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Supplemental Figure 1. Upregulated BHLHE41 in KPL-4 TR and BT-474M1 TR cells does not confer resistance to T-DM1. Supplemental Figure 2. Flow cytometry analysis of HER2 and MDR1 expression in KPL-4 P and TR cells, and tumor derived KPL-4 TR cells. Supplemental Figure 3. qRT-PCR analysis of ErbB receptors and ligands in KPL-4 and BT-474M1 P and TR cells. Supplemental Figure 4. Flow cytometry analysis of HER2 expression in BT-474M1 P and TR cells. Supplemental Figure 5. Immunofluorescence detection of cell surface HER2 on BT-474M1 and T-DM1 resistant cells. Supplemental Figure 6. Analysis of T-DM1 uptake in BT-474M1 P and TR, and KPL-4 P and TR cells using radiolabeled T-DM1. Supplemental Figure 7. Treatment of KPL-4 TR cells with anti-IGF-1R antibody 10H5 or the selective c-met inhibitor PHA665725 does not restore sensitivity to T-DM1. Supplemental Figure 8. Upregulated EGFR does not mediate resistance to T-DM1 in KPL-4 TR cells. Supplemental Figure 9. Potential autocrine receptor-ligand interactions for T-DM1 resistance. Supplemental Figure 10. Comparable shRNA knockdown of PTEN in BT-474M1 cells in 2 different shRNA clones, #1 and #3. Supplemental Figure 11. Molecular alterations in BT-474M1 TR cells that did not confer T-DM1 resistance. Supplemental Figure 12. Expression of drug resistance transporters, assessed by qRT-PCR, in KPL-4 and BT-474M1 parental and TR cells. Supplemental Figure 13. The MDR1 inhibitor XR9051 and the BCRP inhibitor Ko143 do not affect cell viability Supplemental Figure 14. MRP4 siRNA in BT-474M1 TR does not reverse T-DM1 resistance. Supplemental Figure 15. MRP4 siRNA in KPL-4 TR does not reverse T-DM1 resistance. Supplemental Figure 16. Expression of SLC46A3 in KPL-4 parental and TR cells assessed by qRT-PCR. Supplemental Figure 17. SLC46A3 siRNA in BT-474M1 TR cells significantly reverses T-DM1 resistance.

Supplementary Material and methods

Differentially expressed genes in T-DM1-resistant BT-474M1 cells with respect to the parental cell line (fold change > 2 fold and P < 0.05 of individual probes for a given gene in the microarray)

The development of targeted therapies that affect multiple signaling pathways and stimulate antitumor immunity is greatly needed. About 20% of patients with breast cancer overexpress HER2. Small molecules and antibodies targeting HER2 convey some survival benefits; however, patients with advanced disease succumb to the disease under these treatment regimens, possibly because HER2 is not completely necessary for the survival of the targeted cancer cells. In the present study, we show that a polyinosine/polycytosine (pIC) HER2-homing chemical vector induced the demise of HER2-overexpressing breast cancer cells, including trastuzumab-resistant cells. Targeting pIC to the tumor evoked a number of cell-killing mechanisms, as well as strong bystander effects. These bystander mechanisms included type I IFN induction, immune cell recruitment, and activation. The HER2-targeted pIC strongly inhibited the growth of HER2-overexpressing tumors in immunocompetent mice. The data presented here could open additional avenues in the treatment of HER2-positive breast cancer. Cancer Immunol Res; 4(8); 688–97. ©2016 AACR.

MEHD7945A affects the vasculature in tumor xenografts.

