cells were aliquoted into 96-well plates, 3E5 cells per well were centrifuged at 3000rpm for 3min to remove the supernatant, and diluted antibodies (Nb49, Nb66, Nb100, Nb49-Nb66, Nb49-Nb100, control antibody Tiragolumab) and CD155-Biotin protein were added and incubated at 4 ℃ for 20min, with the dilution concentrations of each group of antibodies being 40ug/mL, 20ug/mL, 10ug/mL, 5ug/mL, 2.5ug/mL, 1.25ug/mL, 0.625ug/mL, 0.313ug/mL, 0.156ug/mL, 0.078ug/mL, 0.039ug/mL, 0.019ug/mL, respectively. The supernatant was discarded by centrifugation, and the diluted SA-PE antibody was added and incubated at 4 ℃ for 20 min. The supernatant was centrifuged again, 200uL of PBS was added to each well to resuspend the cells, and the PE signal of the sample was detected by flow cytometry. The results are shown in FIG. 11: compared with single antibodies and control antibodies, the blocking activity of the double-epitope antibodies Nb49-Nb66 and Nb49-Nb100 is obviously improved.
Example 9: activation detection of PBMCs by double-epitope bivalent antibody
Fresh 50uL PBMCs cells were added to corresponding 96-well cell culture plates, 1E5 cells per well. Then, 100uL of the antibody (final concentration: 20ug/mL) and 50uL of SEB (final concentrations: 1ug/mL and 0.1ug/mL) were added to the corresponding wells, respectively, at 37 ℃ with 5% CO2And culturing for 72 h. After the culture is finished, the content of IL-2 in the supernatant is detected by using a BDIL-2ELISA kit. The results are shown in fig. 12, both bi-epitope antibodies showed significant activation of T cells, and the activation was significantly stronger than TIGIT nanobody and the control antibody Tiragolumab alone.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence information of the present invention:
SEQ ID NO:1
GYAYSSNYIG
SEQ ID NO:2
IYPGVGRI
SEQ ID NO:3
AAEVVPALRGGQWYAGRYFSY
SEQ ID NO:4
QVQLQESGGGSAQAGGSLRLSCAAS
SEQ ID NO:5
WFRQAPGKEREGVAT
SEQ ID NO:6
YYADSVKGRFTISQDNAKNTVYLQMTSLKPEDSAMYYC
SEQ ID NO:7
WGQGTQVTVSS
SEQ ID NO:8
QVQLQESGGGSAQAGGSLRLSCAASGYAYSSNYIGWFRQAPGKEREGVATIYPGVGRIYYADSVKGRFTISQDNAKNTVYLQMTSLKPEDSAMYYCAAEVVPALRGGQWYAGRYFSYWGQGTQVTVSS
SEQ ID NO:9
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGCGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATACGCTTACAGTAGCAACTACATAGGATGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAAGGCGTCGCAACTATTTATCCTGGTGTTGGTAGAATATATTATGCCGACTCCGTGAAGGGCCGATTCACCATCTCCCAAGACAACGCCAAGAACACGGTGTATCTGCAAATGACAAGCCTGAAACCTGAGGACTCTGCCATGTACTACTGTGCGGCGGAAGTTGTCCCAGCCTTACGAGGTGGTCAATGGTACGCCGGTCGGTACTTTAGTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO:10
GFSFSMSVMY
SEQ ID NO:11
INSGGRT
SEQ ID NO:12
ARDSRGTF
SEQ ID NO:13
QVQLQESGGGSVQPGGSLRLSCAAS
SEQ ID NO:14
WVRQAPGKELEWVSF
SEQ ID NO:15
YYADSVKGRFTISRDDAKNTLYLQLNSLTTEDTALYYC
SEQ ID NO:16
RGQGTQVTVSS
SEQ ID NO:17
QVQLQESGGGSVQPGGSLRLSCAASGFSFSMSVMYWVRQAPGKELEWVSFINSGGRTYYADSVKGRFTISRDDAKNTLYLQLNSLTTEDTALYYCARDSRGTFRGQGTQVTVSS
SEQ ID NO:18
CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGCCTGGGGGATCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTATGTCGGTCATGTACTGGGTCCGCCAGGCTCCAGGCAAGGAACTCGAGTGGGTCTCATTTATTAATAGTGGTGGTAGGACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACGACGCCAAGAACACGCTGTATCTGCAATTGAACAGCCTGACAACTGAGGACACGGCCTTGTATTACTGTGCAAGAGATTCGCGTGGGACGTTCAGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO:19
GYSICMG
SEQ ID NO:20
IAGLSSK
SEQ ID NO:21
AARGVGYCGIAAGMDY
SEQ ID NO:22
QVQLQESGGGSVQAGGSLRLSCAAS
SEQ ID NO:23
WFRQAPGKEREGVAT
SEQ ID NO:24
TYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAMYYC
SEQ ID NO:25
WGKGTQVTVSS
SEQ ID NO:26
QVQLQESGGGSVQAGGSLRLSCAASGYSICMGWFRQAPGKEREGVATIAGLSSKTYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAMYYCAARGVGYCGIAAGMDYWGKGTQVTVSS
SEQ ID NO:27
CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTCGGTGCAGGCTGGAGGGTCCCTGAGACTGTCCTGTGCAGCCTCTGGATACAGTATTTGTATGGGTTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGGGGGTCGCCACTATTGCTGGTCTTAGTAGCAAGACCTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCCGAGACAACGCCAAGAACACTCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGGCTCGTGGTGTGGGGTACTGCGGAATCGCCGCCGGCATGGACTACTGGGGCAAAGGAACCCAGGTCACCGTCTCCTCA
SEQ ID NO:28
EVQLQESGGGLAQPGGSLRLSCAASGYAYSSNYIGWFRQAPGKGLEGVATIYPGVGRIYYADSVKGRFTISQDNAKNTVYLQMTSLKPEDSAMYYCAAEVVPALRGGQWYAGRYFSYWGQGTLVTVSS
SEQ ID NO:29
EVQLQESGGGLVQPGGSLRLSCAASGFSFSMSVMYWVRQAPGKGLEWVSFINSGGRTYYADSVKGRFTISRDNSKNSLYLQMNSLRTEDTALYYCARDSRGTFRGQGTLVTVSS
SEQ ID NO:30
EVQLQESGGGLVQPGGSLRLSCAASGYSICMGWFRQAPGKGLEGVATIAGLSSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAMYYCAARGVGYCGIAAGMDYWGQGTLVTVSS
SEQ ID NO:31
GAGGTGCAGCTGCAGGAGTCCGGCGGCGGCCTGGCCCAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCCGCCTCCGGCTACGCCTACTCCTCCAACTACATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGGCCTGGAGGGCGTGGCCACCATCTACCCCGGCGTGGGCAGGATCTACTACGCCGACTCCGTGAAGGGCAGGTTCACCATCTCCCAGGACAACGCCAAGAACACCGTGTACCTGCAGATGACCTCCCTGAAGCCCGAGGACTCCGCCATGTACTACTGCGCCGCCGAGGTGGTGCCCGCCCTGAGGGGCGGCCAGTGGTACGCCGGCAGGTACTTCTCCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC
SEQ ID NO:32
CAGGTGCAGCTGCAGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCAGCTTCAGCATGAGCGTGATGTACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCTTCATCAACAGCGGCGGCAGGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGACCGAGGACACCGCCCTGTACTACTGCGCCAGGGACAGCAGGGGCACCTTCAGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC
SEQ ID NO:33
CAGGTGCAGCTGCAGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCCGCCTCCGGCTACTCCATCTGCATGGGCTGGTTCAGGCAGGCCCCCGGCAAGGGCCTGGAGGGCGTGGCCACCATCGCCGGCCTGTCCTCCAAGTACTACGCCGACTCCGTGAAGGGCAGGTTCACCATCTCCAGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCATGTACTACTGCGCCGCCAGGGGCGTGGGCTACTGCGGCATCGCCGCCGGCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC
SEQ ID NO:34
EVQLQESGGGLAQPGGSLRLSCAASGYAYSSNYIGWFRQAPGKGLEGVATIYPGVGRIYYADSVKGRFTISQDNAKNTVYLQMTSLKPEDSAMYYCAAEVVPALRGGQWYAGRYFSYWGQGTLVTVSSGGGGSGGGSEVQLQESGGGLVQPGGSLRLSCAASGFSFSMSVMYWVRQAPGKGLEWVSFINSGGRTYYADSVKGRFTISRDNSKNSLYLQMNSLRTEDTALYYCARDSRGTFRGQGTLVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:35
GAGGTGCAGCTGCAGGAGTCCGGCGGCGGCCTGGCCCAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCCGCCTCCGGCTACGCCTACTCCTCCAACTACATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGGCCTGGAGGGCGTGGCCACCATCTACCCCGGCGTGGGCAGGATCTACTACGCCGACTCCGTGAAGGGCAGGTTCACCATCTCCCAGGACAACGCCAAGAACACCGTGTACCTGCAGATGACCTCCCTGAAGCCCGAGGACTCCGCCATGTACTACTGCGCCGCCGAGGTGGTGCCCGCCCTGAGGGGCGGCCAGTGGTACGCCGGCAGGTACTTCTCCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCCGGCGGCGGCGGCTCCGGCGGCGGCTCCGAGGTGCAGCTGCAGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCCGCCTCCGGCTTCTCCTTCTCCATGTCCGTGATGTACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGTCCTTCATCAACTCCGGCGGCAGGACCTACTACGCCGACTCCGTGAAGGGCAGGTTCACCATCTCCAGGGACAACTCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGACCGAGGACACCGCCCTGTACTACTGCGCCAGGGACTCCAGGGGCACCTTCAGGGGCCAGGGCACCCTGGTGACCGTGTCCTCCGAGCCCAAGTCCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGCTGGGCGGCCCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCTCCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACTCCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCTCCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCAGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCCCCCGGCAAG
SEQ ID NO:36
EVQLQESGGGLAQPGGSLRLSCAASGYAYSSNYIGWFRQAPGKGLEGVATIYPGVGRIYYADSVKGRFTISQDNAKNTVYLQMTSLKPEDSAMYYCAAEVVPALRGGQWYAGRYFSYWGQGTLVTVSSGGGGSGGGSEVQLQESGGGLVQPGGSLRLSCAASGYSICMGWFRQAPGKGLEGVATIAGLSSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAMYYCAARGVGYCGIAAGMDYWGQGTLVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:37
