Anti-trop2 nanobody and its preparation method and application

Abstract

The invention belongs to the field of biotechnology, and specifically relates to an anti-TROP2 nanobody composed of a variable region of a heavy chain antibody, a preparation method and application thereof, and meanwhile, a nucleic acid molecule encoding the antibody, an expression vector, a host cell, and the like are provided. The anti-TROP2 nanobody (TROP2-VHH-01-9F nanobody and TROP2-VHH-01-5A nanobody) prepared by the present invention has high affinity binding to human TROP2 antigen, and has strong antibody endocytic activity and novel sequence , is a new anti-TROP2 nanobody.

Description

technical field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an anti-TROP2 nanobody composed of a variable region of a heavy chain antibody and a preparation method and application thereof. Background technique

[0002] TROP2 is a human trophoblast cell surface glycoprotein antigen, which belongs to the TACSTD family and is a cell surface glycoprotein encoded and expressed by the TACSTD2 gene, also known as tumor-associated calcium ion signal transducer 2 (TACSTD2), epidermal glycoprotein 1 (EGP-1 ), gastrointestinal tumor-associated antigen (GA733-1), surface marker 1 (M1S1) (Li X, et al."TROP2 promotes proliferation, migration and metastasis of gallbladder cancer cells by regulating PI3K / AKT pathway and inducing EMT". Oncotarget. 2017, 8(29)). TROP2 mRNA can be detected in normal tissues, including normal keratinocytes, kidney, lung, ovary and testis. The expression level of TROP2 in normal tissues is very low, mainly expressed...

Examples

Embodiment 1

[0038] Example 1 Construction of camelid phage VHH display library

[0039] 1.1 Camel Immune

[0040] 取1mg人TROP2-Fc(公司自产,氨基酸序列为HTAAQDNCTCPTNKMTVCSPDGPGGRCQCRALGSGMAVDCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDNDGLYDPDCDPEGRFKARQCNQTSVCWCVNSVGVRRTDKGDLSLRCDELVRTHHILIDLRHRPTAGAFNHSDLDAELRRLFRERYRLHPKFVAAVHYEQPTIQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEPLQVERTLIYYLDEIPPKFSMKRLTEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK)蛋白与弗氏完全佐剂1:1等体积混匀并充分乳化,采用皮下多点注射的方式对一只健康成年骆驼进行第一 The first immunization; followed by 4 booster immunizations with 0.5 mg TROP2 protein 1:1 equal volume mixed with incomplete Freund's adjuvant, a total of 5 immunizations, with a single interval of 20 days. Seven days after each immunization, a small amount of camel peripheral blood was drawn for titer detection. After the last detec...

Embodiment 2

[0063] Example 2: Screening of positive clones expressing Nanobody TROP2-VHH

[0064] 2.1 Affinity panning

[0065] 制备固相蛋白ELISA板:用1x carbonate / bicarbonate buffer配制人TROP2-His抗原(公司自产,氨基酸序列为MDMRVPAQLLGLLLLWFPGSRCHTAAQDNCTCPTNKMTVCSPDGPGGRCQCRALGSGMAVDCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDNDGLYDPDCDPEGRFKARQCNQTSVCWCVNSVGVRRTDKGDLSLRCDELVRTHHILIDLRHRPTAGAFNHSDLDAELRRLFRERYRLHPKFVAAVHYEQPTIQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEPLQVERTLIYYLDEIPPKFSMKRLTHHHHHHHHHH),使其终浓度为1μg / ml,以100μl / 孔加液于96孔酶标 Plate (temporarily divided into three areas A, B, and C, each area has 4 wells), overnight at 4°C. After coating, the plate was washed once with PBS solution containing 0.05% Tween 20 (ie 1xPBST), then 200 μl / well of PBST solution containing 2% BSA was added, and placed at 37°C for 2 hours to block. Phage blocking: Add 20 μl of purified Phage and 16 μl of human immunoglobulin IgG (pH4) for intravenous injection (purchased from Taibang, Guizhou) to 364 μl of PBST buffer containing 5% skim...

Embodiment 3

[0076] Example 3: In Vitro Assay for Assaying Anti-TROP2 Nanobody Functional Activity

[0077] 3.1 Reference antibody blocking ELISA

[0078] The clone TROP2-VHH-01-9F and the clone TROP2-VHH-01-5A were expanded and cultivated at 37° C. at 200 rpm until the OD600 value was 0.6-0.8. Add final concentration of 1mM IPTG, induce overnight at 30°C, 150rpm. The cultured cells were ultrasonically disrupted, and then purified using BeaverBeads IDA-Nickel (Beaver Bio, Cat#70501-100) to obtain anti-TROP2 nanobody.

