CN113527498B - anti-Trop 2 nano antibody and application thereof - Google Patents

anti-Trop 2 nano antibody and application thereof Download PDF

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CN113527498B
CN113527498B CN202110182419.3A CN202110182419A CN113527498B CN 113527498 B CN113527498 B CN 113527498B CN 202110182419 A CN202110182419 A CN 202110182419A CN 113527498 B CN113527498 B CN 113527498B
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CN113527498A (en
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万亚坤
朱敏
盖军伟
李光辉
沈晓宁
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Shanghai Novamab Biopharmaceuticals Co Ltd
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    • C12N5/0684—Cells of the urinary tract or kidneys
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    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Abstract

The invention relates to the technical field of antibody medicines, in particular to an anti-Trop 2 nano antibody and application thereof. The invention discloses a group of specific nano antibodies aiming at Trop2, discloses amino acid sequences of a framework region FR and a complementarity determining region CDR of a VHH chain of the nano antibodies, and also discloses a gene sequence for coding the nano antibodies or the VHH chain of the nano antibodies. The anti-Trop 2 nano antibody provided by the invention can be effectively combined with the Trop2 antigen. The gene sequence of the nano antibody disclosed by the invention provides a research and development basis for research based on the Trop2 target spot, including development of antibody coupling drugs and multispecific antibody drugs.

Description

anti-Trop 2 nano antibody and application thereof
The application is a divisional application of Chinese patent application with the application date of 16/4/2020 and the application number of CN202010301498.0, and the invention name of the invention is 'anti-Trop 2 nano antibody and application thereof'.
Technical Field
The invention relates to the technical field of biomedicine or biopharmaceutical, and discloses an anti-Trop 2 nano antibody and application thereof.
Background
Trop2 belongs to the family of tactd, is a cell surface glycoprotein encoded and expressed by tactd 2 gene, and is also named as tumor-associated calcium ion signal transducer 2 (tactd 2), epidermal glycoprotein 1(EGP-1), gastrointestinal tumor-associated antigen (GA733-1), and surface marker 1(M1S 1). TROP-2 promotes the growth, proliferation and metastasis of tumor cells mainly by regulating calcium ion signal pathways, cyclin expression and reducing fibronectin adhesion. Trop2 also interacts with the β -catenin in the Wnt signaling cascade, and thus has effects on transcription of nuclear oncogenes and proliferation of cells.
Research shows that Trop2 has a large amount of expression in tumors such as gastric cancer, cervical cancer, breast cancer, lung cancer, prostatic cancer, colon cancer, uterine mucous papillary carcinoma and the like, has obvious correlation with tumor metastasis and prognosis and plays an important role in tumor generation. Trop2 is a transmembrane protein whose extracellular domain is overexpressed on a variety of tumors and is therefore a natural candidate for targeted therapeutic development. The tissue expression limitations of Trop2 allow for reduced toxicity of the treatment, which is also an advantage of targeted Trop2 therapy. Antibodies, antibody conjugates, combination drugs and other various types of drugs which take Trop2 as a target point are under development. The utility of anti-Trop 2 antibodies conjugated to other chemotherapeutic drugs has been demonstrated in various preclinical studies. Antibody-conjugated drugs (ADC) IMMU-132 for the treatment of Trop 2-overexpressed epithelial malignancies have been in phase II/III clinical trials. The novel antibody coupling drug Sacituzumab virotecan (IMMU-132) takes Trop2 as a target spot, is formed by coupling a humanized antibody hRS7 serving as a targeting carrier with an irinotecan active metabolite SN38, and can be used for treating various epithelial malignant tumors such as breast cancer (triple negative breast cancer), ovarian cancer, small cell lung cancer and the like. In addition, other humanized anti-Trop 2 IgG-SN-38 conjugates, such as anti-Trop 2hRS7-CL2A-SN-38 antibody conjugate drugs, have been shown to have significant specific anti-cancer effects in xenograft models of various tumor cell lines (Calu-3, Capan-1, BxPC-3, and COLO-205).
So far, no nano-antibody drug for Trop2 target has been published in the market, but nano-antibodies (Nb), namely heavy chain single domain antibodies VHH (heavy chain of heavy-chain antibody), exist in camels and are single domain antibodies (hcabs) with naturally deleted light chains, and the single domain antibodies which are obtained by cloning the variable regions thereof and only consist of one heavy chain variable region are the smallest units which can be obtained at present and have complete functions and can stably bind to antigens. The single domain antibody has the characteristics of high stability, good water solubility, simple humanization, high targeting property, strong penetrability and the like, and plays an extremely imaginable huge function in immune experiments, diagnosis and treatment. Single domain antibodies are becoming an emerging force in the diagnosis and treatment of new generation antibodies.
Therefore, the development of an anti-Trop 2 nanobody, particularly an anti-Trop 2 nanobody with good Trop2 antigen binding property is needed in the art.
Disclosure of Invention
The invention provides 7 strains of specific Trop 2-directed nano antibodies, and simultaneously provides a coding sequence and a preparation method of the nano antibodies. Meanwhile, the invention provides a research and development basis for research based on the Trop2 target spot, including development of antibody coupling drugs and multispecific antibody drugs.
In a first aspect of the invention, the invention provides a Complementarity Determining Region (CDR) region of an anti-Trop 2 nanobody.
In another preferred embodiment, the Trop2 nanobody comprises an immunoglobulin single variable domain that specifically binds to Trop 2. In some embodiments, the Trop2 nanobody comprises two or more immunoglobulin single variable domains that specifically bind to Trop 2.
In another preferred embodiment, the at least one immunoglobulin single variable domain comprises a CDRl, a CDR2 and a CDR3 selected from the group consisting of:
(1) CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, CDR3 shown in SEQ ID NO. 3 (corresponding to the CDR of antibody strain Nb1-70 or HuNb 1-70);
(2) CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11, CDR3 shown in SEQ ID NO. 12 (corresponding to the CDR of antibody strain Nb1-78 or HuNb 1-78);
(3) CDR1 shown in SEQ ID NO:18, CDR2 shown in SEQ ID NO:19, CDR3 shown in SEQ ID NO:20 (corresponding to the CDR of antibody strain Nb1-81 or HuNb 1-81);
(4) CDR1 shown in SEQ ID NO:25, CDR2 shown in SEQ ID NO:26, CDR3 shown in SEQ ID NO:27 (corresponding to the CDR of antibody strain Nb2-22 or HuNb 2-22);
(5) CDR1 shown in SEQ ID NO:33, CDR2 shown in SEQ ID NO:34, CDR3 shown in SEQ ID NO:35 (corresponding to the CDR of antibody strain Nb2-38 or HuNb 2-38);
(6) CDR1 shown in SEQ ID NO:41, CDR2 shown in SEQ ID NO:42, CDR3 shown in SEQ ID NO:43 (corresponding to the CDR of antibody strain Nb2-59 or HuNb 2-59);
(7) CDR1 shown in SEQ ID NO:49, CDR2 shown in SEQ ID NO:50, CDR3 shown in SEQ ID NO:51 (corresponding to the CDR of antibody strain Nb6-24 or HuNb 6-24).
