Glutaminyl cyclase is an enzymatic modifier of the CD47- SIRPα axis and a target for cancer immunotherapy

Abstract

†, # These authors contributed equally Ethical Compliance Animal experiments were in compliance with all relevant ethical regulations approved by the IVD committee (Utrecht, the Netherlands). Blood samples from healthy donors was collected after informed consent. The use of human blood samples was in compliance with all relevant ethical regulations approved by the Sanquin Ethics Advisory Council of Sanquin Blood Supply (Amsterdam, the Netherlands). Reporting summary. Further information on experimental design is available in the Nature Research Reporting Summary linked to this article. Data availability All sequencing datasets have been deposited in the NCBI Sequence Read Archive under accession number SRP144590. In addition, all processed screen results are accessible in an interactive database (https://phenosaurus.nki.nl/). All data presented in this manuscript are available from the corresponding authors upon reasonable request Author contributions M.E.W.L. conceived the project, designed and performed experiments, interpreted data and co-wrote the manuscript. M.R., A.F. an T.R.B. designed, performed and interpreted the haploid genetic screens. M.T. and J.N. designed, performed and interpreted biochemical data. J.H.M.J., A.M.B. and J.H.W.L. designed, performed and interpreted anti-Her2 in vitro and in vivo data, and J.H.W.L. co-wrote the manuscript. K.F., H.L.M. and T.K.v.d.B. designed, performed and interpreted in vitro data with human effector cells. S.v.d.S. supported and performed flow cytometry analyses. R.G.-E. and N.A.M.B. designed, performed and interpreted in vitro studies with human T cells. J.H.v.d.B. and J.B.A.G.H. supervised analyses of T cell reactivity. K.A.M. performed and interpreted experiments. M.V. designed experiments and provided reagents. F.A.S. and T.N.S. conceived the project, designed experiments, interpreted data and co-wrote the manuscript.

A homogeneous SIRPα-CD47 cell-based, ligand-binding assay: Utility for small molecule drug development in immuno-oncology

“…We used the LCS assay to validate and extend these findings by treating A2058 melanoma cells with SEN177 to inhibit QPCTL and block the N-terminal pyroglutamate modification of CD47 (Fig 7). As in [36], we observed a reduction in SIRPα binding after 48 h treatment with SEN177 (Fig 7A). This was also associated with a similar reduction in the reactivity of clone CC2C6 antibody, whose epitope is known to be sensitive to the N-terminal pyroglutamate modification [39] (Fig 7B).…”

“…Consistent with interactions inferred from the SIRPα-CD47 co-crystal structure [42,43], SIRPα-CD47 binding critically depends on a CD47 post-translational modification of its Nterminal glutamine to a pyroglutamate [36]. This modification in tumor cells depends on the activity of glutaminyl cyclase (QPCTL) and can be inhibited pharmacologically by the pan-glutaminyl cyclase inhibitory molecule SEN177 [45].…”

“…The use of formalin-fixed as an alternative to live cells adds to the convenience of the assay and potential applications. We used this assay to further validate a number of active SMs from our qHTS program, and quantitatively characterize the impact of the pyro-GLU N-terminal post-translational modification of CD47 on SIRPα binding described by others [36].…”

Scalable, FACS-Free Genome-Wide Phenotypic Screening

“…We applied HT-miCS to a genome-wide CRISPR-Cas9 loss-of-function screen, probing genetic regulators of CD47 and yielding overlapping hits with a parallel FACS-based screen. Most notably, we identified and validated QPCTL as a regulator of CD47 pyro-Glu , corroborating recent findings reporting QPCTL as a potential modifier of CD47-targeted cancer immunotherapy 24 . Supporting this role, high expression of QPCTL has been shown to be a poor prognostic indicator for renal cancer 35 .…”

“…Of note, the three effective CD47 sgRNAs ( Figure 1D) were enriched in the CD47 low population in both HT-miCS and FACS, whereas the ineffective sgRNA (#4) was not (Tables S3, S4). In addition to CD47, five other top-ranked hits (<20% FDR) overlapped between the FACS and HT-miCS CD47 low screens ( Figure 2B), of which QPCTL was the strongest ( Figure S2), in line with a recent report of a gene-trap mutagenesis screen using the same antibody 24 .…”