ANTI-SIRPalpha ANTIBODY

An antibody, CDRL2 technology, applied in the direction of antibodies, anti-tumor drugs, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the problem of no binding

Active Publication Date: 2021-04-16
KOBE UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, OSE-172 is an IgG4Pro antibody that shows binding to SIRPα V1 and SIRPβ1, but not SIRPα V2 and SIRPγ

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0275] Example 1. Production of Rat Anti-SIRPA Antibody

[0276] 1)-1 Preparation of expression construct

[0277] 1) Construction of-1-1SIRPA_V1_ECD expression vector

[0278]By using the In-Fusion HD Cloning Kit (CLONTECH), the DNA encoding the polypeptide that is linked to HHHHHH at the C-terminal side of the 1st to 373rd amino acid sequence of human SIRPA_V1 (NCBI protein database accession number: NP_001035111) was restricted to The vector pcDNA3.3-TOPO / LaxZ (ThermoFisherSCIENTIFIC) digested with the enzyme XbaI and PmeI was combined to produce the SIRPA_V1_ECD expression vector. The amino acid sequence of SIRPA_V1_ECD is shown in SEQ ID NO:45 of the sequence listing, and the nucleotide sequence encoding SIRPA_V1_ECD is shown in SEQ ID NO:44 of the sequence listing.

[0279] 1) Construction of-1-2SIRPA_V1_IgV expression vector

[0280] A SIRPA_V1_IgV expression vector was prepared in the same manner as 1)-1-1 using DNA encoding a polypeptide having HHHHHH linked to the...

Embodiment 2

[0327] Example 2. In Vitro Evaluation of Rat Anti-Human SIRPA Antibodies (7 Kinds)

[0328] 2)-1 Construction of expression vector for antigen-binding antibody screening

[0329] 2) Construction of -1-1FLAG-human SIRPA expression vector (pFLAG V5-DEST-SIRPA_V1-V10)

[0330] The cDNAs encoding 10 human SIRPA variant proteins (excerpted from Nature Immunology 8, 1313-1323, 2007) were cloned into the pFLAG V5-DEST vector, and the vector pFLAG V5-DEST-SIRPA_V1-V10 expressing the respective variant proteins was constructed.

[0331] The amino acid sequence of human SIRPA_V3 is shown in SEQ ID NO:63 of the sequence listing, the amino acid sequence of human SIRPA_V4 is shown in SEQ ID NO:64 of the sequence listing, and the amino acid sequence of human SIRPA_V5 is shown in SEQ ID NO:65 of the sequence listing The amino acid sequence of human SIRPA_V6 is shown in SEQ ID NO:66 of the sequence listing, the amino acid sequence of human SIRPA_V7 is shown in SEQ ID NO:67 of the sequence li...

Embodiment 3

[0385] Example 3. Nucleotide sequence analysis and determination of the amino acid sequence of the cDNA of the variable region of the rat anti-SIRPA antibody (D13, F44, F63)

[0386] 3) Nucleotide sequence analysis and determination of the amino acid sequence of the variable region cDNA of -1D13

[0387] 3) Preparation of total RNA from hybridoma produced by -1-1D13

[0388] To amplify the cDNA encoding the variable region of D13, total RNA was prepared from D13-producing hybridomas using TRIzol reagent (Ambion).

[0389] 3)-1-2 Utilize 5'-RACE PCR to determine the nucleotide sequence analysis and amino acid sequence of the cDNA of the D13 light chain variable region

[0390] The cDNA encoding the light chain variable region was amplified using about 1 μg of total RNA prepared in Example 3)-1-1 and SMARTerRACE 5' / 3' kit (Clontech). As primers for amplifying the cDNA encoding the variable region of the D13 light chain gene by PCR, UPM (Universal Primer A Mix: included in the ...

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Abstract

The present invention addresses the problem of providing: an anti-SIRPalpha antibody which can be used as a tumor agent; and an anti-tumor agent containing the antibody as an active ingredient. The present invention is an antibody capable of binding specifically to human SIRPalpha to inhibit the binding between human SIRPalpha and CD47, the antibody containing (a) a light-chain CDRL1 comprising the amino acid sequence represented by SEQ ID NO: 1, (b) a light-chain CDRL2 comprising the amino acid sequence represented by SEQ ID NO: 2, (c) a light-chain CDRL3 comprising the amino acid sequence represented by SEQ ID NO: 3, (d) a heavy-chain CDRH1 comprising the amino acid sequence represented by SEQ ID NO: 4, (e) a heavy-chain CDRH2 comprising the amino acid sequence represented by SEQ ID NO: 5 and (f) a heavy-chain CDRH3 comprising the amino acid sequence represented by SEQ ID NO: 6, wherein a heavy-chain constant region is a heavy-chain constant region of human IgG4, and an phenylalanine residue at position-234, a leucine residue at position-235 and a serine residue at position-228, as numbered according to the EU index as in Kabat et. al., are substituted by an alanine residue, an alanine residue and a proline residue, respectively.

Description

technical field [0001] The present invention relates to an anti-SIRPα antibody useful for tumor therapy, and an antitumor agent containing the antibody. Background technique [0002] SIRPα (SHPS-1) is a single transmembrane molecule of the Ig superfamily, and is present in myeloid cells such as macrophages, dendritic cells, and neutrophils, as well as glial cells (Non-Patent Document 1). The extracellular region is composed of one IgV domain and two IgC domains, and it has been reported that there are 10 variants of the IgV domain that binds to CD47 in humans (Non-Patent Document 2). On the other hand, the intracellular region contains immunoreceptor tyrosine-based inhibition motifs (ITIMs), which induce SHP-1 and SHP-2 binding to tyrosine phosphatases by binding to CD47. Send inhibitory signals. [0003] As a physiological phenomenon resulting from SIRPα-CD47 interaction, it has been shown that CD47 on erythrocytes binds to SIRPα on macrophages and transmits a "don't eat ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K39/395A61P35/00A61P35/02A61P43/00C12N1/15C12N1/19C12N1/21C12N5/10C12N15/13C12N15/63C12P21/08
CPCA61P35/00A61P35/02A61P43/00C07K16/2803C07K2317/52C07K2317/71C07K2317/76C07K2317/24C07K2317/33C07K2317/34C07K16/32A61K2039/507C07K2317/73C07K16/2896C07K2317/565A61K2039/505C07K16/2863C07K16/2887C07K2317/21C07K2317/72C07K2317/732
Inventor 的崎尚须江真由美中村健介吉村千草
Owner KOBE UNIV
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