SC134-TCB Targeting Fucosyl-GM1, a T Cell-Engaging Antibody with Potent Antitumor Activity in Preclinical Small Cell Lung Cancer Models

Abstract

Small cell lung cancer (SCLC) is an aggressive disease with limited treatment options. Fucosyl-GM1 (FucGM1) is a glycolipid overexpressed in the majority of SCLC tumors but virtually absent from normal healthy tissues. In this study, we validate a FucGM1-targeting T cell-redirecting bispecific (TCB) antibody for the treatment of SCLC. More than 80% of patient-derived xenograft tissues of SCLC expressed FucGM1, whereas only three normal human tissues: pituitary, thymus, and skin expressed low and focal FucGM1. A FucGM1-targeting TCB (SC134-TCB), based on the Fc-silenced humanized SC134 antibody, exhibited nanomolar efficiency in FucGM1 glycolipid and SCLC cell surface binding. SC134-TCB showed potent ex vivo killing of SCLC cell lines with donor-dependent EC50 ranging from 7.2 pmol/L up to 211.0 pmol/L, effectively activating T cells, with picomolar efficiency, coinciding with target-dependent cytokine production such as IFNγ, IL2, and TNFα and robust proliferation of both CD4 and CD8 T cells. The ex vivo SC134-TCB tumor controlling activity translated into an effective in vivo anti-DMS79 tumor therapy, resulting in 100% tumor-free survival in a human peripheral blood mononuclear cell admixed setting and 40% overall survival (55% tumor growth inhibition) with systemically administered human peripheral blood mononuclear cells. Combination treatment with atezolizumab further enhanced survival and tumor growth inhibition (up to 73%). A 10-fold SC134-TCB dose reduction maintained the strong in vivo antitumor impact, translating into 70% overall survival (P < 0.0001). Whole-blood incubation with SC134-TCB, as well as healthy human primary cells analysis, revealed no target-independent cytokine production. SC134-TCB presents an attractive candidate to deliver an effective immunotherapy treatment option for patients with SCLC.

Figure 1.

SC134 specifically binds FucGM1, a SCLC selective target. Glycolipid binding specificity by hybridoma-derived SC134. A, ELISA analysis of FucGM1-binding specificity of SC134. FucGM1 binding is observed, in contrast to lack of binding to closely related gangliosides. Positive control antibody reactivity for the respective gangliosides is shown in Supplementary Fig. S1A. B, Immuno-TLC analysis of SC134 showing reactivity toward DMS79 lipid extract as well as purified FucGM1, combined with lack of GM1 reactivity. C, SC134-binding efficiency for FucGM1 on ELISA. D, SC134 cell surface–binding efficiency on a range of SCLC cell lines. h134 characterization: (E) FucGM1 lipid binding compared with GM1 by ch134 and h134 on ELISA. F, SCLC cell line binding by h134. MFI assessed by flow cytometry. FucGM1 distribution in SCLC tumor tissues: (G) IHC FucGM1 expression analysis using h134 (huIgG1) of frozen patient-derived xenograft SCLC tissues. H, FucGM1 expression by DMS79 in culture. I, FucGM1 expression by DMS79 after growing in NSG mice. J, Restricted FucGM1 expression in frozen normal healthy human tissues, immunostained with h134 (J). The pituitary (a), skin (b), and thymus (c) were the only positive tissues from the 30 normal healthy tissues analyzed. The full arrays are shown in Supplementary Fig. S1D. ch134, chimeric (human IgG1) SC134; MFI, median fluorescence intensity; TLC, thin-layer chromatography.

Figure 2.

SC134-TCB effectively binds SCLC cell lines and T cells. A, Schematic drawing of the SC134-TCB format. Targeting regions of h134 in combination with scFv of huOKT3 were used. Stars denote “short tandem repeat” modification for Fc silencing. SCLC cell binding by SC134-TCB: (B) DMS79 cell binding of SC134-TCB and parental h134. C, Histograms of top concentration (50 nmol/L) SC134-TCB binding (black) to three target-positive cell lines DMS79, DMS153, and H740 compared with secondary antibody–only control (gray). D, Pan–T-cell binding of B12-TCB, SC134-TCB, and parental anti-CD3 (huOKT3). MFI assessed via flow cytometry. MFI, median fluorescence intensity. (A, Created with BioRender.com.)

Figure 3.