Supplementary Figure illustrating the difference in cytokine mRNA and protein expression following treatment of BT-474 with pIC/PPHAffibody and pIC/PPCys

Antibody-dependent cellular cytotoxicity (ADCC) is a key mechanism by which therapeutic antibodies mediate their antitumor effects. The absence of fucose on the heavy chain of the antibody increases the affinity between the antibody and FcγRIIIa, which results in increased in vitro and in vivo ADCC compared with the fucosylated form. However, the cellular and molecular mechanisms responsible for increased ADCC are unknown. Through a series of biochemical and cellular studies, we find that human natural killer (NK) cells stimulated with afucosylated antibody exhibit enhanced activation of proximal FcγRIIIa signaling and downstream pathways, as well as enhanced cytoskeletal rearrangement and degranulation, relative to stimulation with fucosylated antibody. Furthermore, analysis of the interaction between human NK cells and targets using a high-throughput microscope-based antibody-dependent cytotoxicity assay shows that afucosylated antibodies increase the number of NK cells capable of killing multiple targets and the rate with which targets are killed. We conclude that the increase in affinity between afucosylated antibodies and FcγRIIIa enhances activation of signaling molecules, promoting cytoskeletal rearrangement and degranulation, which, in turn, potentiates the cytotoxic characteristics of NK cells to increase efficiency of ADCC. Cancer Immunol Res; 3(2); 173–83. ©2014 AACR.

Kaplan Meier survival curves of UM-SCC6, SCC1483 and NCI-H226 xenografts treated with MEHD7945A in combination with radiation.

NK cells with high affinity FcgRIIIa also exhibit more protein tyrosine phosphorylation in response to afucosylated antibodies compared to fucosylated ones.

Approximates the amount of fucosylated antibody required to elicit similar protein tyrosine phosphorylation signaling compared to afucosylated antibodies.

BT-474M1 TR vs. Parental cells, gene expression changes >5-fold

MEHD7945A was more effective than anti-EGFR or anti-HER3 antibody in inhibiting cell signaling, cell proliferation and clonogenity.

The anti-tumor effects of MEHD7945A and single fraction radiation in NCI-H226 xenografts.

Differentially expressed genes in T-DM1-resistant KPL-4 cells with respect to the parental cell line (fold change > 2-fold and P < 0.05 of individual probes for a given gene in the microarray)

KPL-4 TR vs. Parental cells, gene expression changes > 5-fold

Human epidermal growth factor receptor family members (EGFR, HER2, HER3, and HER4) play important roles in tumorigenesis and response to cancer therapeutics. In this study, we evaluated the capacity of the dual-target antibody MEHD7945A that simultaneously targets EGFR and HER3 to modulate radiation response in lung and head and neck cancer models. Antitumor effects of MEHD7945A in combination with radiation were evaluated in cell culture and tumor xenograft models. Mechanisms that may contribute to increased radiation killing by MEHD7945A, including DNA damage and inhibition of EGFR–HER signaling pathways, were analyzed. Immunohistochemical analysis of tumor xenografts was conducted to evaluate the effect of MEHD7945A in combination with radiation on tumor growth and microenvironment. MEHD7945A inhibited basal and radiation-induced EGFR and HER3 activation resulting in the inhibition of tumor cell growth and enhanced radiosensitivity. MEHD7945A was more effective in augmenting radiation response than treatment with individual anti-EGFR or anti-HER3 antibodies. An increase in DNA double-strand breaks associated γ-H2AX was observed in cells receiving combined treatment with MEHD7945A and radiation. Immunohistochemical staining evaluation in human tumor xenografts showed that MEHD7945A combined with radiation significantly reduced the expression of markers of tumor proliferation and tumor vasculature. These findings reveal the capacity of MEHD7945A to augment radiation response in lung and head and neck cancers. The dual EGFR/HER3–targeting action of MEHD7945A merits further investigation and clinical trial evaluation as a radiation sensitizer in cancer therapy. Mol Cancer Ther; 14(9); 2049–59. ©2015 AACR.

Figures PDF file - 501K

MEHD7945A can inhibit activation of EGFR and HER3 in a dose-dependent manner.