GAGGTGCAGCTGCAGGAGTCCGGCGGCGGCCTGGCCCAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCCGCCTCCGGCTACGCCTACTCCTCCAACTACATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGGCCTGGAGGGCGTGGCCACCATCTACCCCGGCGTGGGCAGGATCTACTACGCCGACTCCGTGAAGGGCAGGTTCACCATCTCCCAGGACAACGCCAAGAACACCGTGTACCTGCAGATGACCTCCCTGAAGCCCGAGGACTCCGCCATGTACTACTGCGCCGCCGAGGTGGTGCCCGCCCTGAGGGGCGGCCAGTGGTACGCCGGCAGGTACTTCTCCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCCGGCGGCGGCGGCTCCGGCGGCGGCTCCGAGGTGCAGCTGCAGGAGTCCGGCGGCGGCCTGGTGCAGCCCGGCGGCTCCCTGAGGCTGTCCTGCGCCGCCTCCGGCTACTCCATCTGCATGGGCTGGTTCAGGCAGGCCCCCGGCAAGGGCCTGGAGGGCGTGGCCACCATCGCCGGCCTGTCCTCCAAGTACTACGCCGACTCCGTGAAGGGCAGGTTCACCATCTCCAGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCATGTACTACTGCGCCGCCAGGGGCGTGGGCTACTGCGGCATCGCCGCCGGCATGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCCGAGCCCAAGTCCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGCTGGGCGGCCCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCTCCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACTCCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCTCCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCAGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGACATCGCCGTG
GAGTGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCCCCCGGCAAG
SEQ ID NO:38
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。
the foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
<110> Shanghai Luoqi biomedical technology, Inc
<120> anti-TIGIT nano antibody and application thereof
<160>38
<170>SIPOSequenceListing 1.0
<210>1
<211>10
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>1
Gly Tyr Ala Tyr Ser Ser Asn Tyr Ile Gly
1 5 10
<210>2
<211>8
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>2
Ile Tyr Pro Gly Val Gly Arg Ile
1 5
<210>3
<211>21
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>3
Ala Ala Glu Val Val Pro Ala Leu Arg Gly Gly Gln Trp Tyr Ala Gly
1 5 10 15
Arg Tyr Phe Ser Tyr
20
<210>4
<211>25
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>4
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Ala Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser
20 25
<210>5
<211>15
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>5
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Thr
1 5 10 15
<210>6
<211>38
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>6
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ala Lys Asn Thr Val Tyr Leu Gln Met Thr Ser Leu Lys Pro Glu Asp
20 25 30
Ser Ala Met Tyr Tyr Cys
35
<210>7
<211>11
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>7
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210>8
<211>128
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>8
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Ala Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ala Tyr Ser Ser Asn
20 25 30
Tyr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Thr Ile Tyr Pro Gly Val Gly Arg Ile Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Ser Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Glu Val Val Pro Ala Leu Arg Gly Gly Gln Trp Tyr Ala Gly
100 105 110
Arg Tyr Phe Ser Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210>9
<211>384
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
caggtgcagc tgcaggagtc tgggggaggc tcggcgcagg ctggagggtc tctgagactc 60
tcctgtgcag cctctggata cgcttacagt agcaactaca taggatggtt ccgccaggct 120
ccagggaagg agcgcgaagg cgtcgcaact atttatcctg gtgttggtag aatatattat 180
gccgactccg tgaagggccg attcaccatc tcccaagaca acgccaagaa cacggtgtat 240
ctgcaaatga caagcctgaa acctgaggac tctgccatgt actactgtgc ggcggaagtt 300
gtcccagcct tacgaggtgg tcaatggtac gccggtcggt actttagtta ctggggccag 360