Potent T cell–mediated tumor cell killing by SC134-TCB. SC134-TCB induced tumor cell killing. A, Cytotoxicity EC₅₀ via LDH release and flow-based analysis in pan-T:DMS79 cocultures. B, Percentage maximum target cell death of DMS79 with SC134-TCB and isotype control B12-TCB at 10 nmol/L. C, Increased killing by SC134-TCB (LDH release) at higher E:T ratios. Open symbols denote background killing by isotype control B12-TCB. D, LDH release from two target cell lines: high-expressor DMS79 and moderate-expressor DMS153. SC134-TCB induced T-cell activation. E, EC₅₀ via CD69 staining shown for DMS79 and DMS153. F, Percentage maximum activation with SC134-TCB and isotype control B12-TCB at 10 nmol/L. G, Activation of CD4 and CD8 T-cell subsets from one effector donor. H, SC134-TCB induced CD4 and CD8 T-cell proliferation (day 3) determined by Ki67 staining. Representative donor shown. I, Target-dependent multifunctional cytokine production by SC134-TCB in DMS79 cocultures with enriched T cells. T cells used at an E:T of 5:1, for all assays. Median or representative data shown for three or more human donors.

Figure 4.

Effective tumor control by SC134-TCB. A, Significant tumor-free survival in admixed antitumor study dosed with 100 μg SC134-TCB for 3 weeks. PBMC implanted with DMS79 cells at 1:1 ratio and a further two doses of PBMCs given on days 8 and 15. Log-rank Mantel–Cox P < 0.0001. Significant DMS79 tumor control by SC134-TCB treatment, with PBMCs dosed on days 3 and 18 (dotted lines), after tumor inoculation, at 5:1 ratio. Gray arrows show biweekly dosing of SC134-TCB (100 μg): (B) tumor volume (individual growth curves, shown in Supplementary Fig. S9A). Significance, mixed-effects model P = 0.013 and (C) OS, log-rank Mantel–Cox P = 0.0042, shown. D, Phenotyping data assessing the percentage of exhausted T cells (PD1⁺TIM3⁺) in terminal tumor samples. T cells were gated based on combined CD4 and CD8 staining (gating strategy depicted in Supplementary Fig. S9B). E, SC134-TCB detection in tumors compared with spleens in the SC134-TCB–dosed group (i.v.), paired t test, P = 0.0069. Each point represents an individual mouse. SC134-TCB–mediated T-cell infiltration and activation coincide with significant antitumor impact. F, Significant tumor control by SC134 with single PBMC administration (day 8) combined with SC134-TCB (100 μg at days 9, 11, and 15). Significance, two-way ANOVA, P = 0.0249; individual growth curves are shown in Supplementary Fig. S9C. G, Significantly increased T-cell presence and T-cell activation: (H) PD1⁺ and (I) CXCR3⁺ in tumors of SC134-TCB–treated mice. The presence of individual populations is evident from the IHC analysis of terminal tumor samples (Supplementary Fig. S9D).

Figure 5.

PDL1 inhibition and dose modulation further improve SC134-TCB antitumor impact. SC134-TCB treatment induces PDL1 expression. A, Dose-dependent PDL1 expression in ex vivo 48-hour cocultures (DMS79: huPBMCs at 5:1). B,In vivo SC134-TCB–induced PDL1 expression on DMS79 cells (at termination) from NSG antitumor study, Mann–Whitney test, P = 0.0220. Antitumor study investigating the efficacy of SC134-TCB on implanted DMS79 tumor in NSG mice. DMS79 cells were implanted on day 1 and PBMCs dosed on days 8 and 22 at an E:T of 5:1. Gray arrows show biweekly administration of SC134-TCB (100 μg); upward ticks show atezolizumab dosing (200 μg). C, Tumor volume analysis, mixed-effects model P < 0.0001. Individual growth curves shown in Supplementary Fig. S10; D, OS analysis, log-rank Mantel–Cox P < 0.01. E, Phenotyping data assessing the percentage of live T cells (Tils) and (F) the percentage PD1/TIM3 double-positive Tils in terminal tumor samples, Mann–Whitney test, P = 0.0142. Antitumor study investigating the efficacy of reduced dose or reduced dose frequency of SC134-TCB on implanted DMS79 tumor in NSG mice. DMS79 cells were implanted on day 1 and PBMCs dosed on days 7 and 21 at an E:T of 5:1. SC134-TCB dosing either biweekly (six doses of 10 or 100 μg) or weekly (three doses of 100 μg). G, Tumor volume analysis, two-way ANOVA, P = 0.0004. Individual growth curves shown in Supplementary Fig. S11. H, OS, log-rank Mantel–Cox, P < 0.0001. Atez, atezolizumab.

Figure 6.

T cell–mediated cytokine production by SC134-TCB. A, Serum cytokine concentration after incubation of whole blood with SC134-TCB or huOKT3 for 5 and 24 hours from one representative donor of four analyzed. B, Cytokine detection in mouse sera 24 hours after one 100 μg SC134-TCB dose (DMS79 inoculation 8 days prior to pan-T and TCB dosing). Primary cell analysis of SC134-TCB–mediated T-cell activation. C, Reactivity measured by IFNγ ELISpot with normal primary human cells derived from the thymus and skin or (D) from cardiac and gastric tissues. DMS79 and PBMC only used as positive and negative controls, respectively. Representative data shown for two healthy PBMC donors.