Genome-scale CRISPR activation screen uncovers tumor-intrinsic modulators of CD3 bispecific antibody efficacy

Abstract

Bispecific antibodies (bsAb) that bridge tumor cells and CD3-positive effector T cells are being developed against many tumor cell targets. While tumor cell factors other than target expression level appear to play a role in determining the efficacy of CD3 bsAb, the identity of such factors remains largely unknown. Using a co-culture system of primary human T cells and B lymphoma cell lines, we demonstrate a range of sensitivities to CD20xCD3 bsAb that is independent of CD20 surface expression. To identify genes that modulate tumor cell sensitivity to CD3 bsAb, we employed a genome-scale CRISPR activation screen in a CD20xCD3-sensitive human B lymphoma cell line. Among the most highly enriched sgRNAs were those targeting genes with predicted effects on cell-cell adhesion, including sialophorin (SPN). Increased expression of SPN impeded tumor cell clustering with T cells, thereby limiting CD3 bsAb-mediated tumor cell lysis. This inhibitory effect of SPN appeared to be dependent on sialylated core 2 O-glycosylation of the protein. While SPN is not endogenously expressed in the majority of B cell lymphomas, it is highly expressed in acute myeloid leukemia. CRISPR-mediated SPN knockout in AML cell lines facilitated T cell-tumor cell clustering and enhanced CD3 bsAb-mediated AML cell lysis. In sum, our data establish that the cell cross-linking mechanism of CD3 bsAb is susceptible to subversion by anti-adhesive molecules expressed on the tumor cell surface. Further evaluation of anti-adhesive pathways may provide novel biomarkers of clinical response and enable the development of effective combination regimens for this promising therapeutic class.

Nuclease-Deficient Clustered Regularly Interspaced Short Palindromic Repeat-Based Approaches for In Vitro and In Vivo Gene Activation

“…The authors also noted that despite depletion of exogenous BAM factors and sgRNAs for endogenous activation after transient transfection, endogenously activated factor genes remained high, suggesting sustained epigenetic remodeling of the targeted genomic loci achieved by the CRISPRa approach. This was further supported by chromatin immunoprecipitation (ChIP) qPCR data showing 61 Sensitivity to CD3 bispecific antibody JeKo-1 cells SAM hSAM Positive CD3 bispecific antibodymediated T cell killing Dukhovny 63 Protective host factors against zika virus Huh7 cells SAM hSAM Positive Resistance to zika virus infection Gilbert 30 Sensitivity to cholera/diphtheria toxin K562 cells SunTag Custom Positive Cholera/diphtheria-induced toxicity and death Konermann 55 Resistance to vemurafenib A375 cells SAM hSAM Positive Cell survival after treatment with vemurafenib Sanson 57 Resistance to vemurafenib A375 cells dCas9-VP64 Calabrese Positive Cell survival after treatment with vemurafenib Gilbert 30 Modulator of cell growth rate K562 cells SunTag Custom Negative Growth inhibition and depletion Horlbeck 56 Modulator significant enrichment in H3K27ac and H3k4me3 surrounding the promoter sequences of Brn2 and Ascl1 locimarkers of histone H3 modifications, not observed in BAM factor transgene overexpression.…”

“…The majority of published CRISPRa screens utilized positive selection strategies, identifying perturbations that confer resistance to selected drugs, 55,57,61 toxins, 30,62 and pathogen infection 63 in resistant cell populations. Results from these positive selection screens all report the discovery of novel genes whose upregulation resulted in rescue from cell death in their respective studies.…”

Naturally Occurring Genetic Alterations in Proximal TCR Signaling and Implications for Cancer Immunotherapy

“…Based on our above appreciation for the heterogenous involvement of various CD3 subunits in T cell function and disease, targeting other CD3 subunits could be used to fine-tune desired T cell recruitment with BiTEs. BiTE efficacy is in part limited by specific tumor cell phenotypes, such as the expression of sialophorin which limits T cell to tumor cell adhesion ( 89 ), and by similar side effects of CRS and neurotoxicity observed in CAR Therapy ( 90 , 91 ). One study also identified a polymorphism in the CD3ζ chain (SNP rs2949655) that correlated with reduced cytotoxicity in response to BiTE treatment ( 92 ), demonstrating how CD3 mutational profiling could be used to help guide personalized treatment approaches targeting this pathway.…”

Data-driven discovery of targets for bipotent anticancer drugs identifies Estrogen Related Receptor Alpha

“…Accordingly, the ESRRAi treatment upregulated genes involved in antigen presentation (Fig S5D ; Since increased MHC-I antigen presentation in tumors enhances the ability of T-cells to kill cancer cells (67,79), we hypothesized that ESRRAi would enhance tumor killing by T cells. We tested this using published CRISPR screens that co-culture cancer cells with T-cells to identify which gene knockouts in cancer cells enhance their T-cell-mediated killing (67,(80)(81)(82)(83). ESRRA KO potentiated the killing of cancer cells by both patient-derived and engineered effector T-cells in various experimental and cell-line contexts (Fig 3C).…”