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Recombinant fusion protein targeting CD47 and CD24 as well as preparation and application of recombinant fusion protein

A fusion protein, CD24 technology, applied in the field of tumor treatment, can solve the problems of affecting the binding force and efficacy, changing the structure of antibodies, etc.

Active Publication Date: 2022-01-21
IMMUNEONCO BIOPHARM (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0021] 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 CD24 as well as preparation and application of recombinant fusion protein
  • Recombinant fusion protein targeting CD47 and CD24 as well as preparation and application of recombinant fusion protein
  • Recombinant fusion protein targeting CD47 and CD24 as well as preparation and application of recombinant fusion protein

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Experimental program
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preparation example Construction

[0084] 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.

[0085] 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

[0098] Example 1. Construction and protein expression of IMM4701, IMM4701C, IMM4702C, IMM4702H expression vectors

[0099] The structures of IMM4701, IMM4701C, IMM4702C, IMM4702H, IMM47, IMM47C and IMM47H are as follows figure 1 shown. The full-length coding sequences of these recombinant fusion proteins were artificially designed.

[0100] Specifically, for the long chain of IMM4701C, 57 nucleotides (SEQ ID NO: 34) encoding the mouse IgG1 heavy chain signal peptide were added to the SIRPαD1-linker-CD24 antibody heavy chain coding sequence (SEQ ID NO: 19) 5' end, and a Kozak sequence (SEQ ID NO: 35) 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 chain of IMM4701C, the same signal peptide sequence and Kozak sequence were added to the 5' end of the light chain coding sequence (SEQ ID NO: 21), and HindIII and XbaI restriction enzyme si...

Embodiment 2

[0105] Example 2. IMM4701, IMM4701C, IMM4702C and IMM4702H bind to CD47 on Jurkat cells

[0106] Adjust the density of Jurkat cells naturally expressing CD47 to 1 × 10 6 / ml, take 100 μl, respectively in 100 μl serial dilution (initial concentration is 30 μg / ml, 3-fold serial dilution) of IMM 4701, IMM4701C, IMM4702C, IMM4702H Incubate with control solution IMM01 and IMM47 at 4°C for 1 hour. Cells were washed twice with cold PBS, and then incubated with 100 μl of FITC-labeled secondary antibody against human IgG-Fc (Cat#F9512, Sigma) for 45 minutes. Cells were washed twice and resuspended in 200 μl PBS. Afterwards, the cells were analyzed by flow cytometry (Merck Millipore, easyCyte 5HT) for FACS analysis.

[0107] Figure 2A and 2B It was shown that IMM4701, IMM4701C, IMM4702C, and IMM4702H can bind to CD47 on Jurkat cells, which is slightly worse than the traditional single antigen targeting protein IMM01.

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Abstract

The invention provides a recombinant fusion protein. The recombinant fusion protein comprises a CD24 antibody or an antibody fragment thereof, wherein at least one paratope of the CD24 antibody or the antibody fragment thereof is linked to an extracellular Ig-like domain of a signal regulatory protein (SIRP) at the N end of a heavy chain or a light chain forming the paratope through a linker, and the recombinant fusion protein can be combined with CD47, CD24 and FcR at the same time. The invention further provides nucleic acid molecules encoding the recombinant fusion protein, expression vectors comprising the nucleic acid molecules, methods for preparing the recombinant fusion protein, and methods for treating diseases associated with CD47 and / or CD24 overexpression by using the recombinant fusion protein.

Description

[0001] field of invention [0002] This application relates to a recombinant fusion protein targeting CD47, CD24 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) Evade the immune surveillance of macrophages, T lymphocytes, B lymphocytes and natural killer cells by highly expressing the membrane protein CD24, in which the membrane protein CD24 can interact with Siglec-10 receptors on the surface of immune cells bind to inhibit immune activation; 2) Evade the immune surveillance of macrophages (Mφ) by overexpressing CD47, which binds to signal regulatory protein α (SIRPα) on the surface of macrophages , thereby triggering the generation of an inhibitory signal that inhibits the phagocytosis of cancer cells by macrophages. It can be seen that cancer cells are quite smart and can proliferate rapidly based on the evasion mechan...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K39/395A61P35/00A61P35/02
CPCC07K16/2896C07K14/00A61P35/00A61P35/02A61K2039/507C07K2317/56C07K2317/52C07K2319/30C07K2319/00C07K2317/73C07K2317/732C07K2317/76C07K14/70503C07K2317/24C07K2317/66C07K2317/92
Inventor 田文志李松陈典泽
Owner IMMUNEONCO BIOPHARM (SHANGHAI) CO LTD
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