Bispecific antibody approach for EGFR-directed blockade of the CD47-SIRPα “don’t eat me” immune checkpoint promotes neutrophil-mediated trogoptosis and enhances antigen cross-presentation

Abstract

Helfrich (2020) Bispecific antibody approach for EGFR-directed blockade of the CD47-SIRPα "don't eat me" immune checkpoint promotes neutrophil-mediated trogoptosis and enhances antigen cross-presentation, OncoImmunology, 9:1, 1824323,

Cancer cells under immune attack acquire CD47-mediated adaptive immune resistance independent of the myeloid CD47-SIRPα axis

“…We previously reported that the capacity of bsAb CD47xEGFR-IgG2s to internalize CD47 dose-dependently relied on the cell surface expression levels of EGFR of the respective cancer cell lines. 8 In particular, bsAb CD47xEGFR-IgG2s showed only low capacity for CD47 internalization when treating EGFR low cell lines like OvCAR-3 and PC-3, but prominent capacity to do so when treating EGFR high cell lines like NCI-H292, FaDu, A431 and SK-RC-52 cells as is shown in Suppl. Figure S4A-C.…”

“…In this respect, we previously reported on bsAb CD47xEGFR-IgG1 which has potent capacity to selectively direct CD47-blockade toward EGFR-overexpressing cancer cells. 8 BsAb CD47xEGFR-IgG1 has a unique mode-of-action in that treatment of CD47 pos /EGFR pos cancer cells resulted in rapid co-internalization of EGFR and CD47, followed by prolonged displacement of both EGFR and CD47 from the cell surface. For the current study, we constructed variant bsAb CD47xEGFR-IgG2s which was equipped with an immune effector function nullified Fc domain.…”

“…For the current study, we constructed variant bsAb CD47xEGFR-IgG2s which was equipped with an immune effector function nullified Fc domain. 8 , 11 Indeed, treatment of CD47 pos /EGFR pos cancer cells with bsAb CD47xEGFR-IgG2s resulted in prolonged cell surface displacement of both EGFR and CD47 and a marked sensitization of these cancer cells to TRAIL- and T cell-induced cytotoxicity.…”

“…A detailed evaluation of bsAb CD47xEGFR-IgG, including its construction and production, was recently published by our group. 8 In short, DNA fragments encoding scFv MABL and scFv 425 were generated by commercial gene synthesis service (GenScript) based on published VH and VL sequence data. 20 , 21 22 For construction and production of bsAb CD47xEGFR-IgG2s, we used eukaryotic expression plasmid pEE14-bsAb, which contains three multiple cloning sites (MCS) for directional and in-frame insertion of DNA fragments: scFv MABL, scFv 425 and IgG2s-Fc.…”

“…The CMV pp65 -based model system has been described in detail elsewhere. 8 In short, PBMCs were freshly isolated from an HLA-B*07:02 CMV-positive healthy donor and stimulated with 5–10 μl of recombinant CMVpp65 protein stock solution (Miltenyi Biotec) per ml of cell suspension for 2–3 d. Next, PBMCs were repeatedly treated with 500–1000 U/ml IL-2 and/or cultured on a feeder layer of OvCAR-3.pp65 cells (HLA-B*07:02 pos ). Flow cytometry indicated that this stimulation protocol yielded >60% CMV pp65 -specific T cells (data not shown).…”