gggacccagg tcaccgtctc ctca 384
<210>10
<211>10
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>10
Gly Phe Ser Phe Ser Met Ser Val Met Tyr
1 5 10
<210>11
<211>7
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>11
Ile Asn Ser Gly Gly Arg Thr
1 5
<210>12
<211>8
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>12
Ala Arg Asp Ser Arg Gly Thr Phe
1 5
<210>13
<211>25
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>13
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser
20 25
<210>14
<211>15
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>14
Trp Val Arg Gln Ala Pro Gly Lys Glu Leu Glu Trp Val Ser Phe
1 5 10 15
<210>15
<211>38
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>15
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
1 5 10 15
Ala Lys Asn Thr Leu Tyr Leu Gln Leu Asn Ser Leu Thr Thr Glu Asp
20 25 30
Thr Ala Leu Tyr Tyr Cys
35
<210>16
<211>11
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>16
Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210>17
<211>114
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>17
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Met Ser
20 25 30
Val Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Glu Leu Glu Trp Val
35 40 45
Ser Phe Ile Asn Ser Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Leu Asn Ser Leu Thr Thr Glu Asp Thr Ala Leu Tyr Tyr Cys Ala
85 90 95
Arg Asp Ser Arg Gly Thr Phe Arg Gly Gln Gly Thr Gln Val Thr Val
100 105 110
Ser Ser
<210>18
<211>342
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>18
caggtgcagc tgcaggagtc tggaggaggc tcggtgcagc ctgggggatc tctgagactc 60
tcctgtgcag cctctggatt ctccttcagt atgtcggtca tgtactgggt ccgccaggct 120
ccaggcaagg aactcgagtg ggtctcattt attaatagtg gtggtaggac atactatgca 180
gactccgtga agggccgatt caccatctcc agagacgacg ccaagaacac gctgtatctg 240
caattgaaca gcctgacaac tgaggacacg gccttgtatt actgtgcaag agattcgcgt 300
gggacgttca ggggccaggg gacccaggtc accgtctcct ca 342
<210>19
<211>7
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>19
Gly Tyr Ser Ile Cys Met Gly
1 5
<210>20
<211>7
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>20
Ile Ala Gly Leu Ser Ser Lys
1 5
<210>21
<211>16
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>21
Ala Ala Arg Gly Val Gly Tyr Cys Gly Ile Ala Ala Gly Met Asp Tyr
1 5 10 15
<210>22
<211>25
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>22
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser
20 25
<210>23
<211>15
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>23
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Thr
1 5 10 15
<210>24
<211>38
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>24
Thr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210>25
<211>11
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>25
Trp Gly Lys Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210>26
<211>119
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>26
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Cys Met Gly
20 25 30
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Thr Ile
35 40 45
Ala Gly Leu Ser Ser Lys Thr Tyr Ala Asp Ser Val Lys Gly Arg Phe
50 55 60
Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn
6570 75 80
Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Arg Gly
85 90 95
Val Gly Tyr Cys Gly Ile Ala Ala Gly Met Asp Tyr Trp Gly Lys Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
<210>27
<211>357
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>27
caggtgcagc tgcaggagtc tggaggaggc tcggtgcagg ctggagggtc cctgagactg 60
tcctgtgcag cctctggata cagtatttgt atgggttggt tccgccaggc tccagggaag 120
gagcgcgagg gggtcgccac tattgctggt cttagtagca agacctacgc agactccgtg 180
aagggccgat tcaccatctc ccgagacaac gccaagaaca ctctgtatct gcaaatgaac 240
agcctgaaac ctgaggacac tgccatgtac tactgtgcgg ctcgtggtgt ggggtactgc 300
