Recombinant fused protein targeting CD47 and CD70 as well as preparation and application of recombinant fused protein

A fusion protein, -CD70 technology, applied in the field of tumor treatment, can solve the problems of affecting the binding force and drug effect, changing the structure of the antibody, etc.

Inactive Publication Date: 2022-01-04
IMMUNEONCO BIOPHARM (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0019] Although conceptually, attaching additional binding groups to conventional antibodies is not very complicated, however, such modification can significantly change the structure of the antibody, and the interaction between the antibody and the additional binding groups may affect the binding force and / or or efficacy (Wang S et al., 2021)

Method used

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  • Recombinant fused protein targeting CD47 and CD70 as well as preparation and application of recombinant fused protein
  • Recombinant fused protein targeting CD47 and CD70 as well as preparation and application of recombinant fused protein
  • Recombinant fused protein targeting CD47 and CD70 as well as preparation and application of recombinant fused protein

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Experimental program
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preparation example Construction

[0085] Another object of the present application is to provide a method for preparing the above-mentioned recombinant fusion protein and a pharmaceutical composition comprising the recombinant fusion protein. In one embodiment, the preparation method comprises the following steps: (1) providing a nucleic acid molecule encoding a fusion protein; (2) constructing an expression vector comprising the nucleic acid molecule of (1); (3) using the expression vector in (2) to transform transfecting or transforming suitable host cells and culturing these host cells to express the protein; and (4) purifying the protein. Preparation can be carried out by techniques well known to those of ordinary skill.

[0086] Another object of the present application is to provide a method of using the pharmaceutical composition of the present application to treat cancer, comprising administering an effective amount of the above pharmaceutical composition to a patient or subject in need. In one embodi...

Embodiment 1

[0095] Embodiment 1. Construction and protein table of IMM40H, IMM40M, IMM4002, IMM4003 and IMM4002M expression vectors reach

[0096] The coding sequences of IMM40H, IMM40M, IMM4002, IMM4003 and IMM4002M were artificially designed.

[0097] Specifically, for the long peptide chain in IMM4002, the SIRPαD1-linker-CD70 antibody heavy chain, 57 nucleotides (SEQ ID NO:25) encoding the mouse IgG1 heavy chain signal peptide were added to SIRPαD1-linker-CD70 The 5' end of the antibody heavy chain coding sequence (SEQ ID NO: 13), and a Kozak sequence (SEQ ID NO: 26) was added to the 5' end of the signal peptide sequence. Finally, HindIII and NheI restriction sites were added to the 5' and 3' ends of the resulting sequence, respectively. For the short peptide chain in IMM4002, that is, the CD70 antibody light chain, the same signal peptide sequence and Kozak sequence are added to the 5' end of the CD70 antibody light chain coding sequence (SEQ ID NO: 15, N1=A, N2=C), And HindIII ...

Embodiment 2

[0101] Example 2. Recombinant fusion protein binds to cell surface CD47 or / and CD70

[0102] Each cell, that is, Chinese hamster ovary cells expressing human CD70, CD47 + CD70 - Jurkat human T lymphoid leukemia cells, CD47+ CD70 + U266 human multiple myeloma cells, CD47 + CD70 + Raji human B lymphoblastoid cells, CD47 + CD70 + U266 human multiple myeloma cells, CD47 + CD70 + Jeko-1 human mantle cell lymphoma cells, and CD47 + CD70 + Daudi human Burkitt lymphoma cells were centrifuged at 1000rpm for 5min to collect the cells, washed once with PBS, and the cell density was adjusted to 1×10 6 / ml. Recombinant fusion proteins IMM4002, IMM4003, IMM4002M, and / or control proteins IMM40H, IMM01 (Fc fusion proteins that bind to CD47, described in US 2021 / 0024598 A1, containing two mutations SIRPαD1 (SEQ ID NO: 1), and Fc two The polymer fragments are connected, wherein the monomer contains the nucleic acid sequence and amino acid sequence shown in SEQ ID NO:24 and SEQ ID N...

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Abstract

The invention provides a recombinant fused protein. The recombinant fused protein comprises a CD70 antibody or an antibody fragment thereof, wherein at least one complementary position of the CD70 antibody or the antibody fragment thereof is connected with an extracellular Ig-like structural domain of a signal regulatory protein (SIRP) at an N-terminal of a heavy chain or a light chain forming the complementary position through a linker, and the recombinant fused protein can be combined with CD47, the CD70 and FcR at the same time. The invention also provides a nucleic acid molecule coding the recombinant fused protein, an expression vector comprising the nucleic acid molecule, a method for preparing the recombinant fused protein and use of the recombinant fused protein in treatment of diseases associated with CD47 and/or CD70 overexpression.

Description

[0001] field of invention [0002] The present application relates to a recombinant fusion protein targeting CD47, CD70 and / or FcR, its preparation and use, especially its use in tumor treatment. Background technique [0003] Cancer cells have developed some mechanisms to evade the host's immune surveillance, including: 1) evading the immune surveillance of T lymphocytes by highly expressing membrane proteins PD-L1 and PD-L2, both of which are associated with The combination of PD-1 on the surface of T cells triggers the apoptosis of T cells; 2) through the expression of CD70, it binds to CD27 on the surface of immune cells to induce T cell apoptosis and T cell exhaustion, and greatly increases the amount of Treg; 3) through high CD47 is expressed to escape the immune surveillance of macrophages (Mφ), and CD47 binds to signal regulatory protein α (SIRPα) on the surface of macrophages, thereby triggering the generation of inhibitory signals, which inhibit macrophages against ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00
CPCC07K16/2875C07K14/70503C12N15/85C12N5/0682A61K47/6849A61K39/39558A61P35/00A61P35/02C07K2317/52C07K2317/56C07K2317/565C07K2317/732C07K2317/734C07K2317/73C07K2317/31C07K2319/03C12N2800/107C12N2510/00C07K2319/02C07K2319/00A61K2039/505
Inventor 田文志李松陈典泽
Owner IMMUNEONCO BIOPHARM (SHANGHAI) CO LTD
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