A kind of TPBG antibody and its preparation method, its conjugate and application

Abstract

The invention discloses a TPBG antibody, its preparation method, its conjugate and application. The TPBG antibody includes one or more of the heavy chain variable region heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 of the TPBG antibody, and / or, the light chain variable region light chain CDR1, light chain CDR1 of the TPBG antibody One or more of CDR2 and light chain CDR3, the amino acid sequences of which are respectively described in the present invention. The TPBG antibody is a humanized antibody with high affinity, and the conjugate prepared after coupling with the small-molecule drug toxin MMAF can have a cytotoxic killing effect on TPBG-positive cells, so it is used in the preparation of drugs for treating tumors and the like middle.

Description

technical field

[0001] The present invention relates to the field of antibodies, in particular to a TPBG antibody, its preparation method, its conjugate and its application. Background technique

[0002] A cell surface molecule, trophoblast-specific glycoprotein (TPBG, also known as 5T4), was found to be a specific protein expressed by embryonic trophoblasts when comparing embryonic stem cell trophoblasts with cancer cells. The human TPBG protein has a molecular weight of about 72kDa and contains 420 amino acids. Its N-terminal oligosaccharide structure has diversity, which can prevent proteolysis and interact with other molecules in the process of cell membrane signal transduction. The TPBG protein contains seven repeating leucine domains (LRRs), which can participate in protein-protein interactions.

[0003] The trophoblast is a layer of special embryonic stem cells between the placenta and the fetus, and TPBG is widely expressed in various trophoblast cells during embryo...

Examples

Embodiment 1

[0198] Example 1 Preparation of TPBG Antibody

[0199] (1) Preparation of Immunogen A

[0200] The nucleotide sequence containing the amino acid sequence 32-355 (Ser32-Ser355) of the extracellular region encoding the human source TPBG protein (wherein the nucleotide sequence encoding the human source TPBG protein is Genebank ID: AAH37161.1 in Genebank) Cloning into pCpC vector (purchased from Invitrogen, V044-50) carrying human IgG Fc fragment (hFc) and plasmid preparation according to established standard molecular biology methods. For specific methods, see Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press). HEK293 cells (purchased from Invitrogen) were transiently transfected (polyetherimide PEI, purchased from Polysciences) and expanded at 37° C. using FreeStyle™ 293 (purchased from Invitrogen). After 4 days, the cell culture fluid was collected, and the ce...

Embodiment 2

[0217] Example 2 Production and Purification of Lead Antibody

[0218] The concentration of antibodies produced by hybridoma cells is low, only about 1-10 μg / mL, and the concentration varies greatly. Moreover, various proteins produced by cell culture in the culture medium and fetal bovine serum components contained in the culture medium have varying degrees of interference with many biological activity analysis methods, so small-scale (1-5 mg) antibody production and purification are required.

[0219] The hybridoma cells obtained in Example 1 were inoculated into T-75 cell culture flasks and acclimatized and passaged for 3 generations with a production medium (Hybridomaserum free medium, purchased from Invitrogen). When the growth state is good, inoculate the cell culture spinner bottle. Add 200mL of production medium to each 2-liter culture spinner bottle, and inoculate the cell density at 1.0╳10 5 / mL. Close the bottle cap tightly, and place the spinner bottle on a spin...

Embodiment 3

[0225] The assay of embodiment 3 leading antibody

[0226] A. Enzyme-linked immunosorbent assay (ELISA) to detect the binding of TPBG antibody to TPBG protein

[0227] The purified TPBG antibody obtained in Example 2 was reacted with human TPBG-hFc protein (immunogen A).

