Supplementary Table 1 from Molecular predictors of response to a humanized anti–insulin-like growth factor-I receptor monoclonal antibody in breast and colorectal cancer

Supplementary Table 2: Details of in vivo efficacy studies (# cells implanted; mouse strain, age and weight; tumor randomization volumes and IHC scores; % tumor growth inhibition and time to doubling versus vehicle).

Supplementary Figure 2: IHC validation of anti-mesothelin antibody SP74.

Supplemental Fig. 1: Characteristics of T cell activation and killing induced by HER2-TDB. Supplemental Fig 2: Activation of T cells by HER2-TDB induces T cell proliferation. Supplemental Figure 3. Activity of HER2-TDB in NOD-SCID mice is dependent on human PBMCs. Supplemental Figure 4. TDB mediated killing by CD3-TG splenic T cells. Supplemental Figure 5. Effect of 4D5 on established mammary tumors in MMTV-huHER2 transgenic mice. CD3-TG T cells express both mouse and human CD3 on approximately 50% level of respective Balb/c mouse or human T cells. Supplemental Figure 6. Anti-tumor activity of HER2-TDB is T cell dependent. Supplemental Figure 7. T cells in CT26-HER2 tumors display CD69 activation marker. Supplemental Figure 8. CT-26-HER2 tumor infiltrating T cells express PD-1. CT-26-HER2 tumor cells express PD-L1.

Commentary on this Article from Molecular predictors of response to a humanized anti–insulin-like growth factor-I receptor monoclonal antibody in breast and colorectal cancer

Supplementary Figure 4: Western blots of cell lines and xenografts used in Figure 5.

Supplementary Figures 1-7 from Molecular predictors of response to a humanized anti–insulin-like growth factor-I receptor monoclonal antibody in breast and colorectal cancer

Supplementary Figure 1: IHC validation of anti-mesothelin antibody 19C3.

Supplementary Table 1: Summary of 5B2 anti-mesothelin IHC literature and TMA details.

Supplementary Figures S1-4 and Tables S1-6. Supplementary Figure S1. LY6E is overexpressed in a subset of breast cancers Supplementary Figure S2. LY6E transcript expression in normal human tissues is shown. Supplementary Figure S3. Endocytosis of anti-LY6E antibody 9B12 Supplementary Figure S4. LY6E ADC activity in MAXF-1162, a HER2+/ T-DM1 resistant breast cancer model. Supplementary Table S1. Table lists the relative LY6E protein expression detected by IHC Supplementary Table S2. Anti-LY6E antibody 9B12 affinity to human and cynomolgus monkey LY6E. Supplementary Table S3. The correlation of LY6E copy number to anti-LY6E ADC killing in a panel of ovarian cancer cell lines is shown Supplementary Table S4. LY6E and GAPDH specific primer and probe sets used for transcript analysis and LY6E specific siRNAs are listed. Supplementary Table S5. Detailed percent tumor growth inhibition data for xenograft efficacy studies presented in Fig. 6 and in Supplementary Fig. S4B. Supplementary Table S6. Statistical analyses of differences between tumor growth in test groups versus vehicle control group

Supplemental ancillary methods (ELISA, surface plasmon resonance and humanization) and 4 supplemental figure legends.

Supplementary Figure 3: In vivo efficacy data from Figure 6 replotted as mean tumor volumes {plus minus} SEM.

Supplementary Figure Legends 1-3 from Armed Antibodies Targeting the Mucin Repeats of the Ovarian Cancer Antigen, MUC16, Are Highly Efficacious in Animal Tumor Models

The anti-FcRH5/CD3 T cell-dependent bispecific antibody (TDB) targets the B cell lineage marker FcRH5 expressed in multiple myeloma (MM) tumor cells. We demonstrate that TDBs trigger T cell receptor activation by inducing target clustering and exclusion of CD45 phosphatase from the synapse. The dimensions of the target molecule play a key role in the efficiency of the synapse formation. The anti-FcRH5/CD3 TDB kills human plasma cells and patient-derived myeloma cells at picomolar concentrations and results in complete depletion of B cells and bone marrow plasma cells in cynomolgus monkeys. These data demonstrate the potential for the anti-FcRH5/CD3 TDB, alone or in combination with inhibition of PD-1/PD-L1 signaling, in the treatment of MM and other B cell malignancies.

Bispecific antibodies that retarget cytotoxic T cell activity to kill cancer cells are currently under clinical evaluation. However, the molecular mechanism for how CD3-bispecific antibodies 'trigger' intracellular T cell signaling is not known. We demonstrate that bispecific antibodies invoke an equivalent biophysical mechanism of TCR triggering as that observed for the TCR/pMHC interaction, including target clustering and exclusion of CD45 phosphatase from the synapse. The dimensions of the target molecule play a key role in the efficiency of the synapse formation. However, we demonstrate that rational epitope selection can overcome the spatial inhibition caused by target molecules with a large extracellular domain and result in efficient synapse formation and highly potent T cell triggering. With this insight, we developed a novel T-cell dependent bispecific (TDB) antibody, anti-FcRH5/CD3 TDB, targeting the B cell lineage marker FcRH5 for multiple myeloma. Anti-FcRH5/CD3 TDB demonstrated cytotoxicity against human plasma cells and patient derived myeloma tumor cells at picomolar doses. Very low target expression level is sufficient to induce anti-FcRH5/CD3 TDB mediated killing, indicating broad activity in multiple myeloma where the prevalence of FcRH5 expression is 100%. In primates, anti-FcRH5/CD3 treatment resulted in complete depletion of tissue B cells and bone marrow plasma cells. Anti-FcRH5/CD3 TDB induces immunosuppressive feedback signaling, including PD1 up-regulation, which can be overcome by PD-L1 antibodies. These data demonstrate the potential for the anti-FcRH5/CD3 TDB, alone or in combination with inhibition of PD1/PDL1 signaling in the treatment of multiple myeloma and other B-cell malignancies.