In another preferred embodiment, any one of the above amino acid sequences further comprises a derivative sequence optionally added, deleted, modified and/or substituted with at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and retaining high affinity binding ability to Trop 2.
In another preferred embodiment, the CDRs 1, 2 and 3 are separated by framework regions FR1, FR2, FR3 and FR4 of the VHH chain.
In a second aspect of the invention, there is provided a VHH chain of an anti-Trop 2 nanobody, the VHH chain comprising one or more of the framework region FRs and the CDR regions according to the first aspect of the invention.
In some embodiments, the at least one immunoglobulin single variable domain comprises FRl, FR2, FR3, and FR4 selected from the group consisting of:
(1) 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 (corresponding to the FR of antibody strain Nb 1-70);
(2) 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. 7 (corresponding to the FR of antibody strain Nb 1-78);
(3) FR1 shown by SEQ ID NO. 13, FR2 shown by SEQ ID NO. 21, FR3 shown by SEQ ID NO. 22, and FR4 shown by SEQ ID NO. 7 (corresponding to the FR of antibody strain Nb 1-81);
(4) FR1 shown by SEQ ID NO:28, FR2 shown by SEQ ID NO:29, FR3 shown by SEQ ID NO:30, and FR4 shown by SEQ ID NO:7 (corresponding to the FR of antibody strain Nb 2-22);
(5) FR1 shown by SEQ ID NO:36, FR2 shown by SEQ ID NO:37, FR3 shown by SEQ ID NO:38, and FR4 shown by SEQ ID NO:7 (corresponding to the FR of antibody strain Nb 2-38);
(6) FR1 shown by SEQ ID NO:44, FR2 shown by SEQ ID NO:45, FR3 shown by SEQ ID NO:46, and FR4 shown by SEQ ID NO:7 (corresponding to the FR of antibody strain Nb 2-59);
(7) FR1 shown by SEQ ID NO. 13, FR2 shown by SEQ ID NO. 52, FR3 shown by SEQ ID NO. 53, and FR4 shown by SEQ ID NO. 7 (corresponding to the FR of antibody strain Nb 6-24);
(8) FR1 shown by SEQ ID NO:56, FR2 shown by SEQ ID NO:57, FR3 shown by SEQ ID NO:58, and FR4 shown by SEQ ID NO:59 (corresponding to the FR of antibody strain HuNb 1-70);
(9) FR1 shown by SEQ ID NO:56, FR2 shown by SEQ ID NO:62, FR3 shown by SEQ ID NO:63, and FR4 shown by SEQ ID NO:64 (corresponding to the FR of antibody strain HuNb 1-78);
(10) FR1 shown by SEQ ID NO:56, FR2 shown by SEQ ID NO:67, FR3 shown by SEQ ID NO:63, and FR4 shown by SEQ ID NO:64 (corresponding to the FR of antibody strain HuNb 1-81);
(11) FR1 shown by SEQ ID NO:56, FR2 shown by SEQ ID NO:70, FR3 shown by SEQ ID NO:71, and FR4 shown by SEQ ID NO:64 (corresponding to the FR of antibody strain HuNb 2-22);
(12) FR1 shown by SEQ ID NO:56, FR2 shown by SEQ ID NO:74, FR3 shown by SEQ ID NO:63, and FR4 shown by SEQ ID NO:59 (corresponding to the FR of antibody strain HuNb 2-38);
(13) FR1 shown by SEQ ID NO:56, FR2 shown by SEQ ID NO:77, FR3 shown by SEQ ID NO:63, and FR4 shown by SEQ ID NO:64 (corresponding to the FR of antibody strain HuNb 2-59);
(14) FR1 shown by SEQ ID NO:56, FR2 shown by SEQ ID NO:80, FR3 shown by SEQ ID NO:81, and FR4 shown by SEQ ID NO:64 (corresponding to the FR of antibody strain HuNb 6-24).
In another preferred embodiment, the VHH chain is selected from one or more of SEQ ID NO 8, SEQ ID NO 16, SEQ ID NO 23, SEQ ID NO 31, SEQ ID NO 39, SEQ ID NO 47, SEQ ID NO 54, SEQ ID NO 60, SEQ ID NO 65, SEQ ID NO 68, SEQ ID NO 72, SEQ ID NO 75, SEQ ID NO 78 or SEQ ID NO 82.
In another preferred embodiment, the VHH chain is selected from one or more of SEQ ID NO 60, SEQ ID NO 65, SEQ ID NO 68, SEQ ID NO 72, SEQ ID NO 75, SEQ ID NO 78 or SEQ ID NO 82.
In a third aspect of the invention, there is provided an anti-Trop 2 antibody comprising one or more VHH chains of an anti-Trop 2 nanobody according to the second aspect of the invention.
In another preferred example, the anti-Trop 2 antibody also encompasses anti-Trop 2 antibody molecules capable of binding to the same epitope on Trop2 as a VHH consisting of the amino acid sequence of any one of SEQ ID NO 8, 16, 23, 31, 39, 47, 54, 60, 65, 68, 72, 75, 78 or 82.
In another preferred embodiment, the antibody may be a monomeric, bivalent, and/or multivalent antibody.
In a fourth aspect of the invention, there is provided an isolated polynucleotide sequence encoding a protein selected from the group consisting of: a CDR region according to the first aspect of the invention or a VHH chain according to the second aspect of the invention, or an anti-Trop 2 antibody according to the third aspect of the invention.
In another preferred embodiment, the polynucleotide sequences are in combination, preferably the polynucleotide sequences comprise one or more of SEQ ID NO 9, SEQ ID NO 17, SEQ ID NO 24, SEQ ID NO 32, SEQ ID NO 40, SEQ ID NO 48, SEQ ID NO 55, SEQ ID NO 61, SEQ ID NO 66, SEQ ID NO 69, SEQ ID NO 73, SEQ ID NO 76, SEQ ID NO 79 or SEQ ID NO 83.
In another preferred embodiment, the invention relates to a nucleic acid molecule encoding the Trop2 nanobody of the invention. The nucleic acid of the present invention may be RNA, DNA or cDNA.
In a fifth aspect of the invention, there is provided an expression vector expressing the polynucleotide of the fourth aspect of the invention.
According to a sixth aspect of the present invention there is provided a host cell comprising an expression vector according to the fifth aspect of the present invention or having a polynucleotide according to the fourth aspect of the present invention integrated into its genome.
In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.
In another preferred embodiment, the host cell is selected from the group consisting of: escherichia coli, yeast cells.
In a seventh aspect of the present invention, there is provided a method for producing an anti-Trop 2 nanobody, comprising the steps of:
(a) culturing the host cell of the sixth aspect of the invention under conditions suitable for the production of nanobodies, thereby obtaining a culture comprising the anti-Trop 2 nanobody; and
(b) isolating or recovering the anti-Trop 2 nanobody from the culture; and optionally
(c) Purifying and/or modifying the Trop2 nanobody obtained in step (b).
