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Recombinant fusion protein targeting CD47 and PD-L1 as well as preparation and application of recombinant fusion protein

A PD-L1, fusion protein technology, applied in the field of tumor treatment, can solve the problems of changing the structure of the antibody, affecting the binding force and efficacy, etc.

Active Publication Date: 2021-12-10
IMMUNEONCO BIOPHARM (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0018] 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 drug efficacy (Wang S et al., 2021)

Method used

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  • Recombinant fusion protein targeting CD47 and PD-L1 as well as preparation and application of recombinant fusion protein
  • Recombinant fusion protein targeting CD47 and PD-L1 as well as preparation and application of recombinant fusion protein
  • Recombinant fusion protein targeting CD47 and PD-L1 as well as preparation and application of recombinant fusion protein

Examples

Experimental program
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preparation example Construction

[0079]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.

[0080] 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 embodim...

Embodiment 1

[0089] Embodiment 1. Construction of IMM2520 and IMM2521 expression vectors

[0090] The structure of IMM2520 and IMM2521 is as Figure 1A and 1B shown. The full-length coding sequences of recombinant fusion proteins IMM2520 and IMM2521 were artificially designed.

[0091] Specifically, for the SIRPαD1-linker-PD-L1 antibody heavy chain in IMM2520, the coding sequence of the mutant SIRPαD1 (SEQ ID NO: 1) was combined with the PD-L1 in IMM2515 via the GS-linker coding sequence (SEQ ID NO: 3) The 5' end of the coding sequence for the antibody heavy chain (SEQ ID NO:5) was ligated; 57 nucleotides (SEQ ID NO:13) encoding the mouse IgG1 heavy chain signal peptide were added to the 5' of the mutant SIRPαD1 coding sequence end, and a Kozak sequence (SEQ ID NO: 14) 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 PD-L1 antibody light chain in IMM25...

Embodiment 2

[0094] Example 2. Protein expression and purification

[0095] To prepare the recombinant proteins IMM2520 and IMM2521, the expression vectors were electroporated into Chinese hamster ovary (CHO) cells (ATCC, Cat#CCL-61), after which these CHO cells were subjected to several rounds of neomycin pressure selection. Selected stable expressing cells were adapted in serum-free Balan CD CHO Growth A medium (Irvine Scientific, Cat#94120). For protein expression, cells were seeded into 3-liter bioreactors and cultured in fed-batch culture. When the cell viability dropped to -80%, the cell culture supernatant in the bioreactor was collected and subjected to protein purification by affinity chromatography. The purity of the recombinant protein is higher than 95%, and the amount of endotoxin is lower than 0.5U / g.

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Abstract

The invention provides a recombinant fusion protein. The recombinant fusion protein comprises a PD-L1 antibody or an antibody fragment thereof, a complementary position of the PD-L1 antibody or the antibody fragment thereof is connected with an extracellular Ig-like domain of a signal regulatory protein (SIRP) at an N end of a heavy chain variable region or a light chain variable region forming the complementary position through a linker, and the recombinant fusion protein can be simultaneously combined with CD47, PD-L1 and FcR. The invention also provides a nucleic acid molecule for coding the recombinant fusion protein, an expression vector containing the nucleic acid molecule, a method for preparing the recombinant fusion protein, and a method for treating diseases related to CD47 and / or PD-L1 overexpression by using the recombinant fusion protein.

Description

[0001] field of invention [0002] This application relates to a recombinant fusion protein targeting CD47, PD-L1 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 PD-1 binding on the surface of T cells triggers apoptosis of T cells; 2) escape the immune surveillance of natural killer (NK) cells by shedding MICA / MICB on the cancer cell membrane, and the shed MICA / MICB binds to NKG2D on the surface of NK cells , to block NK cell response to MICA / MICB + Killing of cancer cells; 3) Evade the immune surveillance of macrophages (Mφ) by highly expressing CD47, CD47 binds to signal regulatory protein α (SIRPα) on the surface of macrophages, thereby triggering the generation of inhibitory signal...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K39/395A61K38/17A61P35/02A61P35/00
CPCC07K16/2827C07K14/70596C12N15/85C12N5/0682A61P35/02A61P35/00C07K2317/56C07K2317/52C07K2317/76C07K2317/92C07K2317/732C07K2317/73C07K2319/00C07K2319/74C12N2800/107C12N2510/00A61K38/00A61K2039/505A61P37/00A61P1/00A61K38/1774C07K14/70503A61K2300/00A61K38/177A61K39/39558C07K16/30C07K16/3046C07K16/3061C07K16/468C07K2317/51C07K2317/515C12N15/63
Inventor 田文志李松
Owner IMMUNEONCO BIOPHARM (SHANGHAI) CO LTD
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