A protein binding nkg2d, cd16 and a tumor-associated antigen

A protein and antigen technology, applied in the direction of anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, anti-tumor drugs, etc., can solve problems such as adverse side effects and ineffectiveness of patients

Pending Publication Date: 2021-03-02
DRAGONFLY THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Current treatment options for these cancers are ineffective in all patients and / or may have significant adverse side effects
Treating other types of cancer with existing treatment options also remains challenging

Method used

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  • A protein binding nkg2d, cd16 and a tumor-associated antigen
  • A protein binding nkg2d, cd16 and a tumor-associated antigen
  • A protein binding nkg2d, cd16 and a tumor-associated antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1-N

[0263] Example 1 - NKG2D binding domains bind to NKG2D

[0264] NKG2D binding domain binds to purified recombinant NKG2D

[0265] The nucleic acid sequence of the extracellular domain of human, mouse or cynomolgus NKG2D is fused to the nucleic acid sequence encoding the human IgG1 Fc domain and introduced into mammalian cells to be expressed. After purification, the NKG2D-Fc fusion protein was adsorbed to the wells of a microplate. After blocking the wells with bovine serum albumin to prevent non-specific binding, the NKG2D-binding domain was titrated and added to the wells pre-adsorbed with NKG2D-Fc fusion protein. Primary antibody binding was detected using a secondary antibody conjugated to horseradish peroxidase and specifically recognizing human kappa light chain to avoid Fc cross-reactivity. The substrate of horseradish peroxidase, 3,3',5,5'-tetramethylbenzidine (TMB), was added to the wells to visually observe the binding signal, its absorbance was measured at 450 n...

Embodiment 2

[0270] Example 2 - NKG2D Binding Domains Block the Binding of Natural Ligands to NKG2D Compete with ULBP-6

[0271] The recombinant human NKG2D-Fc protein was adsorbed to the wells of the microplate, and the wells were blocked with bovine serum albumin to reduce non-specific binding. A saturating concentration of ULBP-6-His-biotin was added to the wells, followed by the addition of NKG2D binding domain clones. After 2 hours of incubation, the wells were washed and ULBP-6-His-biotin still bound to NKG2D-Fc-coated wells was detected by streptavidin and TMB substrate conjugated to horseradish peroxidase . Absorbance was measured at 450 nM and corrected at 540 nM. The specific binding of the NKG2D binding domain to the NKG2D-Fc protein was calculated from the percentage of ULBP-6-His-biotin blocked from binding to the NKG2D-Fc protein in the wells after background subtraction. Positive control antibodies (comprising heavy and light chain variable domains selected from SEQ ID ...

Embodiment 3-N

[0278] Example 3 - NKG2D binding domain cloning activates NKG2D

[0279]The nucleic acid sequences of human and mouse NKG2D were fused to the nucleic acid sequence encoding the CD3ξ signaling domain to obtain chimeric antigen receptor (CAR) constructs. Then, the NKG2D-CAR construct was cloned into a retroviral vector using Gibson assembly and transfected into expi293 cells to generate retrovirus. EL4 cells were infected with virus containing NKG2D-CAR and 8 μg / mL polybrene. 24 hours after infection, the expression level of NKG2D-CAR in EL4 cells was analyzed by flow cytometry, and clones expressing high levels of NKG2D-CAR on the cell surface were selected.

[0280] To determine whether NKG2D-binding domains activate NKG2D, they were adsorbed to wells of microplates and antibody fragments coated in the presence of brefeldin-A (brefeldin-A) and monensin (monensin) NKG2D-CAR EL4 cells were cultured on the wells for 4 hours. Intracellular TNF-[alpha] production, an indicator o...

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Abstract

Multi-specific binding proteins that bind NKG2D receptor, CD16, and P-cadherin, as well as pharmaceutical compositions and therapeutic methods useful for the treatment of cancer.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 667,844, filed May 7, 2018. [0003] sequence listing [0004] This application contains a Sequence Listing, filed electronically in ASCII format and incorporated herein by reference in its entirety. Said ASCII copy, created on April 30, 2019, is named DFY-054WO_SL.txt and is 152,838 bytes in size. technical field [0005] The present invention relates to multispecific binding proteins that bind to P-cadherin (CDH3), NKG2D and CD16. Background technique [0006] Despite numerous research efforts and scientific advances reported in the literature for the treatment of cancer, the disease remains an important health problem. Some of the most commonly diagnosed cancers include prostate, breast, and lung. Prostate cancer is the most common form of cancer in men. Breast cancer remains the leading cause of death in women. C...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/395C07K16/28C07K16/32C12N15/13C40B30/04
CPCC07K16/28C07K16/2851A61K2039/505C07K2317/55C07K2317/92C07K2317/31C07K2317/73C07K2317/70C07K2317/33C07K2317/622C07K16/283A61P35/00
Inventor 格雷戈里·P·常安·F·张杜金燕阿斯亚·格林贝格威廉·哈尼布拉德利·M·伦德比昂卡·普林茨
Owner DRAGONFLY THERAPEUTICS INC
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