In another preferred embodiment, the anti-Trop 2 nanobody has an amino acid sequence shown as SEQ ID NO 8, 16, 23, 31, 39, 47, 54, 60, 65, 68, 72, 75, 78 or 82.
In an eighth aspect of the present invention, there is provided a conjugate comprising:
(a) a CDR region according to the first aspect of the invention or a VHH chain according to the second aspect of the invention, or an anti-Trop 2 antibody according to the third aspect of the invention; and operatively connected
(b) A modifying label selected from the group consisting of: chemical markers and biological markers.
In another preferred embodiment, the chemical label is an isotope, an immunotoxin and/or a chemical drug.
In another preferred embodiment, the biomarker is a biotin, avidin or enzyme label.
In a ninth aspect of the invention there is provided a conjugate prepared by conjugating a CDR region according to the first aspect of the invention or a VHH chain according to the second aspect of the invention, an anti-Trop 2 antibody according to the third aspect of the invention, or a conjugate according to the eighth aspect of the invention to a solid or semi-solid medium.
In another preferred embodiment, the conjugate consists of a conjugate according to the eighth aspect of the invention and a coupling element 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)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles, and the like.
In another preferred embodiment, the conjugate comprises: a multivalent (e.g. bivalent) CDR region according to the first aspect of the invention or a VHH chain according to the second aspect of the invention, or an anti-Trop 2 antibody according to the third aspect of the invention.
In another preferred embodiment, the multivalent is that the immunoconjugate comprises multiple repeats of the CDR regions according to the first aspect of the invention or of the VHH chain according to the second aspect of the invention, or of the anti-Trop 2 antibody according to the third aspect of the invention in its amino acid sequence.
In a tenth aspect of the invention, there is provided the use of a CDR region according to the first aspect of the invention or a VHH chain according to the second aspect of the invention, or an anti-Trop 2 antibody according to the third aspect of the invention, for the preparation of (a) a reagent for the detection of a Trop2 molecule; (b) a medicine for treating tumor.
In another preferred embodiment, the detection comprises flow detection and cell immunofluorescence detection.
In an eleventh aspect of the present invention, there is provided a pharmaceutical composition comprising:
(i) a CDR region according to the first aspect of the invention or a VHH chain according to the second aspect of the invention, an anti-Trop 2 antibody according to the third aspect of the invention, and/or a conjugate according to the eighth aspect of the invention;
(ii) a pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition is in the form of injection.
In another preferred embodiment, the pharmaceutical composition is used for preparing a medicament for treating tumors selected from the group consisting of: tumors of reproductive system, digestive system, head and neck, and nervous system.
In another preferred embodiment, the tumor of the reproductive system is: endometrial, ovarian, cervical, prostate cancer, and the like.
In another preferred embodiment, the tumor of the digestive system is: pancreatic cancer, colon cancer, gastric cancer, esophageal cancer, bile duct cancer, and the like.
In another preferred embodiment, the head and neck tumor is: oral cancer, laryngeal cancer, tongue cancer, gingival cancer, etc.
In another preferred embodiment, the tumor of the nervous system is: brain glioma, medulloblastoma, and the like.
In a twelfth aspect of the invention, there is provided use of one or more of a CDR region according to the first aspect of the invention or a VHH chain according to the second aspect of the invention, or an anti-Trop 2 antibody according to the third aspect of the invention:
(i) for detecting Trop2 molecules;
(ii) for streaming detection;
(iii) for cellular immunofluorescence detection;
(iv) for the treatment of tumors;
(v) can be used for tumor diagnosis.
In another preferred embodiment, the use is non-diagnostic and non-therapeutic.
In a thirteenth aspect of the present invention, there is provided a recombinant protein having:
(i) a CDR region according to the first aspect of the invention or a VHH chain according to the second aspect of the invention, or an anti-Trop 2 antibody according to the third aspect of the invention; and
(ii) optionally a tag sequence to facilitate expression and/or purification.
In another preferred embodiment, the tag sequence comprises a 6His tag and an HA tag.
In another preferred embodiment, the recombinant protein specifically binds to Trop2 protein.
In a fourteenth aspect of the invention, there is provided the use of a CDR region according to the first aspect of the invention or a VHH chain according to the second aspect of the invention, an anti-Trop 2 antibody according to the third aspect of the invention, or a conjugate according to the eighth aspect of the invention, in the manufacture of a medicament, a reagent, a detection plate or a kit;
wherein the reagent, assay plate or kit is for: detecting Trop2 protein in the sample;
wherein the medicament is used for treating or preventing tumors expressing Trop 2.
In a fifteenth aspect of the invention, there is provided a kit comprising CDR regions according to the first aspect of the invention or VHH chains according to the second aspect of the invention, an anti-Trop 2 antibody according to the third aspect of the invention, or a conjugate according to the eighth aspect of the invention.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the SDS-PAGE detection result of the fusion Fc of the extracellular domain of human Trop2 protein. The result shows that the purity of the protein reaches more than 90%.
FIG. 2 shows the result of the library capacity detection of the phage display Trop2 nanobody library. The results showed that the library had a size of 1.0X 109 CFU.
FIG. 3 shows the detection result of the insertion rate of the target gene VHH of the phage display Trop2 nano antibody library. The results showed that the VHH insertion rate of the library was 91.6%.
FIG. 4 shows the results of phage display technology screening for enrichment of Trop 2-specific nanobody phage. The results show that after 3 rounds of screening, the antibody specific phage is enriched by 46 times.
Fig. 5 shows the results of flow cytometry to detect the binding activity of Trop2 nanobody to Colo205 cells. The results show that 7 strains of the 23 strains of the nano-antibodies can effectively bind to the Trop2 protein on the cell surface.
FIG. 6 shows the binding activity of the Trop2 nanobody to Colo205 cells before and after humanization. The result shows that the Trop2 nano antibody still has good binding activity to Colo205 cells (Trop2 positive) after being humanized.
Fig. 7 shows the identification result of the endocytosis property of Trop2 nanometer antibody. The results show that the antibodies HuNb1-70, HuNb2-59 and HuNb2-22 are slightly endocytosed by Colo205 cells, while HuNb6-24, HuNb2-38, HuNb1-78 and HuNb1-81 can be obviously endocytosed by Colo205 cells.
Detailed Description
The inventor successfully obtains a group of anti-Trop 2 nanometer antibodies through extensive and intensive research and a large number of screens. Specifically, the invention utilizes the human Trop2 extracellular segment antigen protein to immunize camels to obtain a high-quality immune nano antibody gene library. Then, the Trop2 protein molecules are coupled on an enzyme label plate to display the correct spatial structure of the Trop2 protein, and the immune nano antibody gene library (camel heavy chain antibody phage display gene library) is screened by the antigen in the form by using a phage display technology, so that the Trop2 specific nano antibody gene is obtained. Experimental results show that the 7-strain anti-Trop 2 nano antibody obtained by the invention can be effectively combined with Trop 2. The present invention has been completed based on this finding.