ggaatcgccg ccggcatgga ctactggggc aaaggaaccc aggtcaccgt ctcctca 357
<210>28
<211>128
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>28
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Ala Gln Pro Gly Gly
15 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ala Tyr Ser Ser Asn
20 25 30
Tyr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Thr Ile Tyr Pro Gly Val Gly Arg Ile Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Ser Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Glu Val Val Pro Ala Leu Arg Gly Gly Gln Trp Tyr Ala Gly
100 105 110
Arg Tyr Phe Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210>29
<211>114
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>29
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Met Ser
20 25 30
Val Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Phe Ile Asn Ser Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu Tyr Tyr Cys Ala
85 90 95
Arg Asp Ser Arg Gly Thr Phe Arg Gly Gln Gly Thr Leu Val Thr Val
100 105 110
Ser Ser
<210>30
<211>119
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>30
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Cys Met Gly
20 25 30
Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val Ala ThrIle
35 40 45
Ala Gly Leu Ser Ser Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe
50 55 60
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn
65 70 75 80
Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Arg Gly
85 90 95
Val Gly Tyr Cys Gly Ile Ala Ala Gly Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210>31
<211>384
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>31
gaggtgcagc tgcaggagtc cggcggcggc ctggcccagc ccggcggctc cctgaggctg 60
tcctgcgccg cctccggcta cgcctactcc tccaactaca tcggctggtt caggcaggcc 120
cccggcaagg gcctggaggg cgtggccacc atctaccccg gcgtgggcag gatctactac 180
gccgactccg tgaagggcag gttcaccatc tcccaggaca acgccaagaa caccgtgtac 240
ctgcagatga cctccctgaa gcccgaggac tccgccatgt actactgcgc cgccgaggtg 300
gtgcccgccc tgaggggcgg ccagtggtac gccggcaggt acttctccta ctggggccag 360
ggcaccctgg tgaccgtgtc ctcc 384
<210>32
<211>342
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>32
caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60
agctgcgccg ccagcggctt cagcttcagc atgagcgtga tgtactgggt gaggcaggcc 120
cccggcaagg gcctggagtg ggtgagcttc atcaacagcg gcggcaggac ctactacgcc 180
gacagcgtga agggcaggtt caccatcagc agggacaaca gcaagaacag cctgtacctg 240
cagatgaaca gcctgaggac cgaggacacc gccctgtact actgcgccag ggacagcagg 300
ggcaccttca ggggccaggg caccctggtg accgtgagca gc 342
<210>33
<211>357
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>33
caggtgcagc tgcaggagtc cggcggcggc ctggtgcagc ccggcggctc cctgaggctg 60
tcctgcgccg cctccggcta ctccatctgc atgggctggt tcaggcaggc ccccggcaag 120
ggcctggagg gcgtggccac catcgccggc ctgtcctcca agtactacgc cgactccgtg 180
aagggcaggt tcaccatctc cagggacaac tccaagaaca ccctgtacct gcagatgaac 240
tccctgaggg ccgaggacac cgccatgtac tactgcgccg ccaggggcgt gggctactgc 300
ggcatcgccg ccggcatgga ctactggggc cagggcaccc tggtgaccgt gtcctcc 357
<210>34
<211>483
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>34
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Ala Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ala Tyr Ser Ser Asn
20 25 30
Tyr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Thr Ile Tyr Pro Gly Val Gly Arg Ile Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Ser Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Glu Val Val Pro Ala Leu Arg Gly Gly Gln Trp Tyr Ala Gly
100 105 110
Arg Tyr Phe Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Gln Glu Ser
130 135 140
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
145 150 155 160
Ala Ser Gly Phe Ser Phe Ser Met Ser Val Met Tyr Trp Val Arg Gln
165 170 175
Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Phe Ile Asn Ser Gly Gly
180 185 190
Arg Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
195 200 205
Asp Asn Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr
210 215 220
Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Arg Asp Ser Arg Gly Thr Phe
225 230 235 240
Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser Glu Pro Lys Ser Cys
245 250 255
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
260 265 270
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
275 280 285
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
290 295 300
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
305 310 315 320
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
325 330 335
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
340 345 350
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
355 360 365
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
370 375 380
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
385 390 395 400
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
405 410 415
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
420 425 430
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
435 440 445
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
450455 460
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
465 470 475 480
Pro Gly Lys
<210>35
<211>1449
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>35
gaggtgcagc tgcaggagtc cggcggcggc ctggcccagc ccggcggctc cctgaggctg 60
tcctgcgccg cctccggcta cgcctactcc tccaactaca tcggctggtt caggcaggcc 120
cccggcaagg gcctggaggg cgtggccacc atctaccccg gcgtgggcag gatctactac 180
gccgactccg tgaagggcag gttcaccatc tcccaggaca acgccaagaa caccgtgtac 240
ctgcagatga cctccctgaa gcccgaggac tccgccatgt actactgcgc cgccgaggtg 300
gtgcccgccc tgaggggcgg ccagtggtac gccggcaggt acttctccta ctggggccag 360
ggcaccctgg tgaccgtgtc ctccggcggc ggcggctccg gcggcggctc cgaggtgcag 420
ctgcaggagt ccggcggcgg cctggtgcag cccggcggct ccctgaggct gtcctgcgcc 480
gcctccggct tctccttctc catgtccgtg atgtactggg tgaggcaggc ccccggcaag 540
ggcctggagt gggtgtcctt catcaactcc ggcggcagga cctactacgc cgactccgtg 600
aagggcaggt tcaccatctc cagggacaac tccaagaact ccctgtacct gcagatgaac 660
tccctgagga ccgaggacac cgccctgtac tactgcgcca gggactccag gggcaccttc 720
aggggccagg gcaccctggt gaccgtgtcc tccgagccca agtcctgcga caagacccac 780
acctgccccc cctgccccgc ccccgagctg ctgggcggcc cctccgtgtt cctgttcccc 840
cccaagccca aggacaccct gatgatctcc aggacccccg aggtgacctg cgtggtggtg 900
gacgtgtccc acgaggaccc cgaggtgaag ttcaactggt acgtggacgg cgtggaggtg 960
cacaacgcca agaccaagcc cagggaggag cagtacaact ccacctacag ggtggtgtcc 1020
gtgctgaccg tgctgcacca ggactggctg aacggcaagg agtacaagtg caaggtgtcc 1080
aacaaggccc tgcccgcccc catcgagaag accatctcca aggccaaggg ccagcccagg 1140
gagccccagg tgtacaccct gcccccctcc agggaggaga tgaccaagaa ccaggtgtcc 1200
ctgacctgcc tggtgaaggg cttctacccc tccgacatcg ccgtggagtg ggagtccaac 1260
ggccagcccg agaacaacta caagaccacc ccccccgtgc tggactccga cggctccttc 1320
ttcctgtact ccaagctgac cgtggacaag tccaggtggc agcagggcaa cgtgttctcc 1380
tgctccgtga tgcacgaggc cctgcacaac cactacaccc agaagtccct gtccctgtcc 1440
cccggcaag 1449
<210>36
<211>488
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>36
Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Ala Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ala Tyr Ser Ser Asn
20 2530
Tyr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Thr Ile Tyr Pro Gly Val Gly Arg Ile Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Ser Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Glu Val Val Pro Ala Leu Arg Gly Gly Gln Trp Tyr Ala Gly
100 105 110
Arg Tyr Phe Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Gln Glu Ser
130 135 140
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
145 150 155 160
