CD3 T cell engagers (TCEs) have transformed hematologic oncology, but dose-liming toxicity and the absence of adequate costimulation have limited TCE success in solid tumors. Consequently, to date, only one classical TCE developed for solid tumors - tarlatamab - has been granted a marketing approval. Here, we report a pioneer combination strategy using a novel CD2-targeted costimulatory bispecific antibody to overcome these limitations. Building on a unique non-blocking CD2 antibody, we developed a HER2×CD2 proof-of-concept bispecific that, combined with an EpCAM×CD3 TCE, provides tumor-dependent costimulation and enhances anti-tumor cytotoxicity mediated by the TCE. We show that HER2×CD2 can be dosed independently to restore optimal anti-tumor cytotoxicity of a sub-efficacious low dose of the EpCAM×CD3 TCE, thus providing a route to avoid TCE-driven toxicity while maintaining efficacy. In a humanized xenograft model, co-treatment with HER2×CD2 achieved complete tumor remission in 8 of 9 mice at a TCE dose that otherwise mediated complete remission in only 1 of 9 mice. We show that HER2×CD2 compensates for the loss of CD58 expression by tumor cells - a well-documented tumor escape mechanism. Notably, unlike CD28-based costimulation, HER2×CD2 effectively also harnessed the anti-tumor cytotoxicity of CD28-negative CD8 T cells - a potent cytotoxic subset prevalent in elderly patients and dominant in solid tumors. Furthermore, HER2×CD2 induced markedly lower cytokine release than a HER2×CD28 bispecific while mediating comparable improvement in anti-tumor cytotoxicity. These findings establish our novel CD2-targeted costimulatory bispecific antibody approach as a promising and potentially safe way to expand and enhance TCE immunotherapy for solid tumors.

Supplementary Figure 4: Human SAR T cells are specifically stimulated by anti-human mesothelin x anti-EGFRv3 2 + 1 BiAb and redirect lysis to mesothelin+ target cells in vitro and in vivo

Supplemental Information. Includes Supplementary Methods section and legends for Supplemental Figures

Supplementary Figure 2. Demonstrates no activity of a control non-binding immunotoxin in vitro and in vivo

Fig S1. Description of DNA constructs Fig S2. Distribution of intra-tumoral injected trypan blue Fig S3. Combination of intra-tumoral immunotoxins with anti-CTLA-4 is well tolerated by mice. Fig S4. Anti-tumor effect of anti-CTLA-4 and LMB-100 depends on CD8+ cells. Fig S5. High dose of SS1P is needed for induction of complete remission. Fig S6A & B. Anti-tumor effect of an immunotoxin targeting human CD22 & (B) Combination of SS1P with anti-CTLA-4 lead to tumor regressionof 66C14 tumors not expressing MSLN. Fig S7. Intra-tumors injection of paclitaxel does not improve the anti-tumor activity of anti-CTLA-4. Table S1. Combination of RIT and anti-CTLA-4 induces long-term anti-tumor immunity.

Supplementary Figure 3. Graph showing growth of xenografted MKN28 gastric tumors following treatment with paclitaxel alone or in combination with RG7787 over a 60 day time course.

PDF file - 470K, Calculation of tumor growth inhibition. Tumor growth inhibition was calculated compared with respective controls using a standard formula. Surface plasmon resonance. Calculation of kinetic parameters ka (1/Ms), kd (1/s) and KD (M) of the antibody/receptor domain. Supplementary Table 1. Surface plasmon resonance - Kinetic data (ka, kd, and KD) for the binding of RG7116 and cetuximab to their respective antigen across the entire temperature gradient investigated. Supplementary Table 2. Surface plasmon resonance - Detailed analysis of the Van't Hoff and Eyring association/dissociation thermodynamic parameters for the binding of RG7116/HER3 and cetuximab/HER1. Supplementary Figure S1. Van't Hoff and Eyring association/dissociation plots showing the linear transformation of affinity (KD) and kinetic (ka and kd) parameters varying with reaction temperature for RG7116/HER3 and cetuximab/HER1. Structure of the HER3-ECD::Fab(RG7116) complex - Preparation and crystallization. Detailed methodology for the preparation and crystallization of the Fab fraction of RG7116 bound to HER3-ECD Structure of the HER3-ECD::Fab(RG7116) complex - Data collection, model building and refinement. Detailed methodology for the processing of X-ray diffraction data and solving of the Supplementary Table 3. Data collection and refinement statistics for the HER3-ECD::Fab(RG7116) complex. Supplementary Figure S2. Comparison of apo- and Fab(RG7116) bound HER3-ECD, 2Fo-Fc electron density composite omit map and electrostatic surface calculation of anti-HER3 binding epitope. Sensitivity of models investigated in vivo to therapeutic agents. Details of the common chemotherapeutic agents that the cell lines/tissue fragments used in this study were resistant to.