As used herein, the terms "nanobody of the invention", "anti-Trop 2 nanobody of the invention", "Trop 2 nanobody of the invention" are used interchangeably and refer to nanobodies that specifically recognize and bind to Trop2 (including human Trop 2). Particularly preferred are nanobodies with the amino acid sequence of the VHH chain as shown in SEQ ID NO 60, SEQ ID NO 65, SEQ ID NO 68, SEQ ID NO 72, SEQ ID NO 75, SEQ ID NO 78 or SEQ ID NO 82.
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 "nanobody(s) (sdAb, or VHH)" and "nanobody" (or "nanobody") have the same meaning, and refer to cloning of the variable region of the heavy chain of an antibody, constructing a nanobody consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with full function. Nanobodies (VHHs) consisting of only one heavy chain variable region are typically constructed by first obtaining an antibody that naturally lacks light and heavy chain constant region 1(CH1) and then cloning the variable region of the antibody heavy chain.
The nano antibody/single domain antibody (Nanobody) is used as a novel small molecule antibody fragment and is obtained by cloning a camel natural heavy chain antibody heavy chain variable region (VHH). The Nanobody (Nb) has excellent biological characteristics, the molecular weight of 12-15kDa, which is one tenth of that of a complete antibody, good tissue penetration, high specificity and good water solubility. Due to the special structural properties, the antibody has the advantages of the traditional antibody and the micromolecule drug, almost perfectly overcomes the defects of long development period, low stability, harsh storage conditions and the like of the traditional antibody, gradually becomes a new force in the treatment of the new generation of antibody, and shows wide application prospect in immunodiagnosis and treatment.
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, which are in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a β -sheet 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-Trop 2 protein antibody or the fragment thereof.
As used herein, the terms "heavy chain variable region" and "VH"may be used interchangeably.
As used herein, the term "variable region" is used interchangeably with "Complementary 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 present invention", "protein of the present invention", or "polypeptide of the present invention" are used interchangeably and refer to a polypeptide that specifically binds to Trop2 protein, 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, called variable regions (CDRs), located in the heavy chain variable region, 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 a Trop2 protein binding activity, including 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 nanobodies or fragments thereof. In addition to nearly full-length polypeptides, fragments of the nanobodies of the invention are also encompassed by the present 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.
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 sequences of the invention are shown in Table 2
TABLE 2
Figure BDA0002941786820000131
Figure BDA0002941786820000141
Figure BDA0002941786820000151
Figure BDA0002941786820000161
Figure BDA0002941786820000171
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 a prokaryote such as Escherichia coli, the sense of DNA uptakeThe cells in the state can be harvested after exponential growth phase and then treated with CaCl2Methods, the steps used are well known in the art. Another method is to use MgCl2. 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, etc.
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. drug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 9. a therapeutic agent (e.g., cisplatin) or any form of nanoparticle, and the like.
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;
(4) CDR1 shown in SEQ ID NO. 28, CDR2 shown in SEQ ID NO. 29, CDR3 shown in SEQ ID NO. 30;
(5) CDR1 shown in SEQ ID NO. 37, CDR2 shown in SEQ ID NO. 38, CDR3 shown in SEQ ID NO. 39;
(6) CDR1 shown in SEQ ID NO. 46, CDR2 shown in SEQ ID NO. 47, CDR3 shown in SEQ ID NO. 48;
(7) CDR1 shown in SEQ ID NO. 55, CDR2 shown in SEQ ID NO. 56, and CDR3 shown in SEQ ID NO. 57.
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: intratumoral, intraperitoneal, intravenous, or topical administration.
The pharmaceutical composition can be directly used for binding Trop2 protein molecules, and thus can be used for treating tumors. In addition, other therapeutic agents may also be used simultaneously.
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 nanobody (or its conjugate) of the present invention as described above 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.
Labeled nanobodies
In a preferred embodiment of the invention, the nanobody carries a detectable label. 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 invention, the nano-antibody against Trop2 is labeled with colloidal gold to obtain a colloidal gold-labeled nano-antibody.
The anti-Trop 2 nano antibody has good binding activity.
Detection method
The invention also relates to a method for detecting the Trop2 protein. The method comprises the following steps: obtaining a cell and/or tissue sample; dissolving the sample in a medium; detecting the level of Trop2 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 level of Trop2, which comprises an antibody for identifying the Trop2 protein, a lysis medium for dissolving a sample, and general reagents and buffers required by 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 nanobody of the present invention has wide biological and clinical application values, and its applications relate to many fields such as diagnosis and treatment of diseases related to Trop2, basic medical research, and biological research. One preferred application is for clinical diagnosis and targeted therapy against Trop 2.
The main advantages of the invention include:
(1) the nano antibody can be effectively combined with Trop2 protein on the surface of a cell.
(2) The nano antibody provides a research and development basis for research based on the Trop2 target spot, including antibody coupling drug and multi-specificity antibody drug development.
(3) The production of the nano antibody is simple and convenient.
(4) The nano antibody is suitable for prokaryotic expression and eukaryotic expression, and has the advantages of extremely high solubility, difficult aggregation, high temperature resistance, strong acid, strong alkali resistance and other denaturing conditions. Is suitable for laboratory and industrial development.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
Example 1: human Trop2 extracellular domain protein expression
Transient expression of human Trop2 extracellular domain protein by using mammalian cell HEK 293F: mixing the pFUSE-IgG recombinant plasmid with the cloned human Trop2 extracellular segment gene with a transfection reagent PEI 1:3, and transfecting the mixture to HEK293F cells; 37 ℃ and 6% CO2Culturing for 6 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 protein expressing and purifying hTar 2(ECD) -Fc is more than 90%, and the protein can be used for camel immunization and antibody screening.
Example 2: construction and screening of human Trop2 extracellular domain protein immune library
Immunizing 1 Xinjiang bactrian camel with the purified hTar 2(ECD) -Fc protein, separating total RNA from camel peripheral blood after 7 times of immunization, performing reverse transcription and PCR amplification to obtain a VHH gene, cloning the VHH gene to a phage vector pMECS, and transforming the pMECS to TG1 host cells to construct a phage display library. The library capacity constructed by detection is 1.0 multiplied by 109CFU (fig. 2), library insertion rate was 91.6% (fig. 3).
Then, the phage display technology is used for library screening, and phage enrichment containing antibody genes is finally obtained by 46 times through 3 rounds of 'adsorption-washing-enrichment' screening processes (FIG. 4). 600 phage clones were randomly selected from the enriched phage clones and subjected to PE-ELISA identification, all the obtained positive clones were subjected to sequencing identification, and all antibodies (23 strains) with different sequences were used as candidates.
Example 3: expression of antihuman Trop2 nano antibody
Cloning the 23 strains of nano antibody genes with different sequences to a pFUSE-IgG vector, mixing the recombinant plasmid with a transfection reagent PEI 1:3, and transfecting to HEK293F cells; 37 ℃ and 6% CO2Culturing for 6 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; and ultrafiltering the eluted protein into PBS solution, and sampling for purity detection after yield determination. The purity of the nano antibody expressed and purified by detection is more than 90 percent, and the nano antibody can be used for candidate functional activity research.