Ala Ser Gly Tyr Ser Ile Cys Met Gly Trp Phe Arg Gln Ala Pro Gly
165 170 175
Lys Gly Leu Glu Gly Val Ala Thr Ile Ala Gly Leu Ser Ser Lys Tyr
180 185 190
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
195 200 205
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
210 215 220
Ala Met Tyr Tyr Cys Ala Ala Arg Gly Val Gly Tyr Cys Gly Ile Ala
225 230 235 240
Ala Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
245 250 255
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
260 265 270
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
275 280 285
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
290 295 300
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
305 310 315 320
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
325 330 335
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
340 345 350
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
355 360 365
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
370 375 380
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
385 390 395 400
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
405 410 415
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
420 425 430
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
435 440 445
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
450 455 460
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
465 470 475 480
Ser Leu Ser Leu Ser Pro Gly Lys
485
<210>37
<211>1464
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>37
gaggtgcagc tgcaggagtc cggcggcggc ctggcccagc ccggcggctc cctgaggctg 60
tcctgcgccg cctccggcta cgcctactcc tccaactaca tcggctggtt caggcaggcc 120
cccggcaagg gcctggaggg cgtggccacc atctaccccg gcgtgggcag gatctactac 180
gccgactccg tgaagggcag gttcaccatc tcccaggaca acgccaagaa caccgtgtac 240
ctgcagatga cctccctgaa gcccgaggac tccgccatgt actactgcgc cgccgaggtg 300
gtgcccgccc tgaggggcgg ccagtggtac gccggcaggt acttctccta ctggggccag 360
ggcaccctgg tgaccgtgtc ctccggcggc ggcggctccg gcggcggctc cgaggtgcag 420
ctgcaggagt ccggcggcgg cctggtgcag cccggcggct ccctgaggct gtcctgcgcc 480
gcctccggct actccatctg catgggctgg ttcaggcagg cccccggcaa gggcctggag 540
ggcgtggcca ccatcgccgg cctgtcctcc aagtactacg ccgactccgt gaagggcagg 600
ttcaccatct ccagggacaa ctccaagaac accctgtacc tgcagatgaa ctccctgagg 660
gccgaggaca ccgccatgta ctactgcgcc gccaggggcg tgggctactg cggcatcgcc 720
gccggcatgg actactgggg ccagggcacc ctggtgaccg tgtcctccga gcccaagtcc 780
tgcgacaaga cccacacctg ccccccctgc cccgcccccg agctgctggg cggcccctcc 840
gtgttcctgt tcccccccaa gcccaaggac accctgatga tctccaggac ccccgaggtg 900
acctgcgtgg tggtggacgt gtcccacgag gaccccgagg tgaagttcaa ctggtacgtg 960
gacggcgtgg aggtgcacaa cgccaagacc aagcccaggg aggagcagta caactccacc 1020
tacagggtgg tgtccgtgct gaccgtgctg caccaggact ggctgaacgg caaggagtac 1080
aagtgcaagg tgtccaacaa ggccctgccc gcccccatcg agaagaccat ctccaaggcc 1140
aagggccagc ccagggagcc ccaggtgtac accctgcccc cctccaggga ggagatgacc 1200
aagaaccagg tgtccctgac ctgcctggtg aagggcttct acccctccga catcgccgtg 1260
gagtgggagt ccaacggcca gcccgagaac aactacaaga ccaccccccc cgtgctggac 1320
tccgacggct ccttcttcct gtactccaag ctgaccgtgg acaagtccag gtggcagcag 1380
ggcaacgtgt tctcctgctc cgtgatgcac gaggccctgc acaaccacta cacccagaag 1440
tccctgtccc tgtcccccgg caag 1464
<210>38
<211>232
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>38
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 7075 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys
225 230

Claims (10)

1. A complementarity determining region CDR of an anti-TIGIT nanobody VHH chain, comprising one or more of the following combinations:
(1) CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 3;
(2) CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11, and CDR12 shown in SEQ ID NO. 12;
(3) CDR1 shown in SEQ ID NO. 19, CDR2 shown in SEQ ID NO. 20, and CDR3 shown in SEQ ID NO. 21.
