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Bispecific antibody with double Her2 sites for tumor immunotherapy

A bispecific antibody and site technology, applied in the field of host cells, can solve problems such as poor internalization and drug resistance, and achieve the effects of improving binding capacity, enhancing permeability, and improving spatial flexibility

Active Publication Date: 2020-10-30
SOUND BIOPHARMACEUTICALS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Poor internalization will also lead to drug resistance in T-DM1 therapy in metastatic breast cancer

Method used

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  • Bispecific antibody with double Her2 sites for tumor immunotherapy
  • Bispecific antibody with double Her2 sites for tumor immunotherapy
  • Bispecific antibody with double Her2 sites for tumor immunotherapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1. Design and purification of bispecific antibody Bp-Bs and its control Bi-Bs

[0059] The structures of the bivalent anti-Her2 bispecific antibody (Bi-Bs) and the bispecific antibody Bp-Bs binding to Her2 double sites, respectively figure 1 A and 1B are shown. DNA shuffling and ligation techniques were used to clone the respective genes. Wherein, Bi-Bs: single chain domain anti-Her2 VHH1 (SEQ ID NO.1, GenBank: JX047590.1; Even-Desrumeaux, K., P. Fourquet, V. Secq, D. Baty and P. Chames ( 2012). "Single-domain antibodies: a versatile and rich source of binders for breast cancer diagnostic approaches." MolBiosyst 8(9): 2385-2394.) VH-CH1 ligated to anti-CD3 UCHT1 clone (with linker: (GGGGS) 3 )) and the C-terminus of VL-CL; and for Bp-Bs: replace the anti-Her2 VHH1 at VH-CH1 of Bi-Bs with another anti-Her2 VHH2 (SEQ ID NO.2; Wu, X ., S. Chen, L. Lin, J. Liu, Y. Wang, Y. Li, et al.(2018). "A Single Domain-Based Anti-Her2 Antibody Has Potent Antitumor Activities...

Embodiment 2

[0060] Example 2. Binding properties of the Bp-Bs antibody

[0061] experimental method:

[0062] Cell lines: CHO, MCF7, LS174T, SKOV3, SKBR3 cells are all from the cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences; the medium used for cell culture, fetal bovine serum, trypsin, penicillin-streptomycin antibiotic mixture and other additives are purchased from from Gibco; consumables used for cell culture were purchased from Corning Costar. All cell lines were incubated at 37°C, 5% CO 2 DMEM (for MCF7, SKBR-3 and SKOV3) or RPMI-1640 (Thermo, China) containing 10% HI fetal bovine serum (Thermo, USA) and 1% penicillin / streptomycin (Hyclone) (for MCF7, SKBR-3 and SKOV3) (with cultured in LS174T and CHO).

[0063]Affinity Determination: The affinity of the anti-Her2 antibody to the extracellular region of Her2 protein was determined using an OctetQKe instrument (Pall Life Sciences). Briefly, Fc-tagged human Her2 (AcroBiosystem, Cat. No. HE2-H5...

Embodiment 3

[0070] Example 3. Bp-Bs antibody induces T cell-mediated cytotoxicity

[0071] experimental method:

[0072] To measure bispecific antibody cytotoxicity in vitro, human peripheral blood mononuclear cells (PBMCs) were freshly prepared from fresh donated blood using Ficoll-Plaque Plus (GE health) gradient centrifugation. Human peripheral blood was collected from healthy volunteers with written permission. Then use EasySep TM Human CD3 Positive Selection Kit (StemcellTechnologies, Inc., Vancouver, BC, Canada) Human CD3 was isolated from PBMC according to the manufacturer's instructions + T cells. Cytotoxicity assays were performed as previously described (Li, L., P. He, C. Zhou, L. Jing, B. Dong, S. Chen, et al. (2015). "A novel bispecific antibody, S- Fab, induces potent cancer cell killing." J Immunother 38(9): 350-356.). Briefly, SKOV3, MCF7, LS174T or CHO cancer cells were trypsinized and seeded at a density of 5000 cells / well in 96-well tissue culture plates as target ...

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Abstract

The invention discloses a bispecific antibody with double Her2 sites, comprising: (a) an anti-CD3 antigen binding fragment Fab having a light chain variable region VL and a light chain constant regionCL, and a heavy chain variable region VH and a heavy chain constant region CH1; (b) an anti-Her2 single domain antigen-binding fragment VHH1, which is linked to the C-terminus of the CL of the Fab and is capable of binding to a first Her2 epitope; and (c) an anti-Her2 single domain antigen-binding fragment VHH2, which is linked to the C-terminus of CH1 of the Fab and capable of binding to a second Her2 epitope; wherein the first Her2 epitope and the second Her2 epitope are non-overlapping epitopes of Her2. The bispecific antibody with the double Her2 sites also has an effect on Her2 tumors with an IHC score of + 1, or can act on trastuzumab resistant tumors.

Description

technical field [0001] The invention relates to a bispecific antibody for tumor immunotherapy, especially a bispecific antibody with double Her2 sites. The present invention also relates to a pharmaceutical composition comprising the bispecific antibody, a polynucleotide encoding the antibody fragment, an expression vector comprising the polynucleotide, and a host cell comprising the expression vector. Background technique [0002] Human epidermal growth factor receptor 2 (Her2, also known as Her2 / neu or ErbB2) is a member of the Her family of transmembrane receptor tyrosine kinases. Her2 comprises a cytoplasmic tyrosine kinase domain, a single transmembrane domain, and an extracellular domain of approximately 630 amino acids that contains four distinct domains (domains I-IV). The Her2 proto-oncogene is overexpressed and functionally important in 25%–30% of human primary breast tumors and various other human cancers (eg, lung, gastric, oral, and colorectal cancers). [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46A61K39/00A61P35/00
CPCC07K16/32C07K16/2809A61P35/00C07K2317/31C07K2317/55C07K2317/569C07K2317/92C07K2317/73C07K2317/94A61K2039/505A61K39/00C07K2317/52C07K2319/00C07K2317/64C07K2317/22C07K2317/522C07K2317/56
Inventor 李庆马梵辛
Owner SOUND BIOPHARMACEUTICALS CO LTD
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