Example 4: detection of binding activity of Trop2 nano antibody and cell surface antigen
Antibody binding function verification was performed using Colo205 cells highly expressing Trop 2: after the cultured Colo205 cells were trypsinized and neutralized with complete medium, the cells were washed once with PBS and then collected; evenly subpackaging into 96-well plates, adding diluted antibodies (the dilution concentration of each group of antibodies is 0.313ug/mL, 0.156ug/mL and 0.078ug/mL respectively), and incubating at 4 ℃ for 20 min; after centrifugation, the cells were washed once with PBS, and goat anti-human IgG-FITC (diluted at 1: 200) was added 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 detecting FITC signal of each sample by flow. The results are shown in fig. 5, in which 7-strain nanobody can effectively bind to Trop2 protein on the cell surface. The sequences of the 7-strain nano-antibodies are shown in Table 1.
TABLE 1 Trop2 Nanobody sequence numbering
Antibody numbering Amino acid sequence numbering Numbering of nucleotide sequences
Nb1-70 SEQ ID NO：8 SEQ ID NO：9
Nb1-78 SEQ ID NO：16 SEQ ID NO：17
Nb1-81 SEQ ID NO：23 SEQ ID NO：24
Nb2-22 SEQ ID NO：31 SEQ ID NO：32
Nb2-38 SEQ ID NO：39 SEQ ID NO：40
Nb2-59 SEQ ID NO：47 SEQ ID NO：48
Nb6-24 SEQ ID NO：54 SEQ ID NO：55
Example 5: antibody humanization design
Placing the amino acid sequences of the 7 strains of nano antibodies in a structure database to search homologous structures, taking the structures with higher sequence isogenity, comparing the structures, finally selecting proteins including 3dwt according to the resolution of the crystal structure and the constructed evolutionary tree, performing multi-template homologous modeling on the target nano antibody sequence, and selecting the structure with the lowest molpdf according to the high-low ranking 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 Trop2 nano antibody is determined, and the sequence of the humanized antibody corresponds to the sequence shown in the following table 2:
TABLE 2 humanized Trop2 Nanobody sequence numbering
Antibody numbering Amino acid sequence numbering Numbering of nucleotide sequences
HuNb1-70 SEQ ID NO：60 SEQ ID NO：61
HuNb1-78 SEQ ID NO：65 SEQ ID NO：66
HuNb1-81 SEQ ID NO：68 SEQ ID NO：69
HuNb2-22 SEQ ID NO：72 SEQ ID NO：73
HuNb2-38 SEQ ID NO：75 SEQ ID NO：76
HuNb2-59 SEQ ID NO：78 SEQ ID NO：79
HuNb6-24 SEQ ID NO：82 SEQ ID NO：83
After the amino acid sequence of the humanized antibody is optimized according to human codons, the nucleotide 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 3.
Example 6: detection of binding activity of humanized Trop2 nano antibody and cell surface antigen
The humanized antibody expressed and purified above and the corresponding humanized pre-antibody are jointly subjected to cell binding activity detection: after the cultured Colo205 cells were trypsinized and neutralized with complete medium, the cells were washed once with PBS and then collected; evenly subpackaging into 96-well plates, adding diluted antibodies (the dilution concentration of each group of antibodies is respectively 20ug/mL, 10ug/mL, 5ug/mL, 2.5ug/mL, 1.25ug/mL, 0.625ug/mL, 0.313ug/mL, 0.156ug/mL and 0.078ug/mL), and incubating at 4 ℃ for 20 min; after centrifugation, the cells were washed once with PBS, 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 detecting FITC signal of each sample by flow. The results are shown in figure 6 which shows,binding Activity of Each antibody to cells EC50Values are summarized in the following table 3, and the humanized Trop2 nano antibody still has better cell binding activity.
TABLE 3 binding Activity of Trop2 Nanobody on Colo205 cells EC50
Figure BDA0002941786820000231
Example 7: detection of endocytic activity of humanized Trop2 nano antibody
Collecting cultured Colo205 cells, washing the cells once by using a PBS solution, counting the cells and subpackaging the cells into 96-well plates; then adding the diluted Trop2 nano antibody (1.25ug/mL, 100uL) into the cells, mixing uniformly, and incubating for 20min at 4 ℃; after washing once with PBS solution, the 96-well plate of the experiment group not endocytosed (0h) was immediately placed on ice, while the experiment group endocytosed (2h) was cultured in an incubator at 37 ℃ with 5% CO2(ii) a After 2h, both plates were removed simultaneously and centrifuged at 4 ℃ and 3000rpm for 4 min; discarding the supernatant, washing the cells once with PBS, and then centrifuging; adding the goat anti human IgG-FITC (diluted according to a ratio of 1: 200), and incubating for 20min at 4 ℃; 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 detecting FITC signal of each sample by flow. As shown in FIG. 7, the antibodies HuNb1-70, HuNb2-59 and HuNb2-22 were less endocytosed by Colo205 cells, while HuNb6-24, HuNb2-38, HuNb1-78 and HuNb1-81 were significantly endocytosed by Colo205 cells.