Supplemental figure legends

Supplementary Figure 1 from Engineered Thio-Trastuzumab-DM1 Conjugate with an Improved Therapeutic Index to Target Human Epidermal Growth Factor Receptor 2–Positive Breast Cancer

Related Article from Toward an Effective Targeted Chemotherapy for Multiple Myeloma

Purpose: Antibody drug conjugates (ADCs) combine the ideal properties of both antibodies and cytotoxic drugs by targeting potent drugs to the antigen-expressing tumor cells, thereby enhancing their antitumor activity. Successful ADC development for a given target antigen depends on optimization of antibody selection, linker stability, cytotoxic drug potency, and mode of linker-drug conjugation to the antibody. Here, we systematically examined the in vitro potency as well as in vivo preclinical efficacy and safety profiles of a heterogeneous preparation of conventional trastuzumab-mcc-DM1 (TMAb-mcc-DM1) ADC with that of a homogeneous engineered thio-trastuzumab-mpeo-DM1 (thioTMAb-mpeo-DM1) conjugate.Experimental Design and Results: To generate thioTMAb-mpeo-DM1, one drug maytansinoid 1 (DM1) molecule was conjugated to an engineered cysteine residue at Ala114 (Kabat numbering) on each trastuzumab-heavy chain, resulting in two DM1 molecules per antibody. ThioTMAb-mpeo-DM1 retained similar in vitro anti–cell proliferation activity and human epidermal growth factor receptor 2 (HER2) binding properties to that of the conventional ADC. Furthermore, it showed improved efficacy over the conventional ADC at DM1-equivalent doses (μg/m2) and retained efficacy at equivalent antibody doses (mg/kg). An improved safety profile of >2-fold was observed in a short-term target-independent rat safety study. In cynomolgus monkey safety studies, thioTMAb-mpeo-DM1 was tolerated at higher antibody doses (up to 48 mg/kg or 6,000 μg DM1/m2) compared with the conventional ADC that had dose-limiting toxicity at 30 mg/kg (6,000 μg DM1/m2).Conclusions: The engineered thioTMAb-mpeo-DM1 with broadened therapeutic index represents a promising antibody drug conjugate for future clinical development of HER2-positive targeted breast cancer therapies. Clin Cancer Res; 16(19); 4769–78. ©2010 AACR.

Purpose: Oncogenic activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is prevalent in breast cancer and has been associated with resistance to HER2 inhibitors in the clinic. We therefore investigated the combinatorial activity of GDC-0941, a novel class I PI3K inhibitor, with standard-of-care therapies for HER2-amplified breast cancer.Experimental Design: Three-dimensional laminin-rich extracellular matrix cultures of human breast cancer cells were utilized to provide a physiologically relevant approach to analyze the efficacy and molecular mechanism of combination therapies ex vivo. Combination studies were done using GDC-0941 with trastuzumab (Herceptin), pertuzumab, lapatinib (Tykerb), and docetaxel, the principal therapeutic agents that are either approved or being evaluated for treatment of early HER2-positive breast cancer.Results: Significant GDC-0941 activity (EC50 70% of breast cancer cell lines that were examined in three-dimensional laminin-rich extracellular matrix culture. Differential responsiveness to GDC-0941 as a single agent was observed for luminal breast cancer cells upon stimulation with the HER3 ligand, heregulin. Combined treatment of GDC-0941, trastuzumab, and pertuzumab resulted in growth inhibition, altered acinar morphology, and suppression of AKT mitogen-activated protein kinase (MAPK) / extracellular signed-regulated kinase (ERK) kinase and MEK effector signaling pathways for HER2-amplified cells in both normal and heregulin-supplemented media. The GDC-0941 and lapatinib combination further showed that inhibition of HER2 activity was essential for maximum combinatorial efficacy. PI3K inhibition also rendered HER2-amplified BT-474M1 cells and tumor xenografts more sensitive to docetaxel.Conclusions: GDC-0941 is efficacious in preclinical models of breast cancer. The addition of GDC-0941 to HER2-directed treatment could augment clinical benefit in breast cancer patients.

Supplementary Figure 5: Control spectrum obtained via quantitative LC-MS for the anti-HER2-VC(S)-MMAE ADC after 3 hours with in buffer with no proteolytic solution.

