A CRISPR Screen Reveals Resistance Mechanisms to CD3-Bispecific Antibody Therapy

Abstract

CD3-bispecific antibodies represent an important therapeutic strategy in oncology. These molecules work by redirecting cytotoxic T cells to antigen-bearing tumor cells. Although CD3-bispecific antibodies have been developed for several clinical indications, cases of cancer-derived resistance are an emerging limitation to the more generalized application of these molecules. Here, we devised whole-genome CRISPR screens to identify cancer resistance mechanisms to CD3-bispecific antibodies across multiple targets and cancer types. By validating the screen hits, we found that deficiency in IFNγ signaling has a prominent role in cancer resistance. IFNγ functioned by stimulating the expression of T-cell killing-related molecules in a cell type-specific manner. By assessing resistance to the clinical CD3-bispecific antibody flotetuzumab, we identified core fucosylation as a critical pathway to regulate flotetuzumab binding to the CD123 antigen. Disruption of this pathway resulted in significant resistance to flotetuzumab treatment. Proper fucosylation of CD123 was required for its normal biological functions. In order to treat the resistance associated with fucosylation loss, flotetuzumab in combination with an alternative targeting CD3-bispecific antibody demonstrated superior efficacy. Together, our study reveals multiple mechanisms that can be targeted to enhance the clinical potential of current and future T-cell-engaging CD3-bispecific antibody therapies.

Figure 1.

Genome-wide CRISPR screens reveal resistance mechanisms to CD3-bispecific antibodies. A, Schematic of the genome-wide CRISPR screen. The cancer cells were engineered to stably express CAS9 and then infected with a sgRNA lentivirus library. NGS, next-generation sequencing. B, Time-course analysis of T cells and MOLM13 cells in the screen coculture. The T cells were CD3⁺, and the sgRNA-transduced MOLM13 cells were RFP⁺. Top row shows isotype antibody treatment, and bottom row shows CD123-DART antibody treatment. Data represent three independent experiments. C, Flowchart of the screen data analysis. Guide calls were quantified from targeted RNA-seq of the guide sequences and subsequently normalized to internal nontargeting controls in the guide library. The data were then normalized to the initial pool sample, and statistical test was performed between the DART group and isotype group. D, Data presentation for the three screens. Each point represents one gene, and the axes show enrichment scores from CD3-bispecific treatment group (DART) or isotype antibody treatment group (Isotype) on the log₂ scale. Genes with high DART enrichment and low isotype enrichment scores were considered as resistance hits and vice versa for sensitizing hits. E and F, Venn diagram of the shared resistance (E) or sensitizing (F) hits from all three screens. Hits were determined by running two-tailed t test between the DART group and isotype group for each cell line, with a cutoff of −log₁₀ P value >2 (Supplementary Table S3).

Figure 2.

The IFNγ signaling pathway is a central regulator of cancer cell resistance. A, MOLM13, SEM, and HCC827 cells were treated with a blocking antibody against IFNGR1 (α-IFNGR) and applied in the RTCC assay with corresponding CD3-bispecific antibodies (n = 6). The RTCC assay used T-cell:target cell ratio of 0.5:1, and target cell killing was measured 48 hours after treatment. B–D, MOLM13, SEM, and HCC827 cells were knocked out for JAK1 (JAK1^KO) and STAT1 (STAT1^KO), and killing by CD3-bispecific antibodies was compared with control groups (Scramble; n = 4). The RTCC assay used T-cell:target cell ratio of 0.5:1, and target cell killing was measured 48 hours after treatment. E, Scramble and PTPN2^KO cells were starved in RPMI1640 overnight and treated with IFNγ for 5 minutes. IFNγ signaling was reported by STAT1 phosphorylation (pSTAT1) in Scramble and PTPN2^KO MOLM13 cells. F, RTCC assay of PTPN2^KO MOLM13 cells (n = 4). The RTCC assay used T-cell:target cell ratio of 0.5:1, and target cell killing was measured 24 hours after treatment. G, The growth of MOLM13 (n = 3), SEM (n = 5), and HCC827 (n = 8) cells 72 hours after IFNγ treatment. The red dashed line indicates the maximum IFNγ release in the corresponding RTCC assay (referring to Supplementary Fig. S2D). H, Heatmap of the overlapped hits from the IFNγ treatment RNA-seq and CRISPR screen datasets of each individual cell line. I, Flow cytometric analysis of ICAM1 and FAS expression in the MOLM13 and SEM cells with IFNγ titration (n = 3). J, Western blot of caspase family protein expression in the three cell lines 24 hours after IFNγ (100 ng/mL) treatment (n = 1). K, RTCC assay of ICAM1^KO MOLM13 cells using the CD123-DART or CD33-BiTE (n = 6). The RTCC assay used T-cell:target cell ratio of 0.5:1, and target cell killing was measured 24 hours after treatment. bsAb, bispecific antibody. L, RTCC assay of FAS^KO SEM cells using the CD123-DART or CD19-BiTE (n = 4). The RTCC assay used T-cell:target cell ratio of 0.5:1, and target cell killing was measured 48 hours after treatment. M, RTCC assay of CASP8^KO HCC827 cells using the PCAD-DART (n = 4). The RTCC assay used T-cell:target cell ratio of 1:1, and target cell killing was measured 48 hours after treatment. For each plot, error bars represent SEM.

Figure 3.