PDF file - 62K, Internalization of GA101 (black squares), rituximab (open diamonds), and ofatumumab (open triangles) upon binding to (A) SU-DHL4 cells and (B, C) whole blood derived from two CLL patients. Samples were incubated for 0.5, 2, 4, or 7 h in (A) and for 0.5, 1, 2, 3 and 5 h in (B, C) with Alexa Fluor (trademark) 488-labeled GA101, rituximab, or ofatumumab at 37{degree sign}C, washed and incubated in the presence or absence of anti-Alexa Fluor 488 for 30 min at 4{degree sign}C. The remaining fluorescence indicates the amount of labeled antibody that is not accessible to the quenching anti-Alexa Fluor 488 antibody and thus corresponds to internalized antibody. The average fluorescence intensity and standard deviations were calculated from duplicates of the experiment with SU-DHL4 cells. Due to low number of primary CLL samples the average fluorescence intensity in B and C correspond to single values.

PDF - 60K, Toxicity studies for RG7787 were performed in athymic nude mice. General health and weight was evaluated during therapy.

Supplementary Figure 3: In contrast to SAR T cells, CAR T cells rely on the release of perforin for efficient killing.

Supplementary Figure 5: Effect of pre-activated SAR T cells on hepatocellular carcinoma and glioblastoma cells


WMV file - 14044K, Time-lapse imaging of direct cell death induction in Z138 lymphoma cells treated with either rituximab, ofatumumab, or GA101. Images were taken over 5.5 h of treatment. Annexin V FLUOS (detects phosphatidylserine exposure, green) and PI (detects loss of membrane integrity, red).

PDF - 207K, A: Untreated KLM-1 tumors stain strongly for mesothelin. B-D: Mesothelin expression is not affected by treatment with RG7787, paclitaxel or both. IHC was done by Histoserv, Inc. according to a standard protocol. Representative pictures (10X) are shown.

PDF - 261K, Supplementary methods related to the IHC protocol and supplementary figure legends.

Supplementary Figure 4. Shows cell killing with combination paclitaxel and RG7787 in vitro

Supplementary Figure 1. Shows evaluation of MSLN expression in the TCGA breast cancer data set

PDF - 66K, A: SYTOX Dead Cell Stains allows to gate out viable cells from a representative dissociated KLM-1 tumor. B: In the subpopulation of viable cells, cell doublets are excluded. C: CD71+ cells (=human KLM-1 tumor cells) are gated out of the subpopulation of viable single cells. D: Based on the Alexa-647 background signal in the CD71+ cells of an untreated tumor (not shown), a gate is set and the percentage of Alexa-647+ cells in an RG7787-Alexa647-treated tumor is determined.

Supplementary Figure 2: Soluble and bound EGFR ligands do not trigger unwanted SAR T cell activation, while antibodies monovalent and bivalent for SAR can trigger T cell activation to the same extent.

PDF file - 307K, Whole-blood B-cell depletion mediated by GA101 (black squares), rituximab (open diamonds), and ofatumumab (open triangles) in lepirudin-treated (A, B) and heat-inactivated (HI) (C) whole-blood samples. GA101 mediated superior B-cell depletion in human whole-blood samples in all experimental conditions (A-C). Heat inactivation of human serum revealed that, in contrast to GA101, ofatumumab and rituximab rely more strongly on complement for efficient B-cell depletion (C). The average B-cell depletion and standard deviations were calculated from the triplicates of each experiment.

Supplementary Figure 1: Murine SAR expression and immunophenotyping of transduced T cells