Antibody-drug conjugates (ADCs), cytotoxic drugs chemically linked to antibodies, provide a means to increase the effectiveness of chemotherapy by targeting the drug to neoplastic cells. Anti-CD138 ADCs show promising preclinical efficacy for the treatment of multiple myeloma; however, the safety aspects of targeting CD138 have yet to be explored.

Purpose: Trastuzumab-emtansine (T-DM1) is an antibody–drug conjugate (ADC) comprising the cytotoxic agent DM1 conjugated to trastuzumab with a stable linker. Thrombocytopenia was the dose-limiting toxicity in the phase I study, and grade ≥3 thrombocytopenia occurred in up to 13% of patients receiving T-DM1 in phase III studies. We investigated the mechanism of T-DM1–induced thrombocytopenia.Experimental Design: The effect of T-DM1 on platelet function was measured by aggregometry, and by flow cytometry to detect the markers of activation. The effect of T-DM1 on differentiation and maturation of megakaryocytes (MK) from human hematopoietic stem cells was assessed by flow cytometry and microscopy. Binding, uptake, and catabolism of T-DM1 in MKs, were assessed by various techniques including fluorescence microscopy, scintigraphy to detect T-[H3]-DM1 and 125I-T-DM1, and mass spectrometry. The role of FcγRIIa was assessed using blocking antibodies and mutant constructs of trastuzumab that do not bind FcγR.Results: T-DM1 had no direct effect on platelet activation and aggregation, but it did markedly inhibit MK differentiation via a cytotoxic effect. Inhibition occurred with DM1-containing ADCs but not with trastuzumab demonstrating a role for DM1. MKs internalized these ADCs in a HER2-independent, FcγRIIa-dependent manner, resulting in intracellular release of DM1. Binding and internalization of T-DM1 diminished as MKs matured; however, prolonged exposure of mature MKs to T-DM1 resulted in a disrupted cytoskeletal structure.Conclusions: These data support the hypothesis that T-DM1–induced thrombocytopenia is mediated in large part by DM1-induced impairment of MK differentiation, with a less pronounced effect on mature MKs. Clin Cancer Res; 21(1); 123–33. ©2014 AACR.

Supplementary Methods

CCR Translation for This Article from Dual Targeting of HER2-Positive Cancer with Trastuzumab Emtansine and Pertuzumab: Critical Role for Neuregulin Blockade in Antitumor Response to Combination Therapy

PDF file 288K, Supp. Table 1: TDM4373g Patient Demographics and Baseline Characteristics; Supp. Table 2: TDM4373g Prior Therapies; Supp. Table 3: TDM4373g Adverse Events

Antibody–drug conjugates (ADC) are designed to selectively bind to tumor antigens via the antibody and release their cytotoxic payload upon internalization. Controllable payload release through judicious design of the linker has been an early technological milestone. Here, we examine the effect of the protease-cleavable valine-citrulline [VC(S)] linker on ADC efficacy. The VC(S) linker was designed to be cleaved by cathepsin B, a lysosomal cysteine protease. Surprisingly, suppression of cathepsin B expression via CRISPR-Cas9 gene deletion or shRNA knockdown had no effect on the efficacy of ADCs with VC(S) linkers armed with a monomethyl auristatin E (MMAE) payload. Mass spectrometry studies of payload release suggested that other cysteine cathepsins can cleave the VC(S) linker. Also, ADCs with a nonprotease-cleavable enantiomer, the VC(R) isomer, mediated effective cell killing with a cysteine-VC(R)-MMAE catabolite generated by lysosomal catabolism. Based on these observations, we altered the payload to a pyrrolo[2,1-c][1,4]benzodiazepine dimer (PBD) conjugate that requires linker cleavage in order to bind its DNA target. Unlike the VC-MMAE ADCs, the VC(S)-PBD ADC is at least 20-fold more cytotoxic than the VC(R)-PBD ADC. Our findings reveal that the VC(S) linker has multiple paths to produce active catabolites and that antibody and intracellular targets are more critical to ADC efficacy. These results suggest that protease-cleavable linkers are unlikely to increase the therapeutic index of ADCs and that resistance based on linker processing is improbable. Cancer Res; 77(24); 7027–37. ©2017 AACR.