[0228] Dilute the purified immunogen A obtained in Example 1 (see Example 1 step (1) for its preparation method) with PBS to a final concentration of 1.0 μg / mL, and then add 100 μL per well to a 96-well ELISA plate. Seal with plastic film Incubate overnight at 4°C, wash the plate twice the next day with plate washing solution [PBS containing 0.01% (v / v) Tween20], add blocking solution [containing 0.01% (v / v) Tween20 and 1% (w / w) PBS of BSA] blocked at room temperature for 2 hours. Pour off the blocking solution, add the purified TPBG antibody 100 μ L per well of the gained Example 2. After incubation for 2 hours at 37° C., wash the plate with the plate washing solution [containing 0.01% (v / v) Tween20 in P...

technical field

[0001] The present invention relates to the field of antibodies, in particular to a TPBG antibody, its preparation method, its conjugate and its application. Background technique

[0002] A cell surface molecule, trophoblast-specific glycoprotein (TPBG, also known as 5T4), was found to be a specific protein expressed by embryonic trophoblasts when comparing embryonic stem cell trophoblasts with cancer cells. The human TPBG protein has a molecular weight of about 72kDa and contains 420 amino acids. Its N-terminal oligosaccharide structure has diversity, which can prevent proteolysis and interact with other molecules in the process of cell membrane signal transduction. The TPBG protein contains seven repeating leucine domains (LRRs), which can participate in protein-protein interactions.

[0003] The trophoblast is a layer of special embryonic stem cells between the placenta and the fetus, and TPBG is widely expressed in various trophoblast cells during embryo...

Examples

Embodiment 1

[0198] Example 1 Preparation of TPBG Antibody

[0199] (1) Preparation of Immunogen A

[0200] The nucleotide sequence containing the amino acid sequence 32-355 (Ser32-Ser355) of the extracellular region encoding the human source TPBG protein (wherein the nucleotide sequence encoding the human source TPBG protein is Genebank ID: AAH37161.1 in Genebank) Cloning into pCpC vector (purchased from Invitrogen, V044-50) carrying human IgG Fc fragment (hFc) and plasmid preparation according to established standard molecular biology methods. For specific methods, see Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press). HEK293 cells (purchased from Invitrogen) were transiently transfected (polyetherimide PEI, purchased from Polysciences) and expanded at 37° C. using FreeStyle™ 293 (purchased from Invitrogen). After 4 days, the cell culture fluid was collected, and the ce...

Embodiment 2

[0217] Example 2 Production and Purification of Lead Antibody

[0218] The concentration of antibodies produced by hybridoma cells is low, only about 1-10 μg / mL, and the concentration varies greatly. Moreover, various proteins produced by cell culture in the culture medium and fetal bovine serum components contained in the culture medium have varying degrees of interference with many biological activity analysis methods, so small-scale (1-5 mg) antibody production and purification are required.

[0219] The hybridoma cells obtained in Example 1 were inoculated into T-75 cell culture flasks and acclimatized and passaged for 3 generations with a production medium (Hybridomaserum free medium, purchased from Invitrogen). When the growth state is good, inoculate the cell culture spinner bottle. Add 200mL of production medium to each 2-liter culture spinner bottle, and inoculate the cell density at 1.0╳10 5 / mL. Close the bottle cap tightly, and place the spinner bottle on a spin...

Embodiment 3

[0225] The assay of embodiment 3 leading antibody

[0226] A. Enzyme-linked immunosorbent assay (ELISA) to detect the binding of TPBG antibody to TPBG protein

[0227] The purified TPBG antibody obtained in Example 2 was reacted with human TPBG-hFc protein (immunogen A).

[0228] Dilute the purified immunogen A obtained in Example 1 (see Example 1 step (1) for its preparation method) with PBS to a final concentration of 1.0 μg / mL, and then add 100 μL per well to a 96-well ELISA plate. Seal with plastic film Incubate overnight at 4°C, wash the plate twice the next day with plate washing solution [PBS containing 0.01% (v / v) Tween20], add blocking solution [containing 0.01% (v / v) Tween20 and 1% (w / w) PBS of BSA] blocked at room temperature for 2 hours. Pour off the blocking solution, add the purified TPBG antibody 100 μ L per well of the gained Example 2. After incubation for 2 hours at 37° C., wash the plate with the plate washing solution [containing 0.01% (v / v) Tween20 in P...

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