Deficiency in the core fucosylation pathway constitutes a novel resistance mechanism to CD123-DART treatment. A, Heatmap showing the enrichment scores of the shared hits from the MOLM13 and SEM CRISPR screen. DART represents CD123-DART. B, Depiction of the core fucosylation pathway. The hits from the screens are highlighted in red. C, RTCC assay of FUT8^KO and CD123^KO cells with CD123-DART treatment (n = 4). The RTCC assay used T-cell:target cell ratio of 1:1, and target cell killing was measured 72 hours after treatment. Knockout of FUT8 was confirmed by the loss of LCA staining (left plots). Data represent two independent experiments. The isotype antibody staining and unstained groups overlapped with the histogram of FUT8^KO cells. D, Scramble, FUT8^KO, and GMDS^KO cells were cultured in media supplemented with 100 mmol/L L-fucose, and core fucosylation was measured by LCA staining (left plots). The cells were then applied to the RTCC assay using CD123-DART (n = 4). The RTCC assay used T-cell:target cell ratio of 1:1, and target cell killing was measured 72 hours after treatment. E, RTCC assay (n = 4) of MOLM13 cells with single fucosyltransferase knockout (left plot) or dual fucosyltransferase knockout (right plot) compared with the killing of the FUT8^KO group (shown in red; n = 3). The RTCC assay used T-cell:target cell ratio of 1:1, and target cell killing was measured 72 hours after treatment. F, Quantification of core fucosylated proteins by LCA pulldown in concatenation with mass spectrometry (n = 3). Cell-surface proteins with significant enrichment (see Materials and Methods; Supplementary Table S5) in the Scramble group are listed (right plot). G, RTCC assay (n = 4) of MOLM13 cells with knockout of selected genes from the protein list in F. The RTCC assay used T-cell:target cell ratio of 1:1, and target cell killing was measured 72 hours after treatment. For each plot, error bars represent SEM.

Figure 4.

Loss of core fucosylation impedes the binding of CD123 to CD123-DART. A, Flow cytometric analysis of 6H6, CD123-DART_IgG, and CSL362 binding to Scramble, FUT8^KO, and CD123^KO MOLM13 cells. Cells were incubated with the antibodies for 30 minutes at 4°C, followed by flow cytometry analysis. Data represent three independent experiments. B, Freestyle 293 cells with Scramble and FUT8^KO were generated by RNP knockout, and the knockout efficiency was evaluated by LCA staining. Data represent three independent experiments. C, SPR analysis on the binding of purified CD123His-Scramble and CD123His-FUT8^KO proteins to 6H6 and CD123-DART_IgG. The antibodies were captured on the chip, and the proteins were subsequently flowed through. The association and dissociation times were set up as indicated in the plots. D, Models generated using SASREF for CD123His-Scramble (blue and green chains) and CD123His-FUT8^KO (gray) superimposed using SUPCOMB as described in the Materials and Methods. Rg, Dmax, and χ² values are displayed in the table (bottom). Chains A and G were from the dimeric 5UV8 structure used to generate the SASREF models.

Figure 5.

Loss of core fucosylation impairs IL3 signaling and its biological functions. A, Growth and IL3 signaling of Scramble, FUT8^KO, and CD123^KO TF-1 cells. The cell growth was measured by cell titer glow 3 days after IL3 treatment (left plot; n = 3), and the IL3 signaling was assessed by STAT5 phosphorylation 15 minutes after IL3 treatment (right plot; n = 3). B, Colony formation of Scramble, FUT8-g3, and IL3RA-g3 stem cells cultured in Methocult media for 10 days, supplied with IL3, SCF, and Flt3L (n = 7). C, IL3 signaling was reported by pSTAT5. CD34⁺ stem cells of different genotypes were treated by titrated dose of IL3 for 30 minutes, and pSTAT5 was measured by flow cytometry (n = 1). D, pDC differentiation of CD34⁺ stem cells. The proportion and absolute count of pDCs (CD303⁺CD123⁺) were measured at day 21 (n = 8). *, P < 0.05; **, P < 0.01; ***, P < 0.001; and n.s., not significant. For each plot, error bars represent SEM.

Figure 6.

FUT8^KO-induced resistance can be managed by dual CD3-bispecific antibody treatment. A and B, Mini-pool validation of MOLM13 (A) and SEM (B) cells in response to CD123-DART and CD33-BiTE/CD19-BiTE. Cells of different genotypes were spiked into the Scramble cells, all with different fluorescent labels (as shown in Supplementary Fig. S6A). The cell mixture was dosed with CD123-DART for the initial 7 days, followed by continued CD123-DART treatment or switched to CD33-BiTE (for MOLM13) or CD19-BiTE (for SEM) for another 10 days. The relative proportion of each genotype was tracked by flow cytometry along the time course (n = 4). C, RTCC assay with single or dual CD3-bispecific antibody. Scramble and FUT8^KO MOLM13 or SEM cells were treated with 10 pmol/L CD123-DART in combination with titrated doses of CD33-BiTE or CD19-BiTE, respectively. The killing was normalized to untreated groups (n = 4). The RTCC assay used T-cell:target cell ratio of 1:1, and target cell killing was measured 72 hours after treatment. D, In vivo efficacy study of CD123-DART. MOLM13 of different genotypes (Scramble/FUT8^KO/CD123^KO) was implanted, and the mice were treated with CD123-DART for 6 consecutive days. The tumor volume was measured on indicated days. E, Comparison of single-agent (CD123-DART) and dual-agent (CD123-DART/CD33-BiTE) bispecific antibody efficacy in vivo (left plot). The tumor growth in each individual mouse implanted with either Scramble or FUT8^KO tumors is presented in the right plots. T test was performed comparing FUT8^KO tumors with single- and dual-agent treatment. n = 5 for each group. *, P < 0.05 and **, P < 0.01. For each plot, error bars represent SEM.