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 listing
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<213> Artificial Sequence (Artificial Sequence)
<400> 3
Ala Ala Ala Tyr Pro Trp Thr Ala Cys Tyr Gly Gly Tyr Ala Met Ala
1 5 10 15
Ala Asp Phe Pro 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 Val Gln Val 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 Gly Met
1 5 10 15
<210> 6
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Ser Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn
1 5 10 15
Ala Lys Asn Thr Leu Tyr Leu Glu Met Asp Ser Leu Glu Pro Glu Asp
20 25 30
Thr Ala Met Tyr Phe 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> 127
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Val Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Ser Leu Ser Ser Thr
20 25 30
Cys Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Gly Met Ile Ala Ser Asp Gly Ser Thr Ser Tyr Thr Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Glu Met Asp Ser Leu Glu Pro Glu Asp Thr Ala Met Tyr Phe Cys Ala
85 90 95
Ala Ala Tyr Pro Trp Thr Ala Cys Tyr Gly Gly Tyr Ala Met Ala Ala
100 105 110
Asp Phe Pro Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 9
<211> 381
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
caggtgcagc tgcaggagtc tggaggaggc tcggtgcagg ttggagggtc tctgagactc 60
tcctgtgcag cctctggagt cagtttaagt agcacttgta tgagctggtt ccgccaggct 120
ccagggaagg agcgcgaggg ggtcggaatg attgcgagtg acggcagcac aagctacaca 180
gactccgtga agggccgatt caccatctcc aaagacaacg ccaagaacac tctgtatctt 240
gaaatggaca gtctggaacc tgaggacact gccatgtatt tctgtgcggc agcgtaccct 300
tggaccgcct gctatggggg ttatgcgatg gcggctgact ttccttactg gggccagggg 360
acccaggtca ccgtctcctc a 381
<210> 10
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Asp Tyr Ser Thr Gly Arg Arg Cys Met Ala
1 5 10
<210> 11
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Ile Asp Tyr Ala Gly Thr Ser Thr
1 5
<210> 12
<211> 24
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Ala Ala Ala Ala Ile Pro Tyr Glu Arg Cys Pro Asp Ile Ser Asp Pro
1 5 10 15
Gln Ile Leu Val Ser Phe Pro Tyr
20
<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 Ala 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 Phe Arg Gln Thr Leu Gly Lys Glu Arg Glu Gly Val Ala Ile
1 5 10 15
<210> 15
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly
1 5 10 15
Thr Lys Asn Thr Met Ser Leu Gln Met Asn Ser Leu Glu Pro Ala Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 16
<211> 131
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
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 Asp Tyr Ser Thr Gly Arg Arg
20 25 30
Cys Met Ala Trp Phe Arg Gln Thr Leu Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ile Ile Asp Tyr Ala Gly Thr Ser Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Thr Lys Asn Thr Met Ser
65 70 75 80
Leu Gln Met Asn Ser Leu Glu Pro Ala Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Ala Ala Ile Pro Tyr Glu Arg Cys Pro Asp Ile Ser Asp Pro
100 105 110
Gln Ile Leu Val Ser Phe Pro Tyr Trp Gly Gln Gly Thr Gln Val Thr
115 120 125
Val Ser Ser
130
<210> 17
<211> 393
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
caggtgcagc tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtgcag cctctgatta cagcacgggt aggcgatgca tggcctggtt tcgccagact 120
ctggggaagg agcgcgaagg ggtcgcaatt atcgattatg ctggtactag cacccggtat 180
gccgactccg tcaagggccg attcaccatc tcccgtgacg gcaccaagaa cacgatgtct 240
ctgcaaatga acagcctcga acctgcggac actgccatgt actactgtgc ggcggcagcg 300
atcccatatg aacggtgccc cgatatctcc gatccgcaga tccttgtgag ttttccttac 360
tggggccagg ggacccaggt caccgtctcc tca 393
<210> 18
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Gly Val Asn Tyr Arg Arg Asn Cys Leu Ala
1 5 10
<210> 19
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Leu Tyr Thr Gly Asp Gly Ser Thr
1 5
<210> 20
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Ala Ala Gln Glu Tyr Gly Ser Arg Cys Tyr Thr Phe Thr Leu Ser Ala
1 5 10 15
Thr Asp Phe Gly Tyr
20
<210> 21
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
Trp Phe Arg Gln Ala Pro Gly Glu Glu Arg Glu Gly Val Ala Gln
1 5 10 15
<210> 22
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 22
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ala Lys Asn Thr Met Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 23
<211> 128
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
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 Val Asn Tyr Arg Arg Asn
20 25 30
Cys Leu Ala Trp Phe Arg Gln Ala Pro Gly Glu Glu Arg Glu Gly Val
35 40 45
Ala Gln Leu Tyr Thr Gly Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Met Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Gln Glu Tyr Gly Ser Arg Cys Tyr Thr Phe Thr Leu Ser Ala
100 105 110
Thr Asp Phe Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 24
<211> 384
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
caggtgcagc tgcaggagtc tggaggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtgcag cctctggagt caactacagg cgcaactgct tggcgtggtt ccgccaggct 120
ccaggggagg agcgcgaggg ggtcgcacaa ctttatactg gtgatggtag cacatactat 180
gccgactccg tgaagggccg attcaccatc tcccaagaca acgccaagaa cacgatgtat 240
ctgcaaatga acagcctgaa acctgaggac actgccatgt actactgtgc ggcccaggaa 300
tacggtagtc gctgctacac cttcaccctg tccgcgactg actttggtta ctggggccag 360
gggacccagg tcaccgtctc ctca 384
<210> 25
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 25
Gly Tyr Thr Tyr Ser Cys Met Gly
1 5
<210> 26
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 26
Ile Tyr Gly Gly Arg Thr
1 5
<210> 27
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 27
Ala Ala Met Trp Pro Tyr Asp Arg Cys Gly Met Asp Ala Arg Asp Tyr
1 5 10 15
Val Tyr
<210> 28
<211> 25
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 28
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 Glu Ala Ser
20 25
<210> 29
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 29
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Ile Ala Asn
1 5 10 15
<210> 30
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 30
Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Val Ser Gln Asp Asn
1 5 10 15
Ala Arg Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Gly Asp
20 25 30
Thr Ala Met Tyr Phe Cys
35
<210> 31
<211> 121
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 31
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 Glu Ala Ser Gly Tyr Thr Tyr Ser Cys Met
20 25 30
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Ile Ala Asn
35 40 45
Ile Tyr Gly Gly Arg Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe
50 55 60
Thr Val Ser Gln Asp Asn Ala Arg Asn Thr Val Tyr Leu Gln Met Asn
65 70 75 80
Ser Leu Lys Pro Gly Asp Thr Ala Met Tyr Phe Cys Ala Ala Met Trp
85 90 95
Pro Tyr Asp Arg Cys Gly Met Asp Ala Arg Asp Tyr Val Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 32
<211> 363
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
caggtgcagc tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtgaag cctctggata cacgtacagt tgcatgggct ggttccgcca ggctccaggg 120
aaggagcgcg aggcgatcgc aaatatatac ggtggtcgca caaactatgc cgactccgtg 180
aagggccgat tcacagtctc ccaagacaac gccaggaata cggtctatct gcaaatgaac 240
agcctgaaac ctggggacac agccatgtac ttctgtgcgg caatgtggcc atatgatcga 300
tgtggaatgg atgcgcgcga ctatgtctat tggggccagg ggacccaggt caccgtctcc 360
tca 363
<210> 33
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 33
Gly Tyr Thr Ile Ser Arg Arg Cys Met Gly
1 5 10
<210> 34
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 34
Ile Tyr Ser Ser Gly Gly Ser Thr
1 5
<210> 35
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 35
Ala Ala Arg Asp Tyr Gly Ser Ser Cys Tyr Gly Ile Pro Glu Ser Ala
1 5 10 15
Asp Phe Gly Tyr
20
<210> 36
<211> 25
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 36
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 Val Ala Ser
