Humanized anti-TPBG antibody, preparation method thereof, conjugate and application thereof

A humanized and human antibody technology, applied in biochemical equipment and methods, chemical equipment and methods, botany equipment and methods, etc., can solve problems such as the limitation of killing effect and the damage of non-cancer cells

Active Publication Date: 2018-10-23
KAIHUI SCI & TECH DEV SHANGHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In clinical practice, although therapeutic antibodies have good targeting, their killing effects are limited
Although small molecule chemical drugs have a highly effective killing effect on cancer cells, they can also cause the same damage to non-cancer cells

Method used

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  • Humanized anti-TPBG antibody, preparation method thereof, conjugate and application thereof
  • Humanized anti-TPBG antibody, preparation method thereof, conjugate and application thereof
  • Humanized anti-TPBG antibody, preparation method thereof, conjugate and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0226] Example 1 Preparation of Chimeric Antibody 12B12 and 28D4 and Humanized TPBG Antibody

[0227] 1. Preparation of murine antibodies 12B12 and 28D4

[0228] (1) Preparation of immunogen A (human TPBG-hFc protein)

[0229] 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 ATCC) were transiently transfected (polyetherimide PEI, purchased from Polysciences) and expan...

Embodiment 2

[0346] Example 2 Enzyme-linked immunosorbent assay (ELISA) detects the binding of humanized TPBG antibody to TPBG protein

[0347] The purified humanized TPBG antibody obtained in Example 1 was reacted with the human TPBG-hFc protein. For the method, see (5) the assay of the lead antibody in Example 1. The results are shown in Figures 7-8 and Tables 11-12 Table 11 and Table 12 are the OD of h12B12 variant and h28D4 variant respectively 450nm The EC50 value calculated by the value shows that the purified humanized TPBG antibody variant has a good combination with the TPBG recombinant protein at the ELISA level. in, Figure 7A and 7B For the binding reaction of purified humanized h12B12 variants to human TPBG-hFc protein, Figure 8A and 8B Binding reaction of purified humanized h28D4 variants to human TPBG-hFc protein.

[0348] Table 11. ELISA detection of binding reaction of humanized h12B12 antibody variants to human TPBG-hFc protein

[0349]

[0350]

[0351] Tabl...

Embodiment 3

[0353] Characterization analysis (Biacore) of embodiment 3 humanized TPBG antibodies

[0354] To evaluate the binding specificity and affinity of humanized anti-TPBG antibodies, Biacore analysis was performed using human TPBG antigen immobilized on a CM5 chip. Biacore technology utilizes the change in the refractive index on the surface after the antibody binds to the TPBG antigen immobilized on the surface. Binding is detected by surface plasmon resonance (SPR) of laser light refracted from the surface. Analysis of signaling kinetics on-rate and off-rate allows the distinction between non-specific and specific interactions. Chimeric antibodies 12B12 and 28D4 were used as controls.

[0355] Table 13. Biacore test results

[0356] Antibody

[0357] Biacore results showed that the variants of humanized anti-TPBG antibodies 12B12 and 28D4 had very similar KD values ​​to chimeric antibodies 12B12 and 28D4, which verified that humanized anti-TPBG antibodies did not sig...

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Abstract

The invention discloses a humanized anti-TPBG antibody, a preparation method thereof, a conjugate thereof and application thereof. The humanized anti-TPBG antibody comprises (a) a framework region comprising residues of a human antibody framework region; and (b) one or more CDRs in a light chain variable region shown as SEQ ID NO.4 or 8 or one or more CDRs in a heavy chain variable region shown asSEQ ID NO.2 or 6. The humanized anti-TPBG antibody has a binding affinity of at least about 1 * 10<-7> M to about 1 * 10<-12>M for a human TPBG antigen. The humanized anti-TPBG antibody is conjugatedwith a small molecule compound such as MC-MMAF to obtain the conjugate which is effective in cytotoxic killing effect on TPBG positive cells. Therefore, the humanized anti-TPBG antibody is used for preparing an antibody cross-linking drug, which can effectively kill tumor cells and treat tumors.

Description

technical field [0001] The present invention relates to the field of antibodies, in particular to a humanized anti-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 cell...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/30C12N15/13C12N15/63G01N33/569A61K47/68A61K45/06A61P35/00
CPCA61K45/06A61K47/6851A61P35/00G01N33/56966C12N15/63C07K16/30C07K2317/56C07K2317/626C07K2317/24A61K39/395A61K47/68C07K16/00C12N15/00G01N33/569A61K2039/505C07K16/2818C07K2317/92C07K2317/33A61K39/3955A61K2300/00
Inventor 张莹宫世勇孙晓岚徐腾蛟彭菲陈雨竹刘礼乐
Owner KAIHUI SCI & TECH DEV SHANGHAI
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