[0079] The blocking ability of anti-TROP2 Nanobodies to block reference antibody / TROP2 antigen binding was evaluated by competition ELISA method. The brief description is as follows, the reference antibody (Benchmark1) was prepared with 1x carbonate / bicarbonate buffer to a final concentration of 1 μg / ml, added to a 96-well ELISA plate at 100 μl / well, and coated overnight at 4°C. The next day, after washing the plate once with PBST, add 200 μl / well of PBST solution contain...

technical field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an anti-TROP2 nanobody composed of a variable region of a heavy chain antibody and a preparation method and application thereof. Background technique

[0002] TROP2 is a human trophoblast cell surface glycoprotein antigen, which belongs to the TACSTD family and is a cell surface glycoprotein encoded and expressed by the TACSTD2 gene, also known as tumor-associated calcium ion signal transducer 2 (TACSTD2), epidermal glycoprotein 1 (EGP-1 ), gastrointestinal tumor-associated antigen (GA733-1), surface marker 1 (M1S1) (Li X, et al."TROP2 promotes proliferation, migration and metastasis of gallbladder cancer cells by regulating PI3K / AKT pathway and inducing EMT". Oncotarget. 2017, 8(29)). TROP2 mRNA can be detected in normal tissues, including normal keratinocytes, kidney, lung, ovary and testis. The expression level of TROP2 in normal tissues is very low, mainly expressed...

Examples

Embodiment 1

[0038] Example 1 Construction of camelid phage VHH display library

[0039] 1.1 Camel Immune

[0040] 取1mg人TROP2-Fc(公司自产,氨基酸序列为HTAAQDNCTCPTNKMTVCSPDGPGGRCQCRALGSGMAVDCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDNDGLYDPDCDPEGRFKARQCNQTSVCWCVNSVGVRRTDKGDLSLRCDELVRTHHILIDLRHRPTAGAFNHSDLDAELRRLFRERYRLHPKFVAAVHYEQPTIQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEPLQVERTLIYYLDEIPPKFSMKRLTEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK)蛋白与弗氏完全佐剂1:1等体积混匀并充分乳化,采用皮下多点注射的方式对一只健康成年骆驼进行第一 The first immunization; followed by 4 booster immunizations with 0.5 mg TROP2 protein 1:1 equal volume mixed with incomplete Freund's adjuvant, a total of 5 immunizations, with a single interval of 20 days. Seven days after each immunization, a small amount of camel peripheral blood was drawn for titer detection. After the last detec...

Embodiment 2

[0063] Example 2: Screening of positive clones expressing Nanobody TROP2-VHH

[0064] 2.1 Affinity panning

[0065] 制备固相蛋白ELISA板:用1x carbonate / bicarbonate buffer配制人TROP2-His抗原(公司自产,氨基酸序列为MDMRVPAQLLGLLLLWFPGSRCHTAAQDNCTCPTNKMTVCSPDGPGGRCQCRALGSGMAVDCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDNDGLYDPDCDPEGRFKARQCNQTSVCWCVNSVGVRRTDKGDLSLRCDELVRTHHILIDLRHRPTAGAFNHSDLDAELRRLFRERYRLHPKFVAAVHYEQPTIQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEPLQVERTLIYYLDEIPPKFSMKRLTHHHHHHHHHH),使其终浓度为1μg / ml,以100μl / 孔加液于96孔酶标 Plate (temporarily divided into three areas A, B, and C, each area has 4 wells), overnight at 4°C. After coating, the plate was washed once with PBS solution containing 0.05% Tween 20 (ie 1xPBST), then 200 μl / well of PBST solution containing 2% BSA was added, and placed at 37°C for 2 hours to block. Phage blocking: Add 20 μl of purified Phage and 16 μl of human immunoglobulin IgG (pH4) for intravenous injection (purchased from Taibang, Guizhou) to 364 μl of PBST buffer containing 5% skim...

Embodiment 3

[0076] Example 3: In Vitro Assay for Assaying Anti-TROP2 Nanobody Functional Activity

[0077] 3.1 Reference antibody blocking ELISA

[0078] The clone TROP2-VHH-01-9F and the clone TROP2-VHH-01-5A were expanded and cultivated at 37° C. at 200 rpm until the OD600 value was 0.6-0.8. Add final concentration of 1mM IPTG, induce overnight at 30°C, 150rpm. The cultured cells were ultrasonically disrupted, and then purified using BeaverBeads IDA-Nickel (Beaver Bio, Cat#70501-100) to obtain anti-TROP2 nanobody.

[0079] The blocking ability of anti-TROP2 Nanobodies to block reference antibody / TROP2 antigen binding was evaluated by competition ELISA method. The brief description is as follows, the reference antibody (Benchmark1) was prepared with 1x carbonate / bicarbonate buffer to a final concentration of 1 μg / ml, added to a 96-well ELISA plate at 100 μl / well, and coated overnight at 4°C. The next day, after washing the plate once with PBST, add 200 μl / well of PBST solution contain...

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