20 25
<210> 37
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 37
Trp Phe Arg Gln Ala Pro Gly Lys Ala Arg Glu Gly Val Ala Ala
1 5 10 15
<210> 38
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 38
Tyr Tyr Ser 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 Asn Ser Leu Arg Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 39
<211> 127
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 39
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 Val Ala Ser Gly Tyr Thr Ile Ser Arg Arg
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Ala Arg Glu Gly Val
35 40 45
Ala Ala Ile Tyr Ser Ser Gly Gly Ser Thr Tyr Tyr Ser 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 Asn Ser Leu Arg Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Arg Asp Tyr Gly Ser Ser Cys Tyr Gly Ile Pro Glu Ser Ala
100 105 110
Asp Phe Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 40
<211> 381
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
caggtgcagc tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtgtag cctctgggta caccattagt agacgctgca tgggctggtt ccgccaggct 120
ccagggaagg cgcgcgaggg ggtcgcagca atttattcta gtggtggtag cacatactat 180
tccgactccg tgaagggccg attcaccatc tcccaagaca acgccaagaa cacggtgtat 240
ctgcaaatga acagcctgag acctgaggac actgccatgt actactgtgc ggcacgtgac 300
tacgggagta gctgctacgg gatacccgag tcagctgact ttggttactg gggccagggg 360
acccaggtca ccgtctcctc a 381
<210> 41
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 41
Gly Ser Thr Tyr Ser Arg Arg Tyr Met Gly
1 5 10
<210> 42
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 42
Ile Ser Pro Tyr Gly Asp Ser Thr
1 5
<210> 43
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 43
Ala Ala Ser Trp Pro Tyr Gln Thr Ala Ser Leu Asp Ala Met Asp Phe
1 5 10 15
Thr Asp
<210> 44
<211> 25
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 44
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 Val Asp Ser
20 25
<210> 45
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 45
Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Gly Val Ser Val
1 5 10 15
<210> 46
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 46
Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 47
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 47
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 Val Asp Ser Gly Ser Thr Tyr Ser Arg Arg
20 25 30
Tyr Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Val Ile Ser Pro Tyr Gly Asp Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Ser Trp Pro Tyr Gln Thr Ala Ser Leu Asp Ala Met Asp Phe
100 105 110
Thr Asp Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 48
<211> 375
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
caggtgcagc tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtgtag actctggatc gacctacagt cgtcgctaca tgggctggtt ccgccgtgct 120
cctggcaagg agcgcgaggg ggtctcagtt atttctcctt atggtgatag cacatactat 180
gccgactccg tgaaggaccg attcaccatc tcccgagaca acgccaagaa cacggtgtat 240
ctacaaatga acagcctgaa acctgaggat actgccatgt actactgtgc ggcttcctgg 300
ccgtatcaaa cagcgtctct tgacgcaatg gattttactg actggggcca gggaacccag 360
gtcaccgtct cctca 375
<210> 49
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 49
Gly Tyr Thr Tyr Ser Ser Tyr Tyr Met Gly
1 5 10
<210> 50
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 50
Ile Glu Ser Asp Gly Ser Thr
1 5
<210> 51
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 51
Ala Ala Asp Glu Tyr Gly Gly Ser Trp Tyr Pro Arg Tyr Leu Gly Thr
1 5 10 15
Pro Glu Tyr Asn Tyr
20
<210> 52
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 52
Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala
1 5 10 15
<210> 53
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 53
Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys 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> 54
<211> 127
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 54
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 Thr Tyr Ser Ser Tyr
20 25 30
Tyr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Glu Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ala Asp Glu Tyr Gly Gly Ser Trp Tyr Pro Arg Tyr Leu Gly Thr Pro
100 105 110
Glu Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 55
<211> 381
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
caggtgcagc tgcaggagtc tggaggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtgcag cctctggata cacctacagt agctactaca tgggctggta ccgccaggct 120
ccaggaaagg agcgcgaggg ggtcgcagct attgagagtg atggtagcac aagctacgca 180
gactccgtga agggccgatt caccatctcc aaagacaacg ccaagaacac tctgtatctg 240
caaatgaaca gcctgaaacc tgaggacact gccatgtact actgtgcggc cgacgagtac 300
ggtggtagct ggtacccgcg atacctaggg acgccggagt ataactactg gggccagggg 360
acccaggtca ccgtctcctc a 381
<210> 56
<211> 25
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 56
Gln 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
20 25
<210> 57
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 57
Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val Gly Met
1 5 10 15
<210> 58
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 58
Tyr His Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
20 25 30
Thr Ala Val Tyr Phe Cys
35
<210> 59
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 59
Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser
1 5 10
<210> 60
<211> 127
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 60
Gln 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 Val Ser Leu Ser Ser Thr
20 25 30
Cys Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Gly Met Ile Ala Ser Asp Gly Ser Thr Tyr His Thr Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala
85 90 95
Ala Ala Tyr Pro Trp Thr Ala Cys Tyr Gly Gly Tyr Ala Met Ala Ala
100 105 110
Asp Phe Pro Tyr Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 61
<211> 381
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60
agctgcgccg ccagcggcgt gagcctgagc agcacctgca tgagctggtt caggcaggcc 120
cccggcaagg gcctggaggg cgtgggcatg atcgccagcg acggcagcac ctaccacacc 180
gacagcgtga agggcaggtt caccatcagc agggacaaca gcaagaacac cctgtacctg 240
cagatgaaca gcctgagggc cgaggacacc gccgtgtact tctgcgccgc cgcctacccc 300
tggaccgcct gctacggcgg ctacgccatg gccgccgact tcccctactg gggcaagggc 360
accctggtga ccgtgagcag c 381
<210> 62
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 62
Trp Phe Arg Gln Thr Leu Gly Lys Gly Leu Glu Gly Val Ala Ile
1 5 10 15
<210> 63
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 63
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn
1 5 10 15
Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 64
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 64
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
1 5 10
<210> 65
<211> 131
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 65
Gln 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 Asp Tyr Ser Thr Gly Arg Arg
20 25 30
Cys Met Ala Trp Phe Arg Gln Thr Leu Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ile Ile Asp Tyr Ala Gly Thr Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Ala Ala Ile Pro Tyr Glu Arg Cys Pro Asp Ile Ser Asp Pro
100 105 110
Gln Ile Leu Val Ser Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr
115 120 125
Val Ser Ser
130
<210> 66
<211> 393
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60
agctgcgccg ccagcgacta cagcaccggc aggaggtgca tggcctggtt caggcagacc 120
ctgggcaagg gcctggaggg cgtggccatc atcgactacg ccggcaccag cacctactac 180
gccgacagcg tgaagggcag gttcatcatc agcagggaca acagcaagaa caccctgtac 240
ctgcagatga acagcctgag gcccgaggac accgccatgt actactgcgc cgccgccgcc 300
atcccctacg agaggtgccc cgacatcagc gacccccaga tcctggtgag cttcccctac 360
tggggccagg gcaccctggt gaccgtgagc agc 393
<210> 67
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 67
Trp Phe Arg Gln Ala Pro Gly Glu Gly Leu Glu Gly Val Ala Gln
1 5 10 15
