United Kingdom (UK)
Recombinant monoclonal antibody to CD47. Manufactured using AbAb’s Recombinant Platform with variable regions (i.e. specificity) from the synthetic clone h4C1.
UniProt Accession Number of Target Protein: Q08722
Alternative Name(s) of Target: IAP; MER6; OV-3; Leukocyte surface antigen CD47; Antigenic surface determinant protein OA3; Integrin-associated protein; Protein MER6; ovarian cancer marker; 4C1
Immunogen: The original mouse antibody was generated by immunizing BALB/c mice with human CD47-ecto recombinant protein emulsified with complete Freund's adjuvant. Later on the original mouse antibody was used to generate the humanized version of the antibody using sequence homology and computational prediction.
Specificity: This antibody binds the FG loop of the human CD47, which interacts with SIRPα and several other known CD47/SIRPα-blocking mAb. CD47 is broadly distributed on normal adult tissues, as well as ovarian tumors, and is especially abundant in some epithelia and the brain. IAP or integrin assocaited protein has a role in both cell adhesion by acting as an adhesion receptor for THBS1 on platelets, and in the modulation of integrins. It further plays an important role in memory formation and synaptic plasticity in the hippocampus. It also acts as a receptor for SIRPA. Binding to SIRPA prevents maturation of immature dendritic cells and inhibits cytokine production by mature dendritic cells.
Application Notes: This antibody specifically binds to the CD47 molecule to block the binding of CD47 to SIRPα, thereby activating macrophages and killing tumor cells. The blocking activity was demonstrated using flow cytometry assay. This antibody also bound CD47 ectodomain in an ELISA. Surface plasmon resonance (SPR)-binding assays demonstrated that h4C1 bound to the human CD47-ECD antigen with a KD of 0.85 nM. Macrophage-mediated phagocytosis activity was investigated by a tumor cell phagocytosis assay with bone marrow-derived macrophages (BMDMs) and tt was reported that h4C1 exhibited significant phagocytosis in Raji cells. In in vivo experiments this antibody improved survival and enhanced suppression of Raji cell xenografts in mice (PMID: 32296041). This antibody was also used in the generation of a bispecific antibody (BsAb) in a dual-variable-domain immunoglobulin (DVD-Ig) format targeting CD47 and PD-L1. The variable-domain from the PD-L1-blocking mAb #18 was introduced in tandem onto h4C1 to generate a dual variable domain bsAb that binds bivalently to both CD47 and PD-L1. This BsAb retained retained blocking activity for both interactions but lacked the hemagglutinating activity of the parental m4C1 (PMID: 32296041; 33244513).
Antibody first published in:
Shi et al. The identification of a CD47-blocking "hotspot" and design of a CD47/PD-L1 dual-specific antibody with limited hemagglutination. Signal Transduct Target Ther. 2020 Mar 6;5(1):16. PMID:32296041
Note on publication:
Describes the generation of a bispecific antibody targeting CD47 and PD-L1 using this antibody.
Immunofluorescence staining of Jurkat cells with Anti-CD47 (Ab03104) h4C1 Immunofluorescence analysis of paraformaldehyde fixed Jurkat cells immobilized on Shi-fix™ plus cover-slips stained with the chimeric rabbit IgG version of h4C1 (Ab03104-23.0) (1:100 dilution) for 1h followed by Alexa Fluor® 488 secondary antibody (1:1000 dilution), showing membrane staining. The nuclear stain is DAPI (blue). Panels show from left-right, top-bottom Ab03104-23.0, DAPI, merged channels and an isotype control. The isotype control was an unknown specificity antibody (Ab00178-23.0) followed by staining with Alexa Fluor® 488 secondary antibody.
Flow cytometry using the Anti-CD47 antibody h4C1 (Ab03104). Paraformaldehyde fixed Jurkat cells were stained with anti-unknown specificity antibody (Ab00178-23.0; isotype control, black line) or the rabbit IgG version of h4C1 (Ab03104-23.0, blue line) at a dilution of 1:100 for 1h at RT. After washing, the bound antibody was detected using a goat anti-rabbit IgG AlexaFluor® 488 antibody at a dilution of 1:1000 and cells analyzed using a FACSCanto flow-cytometer.