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Novel bispecific pd-1/lag-3 antibody molecules

A bispecific antibody, LAG-3 technology, applied in the direction of antibodies, specific peptides, anti-tumor drugs, etc., can solve problems such as defects in down-regulation

Pending Publication Date: 2021-01-15
SHANGHAI WUXI BIOLOGIC TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Blockade of LAG-3 in vitro increases T cell proliferation and cytokine production, and LAG-3-deficient mice are defective in the downregulation of T cell responses induced by superantigen staphylococcal enterotoxin B, peptide, or Sendai virus infection

Method used

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  • Novel bispecific pd-1/lag-3 antibody molecules
  • Novel bispecific pd-1/lag-3 antibody molecules
  • Novel bispecific pd-1/lag-3 antibody molecules

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0324] Example 1: Generation and Characterization of W3055-1.153.7 Monoclonal Antibody

[0325] Fully human W3055-1.153.7 with a heavy chain variable region of SEQ ID NO: 17, a kappa light chain variable region of SEQ ID NO: 18, and human IgG4 was obtained as described in PCT Application No.: PCT / CN2016 / 094624 constant region. As disclosed in PCT Application No. PCT / CN2016 / 094624, the affinity of W3055-1.153.7 for recombinant human PD-1 by SPR is 2.79 nM. W3055-1.153.7 bound cynomolgus monkey PD-1 but not murine PD-1 as determined by FACS. W3055-1.153.7 specifically binds PD-1, but not CD28 and CTLA4 of the PD-1 family. The results of SPR assay and FACS of binning test showed that the epitope on human PD-1 bound by W3055-1.153.7 was different from that of existing PD-1 antibody (i.e. the benchmark antibody nivolumab (5C4 clone of BMS patent US9084776B2) and pembrolizumab (disclosed as clone hPD-1.09A in US8354509B2 and WO2008156712A1).[ 3H] Thymidine incorporation assay sh...

Embodiment 3

[0328] Example 3: Generation and Characterization of Human W3395-3.40.19LAG-3Ab Monoclonal Antibody

[0329] Monoclonal human LAG-3 antibody W3395-3.40.19 was generated as described in PCT / CN2019 / 076356. Typically, OMT rats (transgenic rats with recombinant immunoglobulin loci, as described and produced in US8,907,157B2) are immunized with the human LAG-3 antigen to obtain an immunization in which both the framework and CDR regions are derived from the human germline Antibodies against globulin sequences. Hybridomas generated by fusion of immunized rat lymph nodes and spleens with myeloma cells were isolated, selected and subcloned. Total RNA of hybridomas was extracted, and cDNA was synthesized and amplified. The VH and VL genes were reamplified and cloned into expression vectors to create corresponding clones of the antibodies.

[0330] By FACS, the binding affinity of W3395-3.40.19 to cell surface human LAG-3 had an EC50 value of 0.13 nM, which was much lower than that o...

Embodiment 4

[0335] Example 4. Construction and characterization of bispecific antibodies

[0336] 1. Production of antigens and other proteins

[0337] 1.1 Antigen production

[0338] Nucleic acids encoding human PD-1, human and mouse LAG-3 ECD (extracellular domain) were synthesized by Sangon Biotech. PD-1 or LAG-3 gene fragments were amplified from the synthetic nucleic acids and inserted into the expression vector pcDNA3.3 (ThermoFisher). The inserted PD-1 or LAG-3 gene fragment was further confirmed by DNA sequencing. By transfecting human PD-1 or LAG-3 gene into 293F cells (ThermoFisher), a fusion protein containing human LAG-3 ECD and various tags, including human Fc and mouse Fc, was obtained. Incubate cells at 37 °C, 5% CO 2 Cultured in FreeStyle 293 expression medium. After 5 days of culture, supernatants from cultures of transiently transfected cells were harvested for protein purification. Fusion proteins were purified by protein A and / or SEC columns. Untagged LAG-3 ECD ...

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Abstract

Anti-LAG-3 / PD-1 bispecific antibody molecules, isolated polynucleotides encoding the same, pharmaceutical compositions comprising the same, and the uses thereof are provided.

Description

[0001] priority claim [0002] This application claims priority to PCT Application No. PCT / CN2018 / 079691 filed on March 20, 2018. technical field [0003] The present disclosure generally relates to novel bispecific antibody molecules directed against human PD-1 and human LAG-3. Background technique [0004] Bispecific antibodies are emerging as a new class of therapeutic antibodies. They can bind two different targets or two different epitopes on a target, resulting in an additive or synergistic effect that is better than the effects of individual antibodies. Significant antibody engineering efforts have been devoted to designing new bispecific formats, such as DVD-Ig, CrossMab, BiTE, etc. (Spiess et al., Molecular Immunology, 67(2), pp.95-106 (2015)). However, these forms may have various limitations in terms of stability, solubility, short half-life and immunogenicity. [0005] Accumulating evidence from preclinical and clinical results suggests that targeting immune c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46A61K39/395
CPCC07K2317/31C07K16/2818C07K16/2803C07K2317/21C07K2317/92C07K2317/76C07K2317/33C07K2317/94C07K2317/73A61K2039/505A61K2039/507A61P35/00C07K2317/55C07K2317/622
Inventor 王卓智郑勇李竞吴琼
Owner SHANGHAI WUXI BIOLOGIC TECH CO LTD
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