<210> 68
<211> 128
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 68
Gln 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 Val Asn Tyr Arg Arg Asn
20 25 30
Cys Leu Ala Trp Phe Arg Gln Ala Pro Gly Glu Gly Leu Glu Gly Val
35 40 45
Ala Gln Leu Tyr Thr Gly Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Gln Glu Tyr Gly Ser Arg Cys Tyr Thr Phe Thr Leu Ser Ala
100 105 110
Thr Asp Phe Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 69
<211> 384
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60
agctgcgccg ccagcggcgt gaactacagg aggaactgcc tggcctggtt caggcaggcc 120
cccggcgagg gcctggaggg cgtggcccag ctgtacaccg gcgacggcag cacctactac 180
gccgacagcg tgaagggcag gttcatcatc agcagggaca acagcaagaa caccctgtac 240
ctgcagatga acagcctgag gcccgaggac accgccatgt actactgcgc cgcccaggag 300
tacggcagca ggtgctacac cttcaccctg agcgccaccg acttcggcta ctggggccag 360
ggcaccctgg tgaccgtgag cagc 384
<210> 70
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 70
Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Ala Ile Ala Asn
1 5 10 15
<210> 71
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 71
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Val Ser Arg Asp Asn
1 5 10 15
Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 72
<211> 121
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 72
Gln 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 Thr Tyr Ser Cys Met
20 25 30
Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Ala Ile Ala Asn
35 40 45
Ile Tyr Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe
50 55 60
Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn
65 70 75 80
Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Met Trp
85 90 95
Pro Tyr Asp Arg Cys Gly Met Asp Ala Arg Asp Tyr Val Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 73
<211> 363
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60
agctgcgccg ccagcggcta cacctacagc tgcatgggct ggttcaggca ggcccccggc 120
aagggcctgg aggccatcgc caacatctac ggcggcagga cctactacgc cgacagcgtg 180
aagggcaggt tcaccgtgag cagggacaac agcaagaaca ccctgtacct gcagatgaac 240
agcctgaggc ccgaggacac cgccgtgtac tactgcgccg ccatgtggcc ctacgacagg 300
tgcggcatgg acgccaggga ctacgtgtac tggggccagg gcaccctggt gaccgtgagc 360
agc 363
<210> 74
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 74
Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val Ala Ala
1 5 10 15
<210> 75
<211> 127
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 75
Gln 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 Thr Ile Ser Arg Arg
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ala Ile Tyr Ser Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Arg Asp Tyr Gly Ser Ser Cys Tyr Gly Ile Pro Glu Ser Ala
100 105 110
Asp Phe Gly Tyr Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 76
<211> 381
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60
agctgcgccg ccagcggcta caccatcagc aggaggtgca tgggctggtt caggcaggcc 120
cccggcaagg gcctggaggg cgtggccgcc atctacagca gcggcggcag cacctactac 180
gccgacagcg tgaagggcag gttcatcatc agcagggaca acagcaagaa caccctgtac 240
ctgcagatga acagcctgag gcccgaggac accgccatgt actactgcgc cgccagggac 300
tacggcagca gctgctacgg catccccgag agcgccgact tcggctactg gggcaagggc 360
accctggtga ccgtgagcag c 381
<210> 77
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 77
Trp Phe Arg Arg Ala Pro Gly Lys Gly Leu Glu Gly Val Ser Val
1 5 10 15
<210> 78
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 78
Gln 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 Ser Thr Tyr Ser Arg Arg
20 25 30
Tyr Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ser Val Ile Ser Pro Tyr Gly Asp Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Ser Trp Pro Tyr Gln Thr Ala Ser Leu Asp Ala Met Asp Phe
100 105 110
Thr Asp Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 79
<211> 375
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60
agctgcgccg ccagcggcag cacctacagc aggaggtaca tgggctggtt caggagggcc 120
cccggcaagg gcctggaggg cgtgagcgtg atcagcccct acggcgacag cacctactac 180
gccgacagcg tgaagggcag gttcatcatc agcagggaca acagcaagaa caccctgtac 240
ctgcagatga acagcctgag gcccgaggac accgccatgt actactgcgc cgccagctgg 300
ccctaccaga ccgccagcct ggacgccatg gacttcaccg actggggcca gggcaccctg 360
gtgaccgtga gcagc 375
<210> 80
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 80
Trp Tyr Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val Ala Ala
1 5 10 15
<210> 81
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 81
Ser 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 Arg Ala Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 82
<211> 127
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 82
Gln 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 Thr Tyr Ser Ser Tyr
20 25 30
Tyr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ala Ile Glu Ser Asp Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Asp Glu Tyr Gly Gly Ser Trp Tyr Pro Arg Tyr Leu Gly Thr Pro
100 105 110
Glu Tyr Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 83
<211> 381
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60
agctgcgccg ccagcggcta cacctacagc agctactaca tgggctggta caggcaggcc 120
cccggcaagg gcctggaggg cgtggccgcc atcgagagcg acggcagcac cagctacgcc 180
gacagcgtga agggcaggtt caccatcagc agggacaacg ccaagaacac cctgtacctg 240
cagatgaaca gcctgagggc cgaggacacc gccgtgtact actgcgccgc cgacgagtac 300
ggcggcagct ggtaccccag gtacctgggc acccccgagt acaactactg gggccagggc 360
accctggtga ccgtgagcag c 381

Claims (10)

1. An anti-Trop 2 nanobody, which is capable of specifically binding to Trop2, and the Complementarity Determining Regions (CDRs) of the VHH chain in the nanobody are shown as follows:
CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 11, and CDR3 shown in SEQ ID NO. 12.
2. The anti-Trop 2 nanobody of claim 1, wherein the VHH chain of the anti-Trop 2 nanobody further comprises framework regions FR as shown below:
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. 7; or
FR1 shown by SEQ ID NO. 56, FR2 shown by SEQ ID NO. 62, FR3 shown by SEQ ID NO. 63, and FR4 shown by SEQ ID NO. 64.
3. The anti-Trop 2 nanobody of claim 1, wherein the amino acid sequence of the VHH chain of the anti-Trop 2 nanobody is as set forth in SEQ ID NO 16 or SEQ ID NO 65.
4. An anti-Trop 2 antibody comprising one or more anti-Trop 2 nanobodies of claim 1.
5. The antibody of claim 4, wherein the antibody comprises a monomeric, bivalent, and/or multivalent antibody.
6. The antibody of claim 4, wherein the antibody comprises one or more VHH chains having the amino acid sequence set forth in SEQ ID NO 16 or SEQ ID NO 65.
7. A polynucleotide encoding a protein selected from the group consisting of: the anti-Trop 2 nanobody of claim 1 or the antibody of claim 4.
8. The polynucleotide of claim 7, wherein the nucleotide sequence of said polynucleotide comprises the nucleotide sequence set forth in SEQ ID No. 17 or SEQ ID No. 66.
9. An expression vector comprising the polynucleotide of claim 8.
10. A host cell comprising the expression vector of claim 9, or having the polynucleotide of claim 7 integrated into its genome.
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WO2023049999A1 (en) * 2021-09-29 2023-04-06 Kisoji Biotechnology Inc. Binding agents targeting trop2-expressing tumor cells
CN115232212B (en) * 2022-05-27 2024-02-23 南开大学 TROP-2 specific nano antibody and application thereof
CN114805582B (en) * 2022-06-29 2022-10-04 上海恒润达生生物科技股份有限公司 anti-Trop 2 nano antibody and application thereof
CN115873104B (en) * 2022-08-09 2023-07-14 华南农业大学 Nanometer antibody M124 for H7 subtype avian influenza virus and application thereof
WO2024051383A1 (en) * 2023-07-28 2024-03-14 上海洛启生物医药技术有限公司 Anti-trop2 antibody, conjugate comprising said antibody, and use thereof

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