2. An anti-TIGIT nanobody VHH chain comprising the framework region FR comprising one or more of the following combinations and the complementarity determining region CDR of claim 1:
(a) FR1 shown by SEQ ID NO. 4, FR2 shown by SEQ ID NO. 5, FR3 shown by SEQ ID NO. 6, and FR4 shown by SEQ ID NO. 7;
(b) FR1 shown by SEQ ID NO. 13, FR2 shown by SEQ ID NO. 14, FR3 shown by SEQ ID NO. 15, and FR4 shown by SEQ ID NO. 16;
(c) FR1 shown by SEQ ID NO. 22, FR2 shown by SEQ ID NO. 23, FR3 shown by SEQ ID NO. 24, and FR4 shown by SEQ ID NO. 25.
3. An anti-TIGIT nanobody, which is a nanobody against a TIGIT epitope and has the VHH chain of claim 2.
4. An anti-TIGIT nano antibody Fc fusion protein is characterized in that the structure of the fusion protein from the N end to the C end is shown as the formula Ia or Ib:
A-L-B(Ia)；
B-L-A(Ib)；
wherein the content of the first and second substances,
a is the anti-TIGIT nanobody of claim 3;
b is an Fc fragment of IgG; and
l is a non-or flexible linker.
5. The anti-TIGIT nanobody Fc fusion protein of claim 4, wherein the anti-TIGIT nanobody comprises a monomer, a bivalent antibody, and/or a multivalent antibody.
6. A polynucleotide encoding a protein selected from the group consisting of: the Complementarity Determining Region (CDR) of the anti-TIGIT nanobody VHH chain of claim 1, or the anti-TIGIT nanobody VHH chain of claim 2, or the anti-TIGIT nanobody of claim 3, or the anti-TIGIT nanobody Fc fusion protein of any one of claims 4 and 5.
7. An expression vector comprising the polynucleotide of claim 6.
8. A host cell comprising the expression vector of claim 7, or having the polynucleotide of claim 6 integrated into its genome.
9. A method for producing anti-TIGIT nano antibody and Fc fusion protein thereof is characterized by comprising the following steps:
(a) culturing the host cell of claim 8 under conditions suitable for production of the anti-TIGIT nanobody of claim 3, thereby obtaining a culture comprising the anti-TIGIT nanobody of claim 3; or culturing the host cell of claim 8 under conditions suitable for production of the anti-TIGIT nanobody Fc fusion protein of any one of claims 4 and 5, thereby obtaining a culture comprising the anti-TIGIT nanobody Fc fusion protein of any one of claims 4 and 5; and
(b) isolating and/or recovering the anti-TIGIT nanobody or the anti-TIGIT nanobody Fc fusion protein from the culture; and
(c) optionally, purifying and/or modifying the anti-TIGIT nanobody or the anti-TIGIT nanobody Fc fusion protein obtained in step (b).
10. An immunoconjugate, comprising:
(a) anti-TIGIT nanobody VHH chain of claim 2, or anti-TIGIT nanobody of claim 3, or anti-TIGIT nanobody Fc fusion protein of any one of claims 4, 5; and
(b) a coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle/nanorod, a nanomagnet, a viral coat protein, or a VLP, or a combination thereof.
CN202010632294.5A 2020-07-03 2020-07-03 anti-TIGIT nano antibody and application thereof Active CN111718415B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010632294.5A CN111718415B (en) 2020-07-03 2020-07-03 anti-TIGIT nano antibody and application thereof

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CN113307870A (en) * 2020-10-30 2021-08-27 上海洛启生物医药技术有限公司 anti-IL 5 nano antibody and application thereof
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