Supplementary Methods, Figure Legends 1-3, Tables 1A-B from Engineered Thio-Trastuzumab-DM1 Conjugate with an Improved Therapeutic Index to Target Human Epidermal Growth Factor Receptor 2–Positive Breast Cancer

Supplementary Figure 3C-D from Engineered Thio-Trastuzumab-DM1 Conjugate with an Improved Therapeutic Index to Target Human Epidermal Growth Factor Receptor 2–Positive Breast Cancer

Supplementary Figure 2 from Engineered Thio-Trastuzumab-DM1 Conjugate with an Improved Therapeutic Index to Target Human Epidermal Growth Factor Receptor 2–Positive Breast Cancer

PDF file 67K, TDM4373g Phase Ib Clinical Study Design

Supplementary Figure 3A-B from Engineered Thio-Trastuzumab-DM1 Conjugate with an Improved Therapeutic Index to Target Human Epidermal Growth Factor Receptor 2–Positive Breast Cancer

Supplementary Data from Suppression of HER2/HER3-Mediated Growth of Breast Cancer Cells with Combinations of GDC-0941 PI3K Inhibitor, Trastuzumab, and Pertuzumab

PDF file 238K, Kaplan-Meier plots of time-to-tumor doubling in Calu-3 (A) and KPL-4 (B) xenograft models after treatment with T-DM1 and pertuzumab

Purpose: The mechanisms by which trastuzumab imparts clinical benefit remain incompletely understood. Antibody-dependent cellular cytotoxicity via interactions with Fcγ receptors (FcγR) on leukocytes may contribute to its antitumor effects. Single-nucleotide polymorphisms (SNP) in FCGR3A and FCGR2A genes lead to amino acid substitutions at positions 158 and 131, respectively, and affect binding of antibodies to FcγR such that 158V/V and 131H/H bind with highest affinity. This study aimed to determine whether high-affinity SNPs are associated with disease-free survival (DFS) among patients with HER2-positive nonmetastatic breast cancer.Experimental Design: Genomic DNA was isolated from 1,286 patients enrolled in a trial of adjuvant trastuzumab-based chemotherapy. Genotyping was conducted using Sanger sequencing and Sequenom mass spectrometry.Results: Patient samples (N = 1,189) were successfully genotyped for FCGR3A and 1,218 for FCGR2A. Compared with the overall results of the BCIRG006 study, in the subset of patients genotyped in this analysis, a less robust improvement in DFS was observed for the trastuzumab arms than control arm (HR, 0.842; P = 0.1925). When stratified for prognostic features, the HR in favor of trastuzumab was consistent with that of the overall study (HR, 0.74; P = 0.036). No correlation between DFS and FCGR3A/2A genotypes was seen for trastuzumab-treated patients (158V/V vs. V/F vs. F/F, P = 0.98; 131H/H vs. H/R vs. R/R, P = 0.76; 158V/V and/or 131H/H vs. others, P = 0.67).Conclusion: This analysis evaluating the association between FCGR3A/2A genotypes and trastuzumab efficacy in HER2-positive breast cancer did not show a correlation between FCGR3A-V/F and FCGR2A-H/R SNPs and DFS in patients treated with trastuzumab. Clin Cancer Res; 18(12); 3478–86. ©2012 AACR.

PDF file 12K, Kaplan-Meier plot of time-to-tumor doubling in the MDA-175 breast cancer xenograft model after treatment with T-DM1 and pertuzumab

Supplementary Methods, Figures S1-S7 Suppl. Fig. S1, Differentiation of hematopoietic stem cells (CD133+/CD34+) into immature megakarytocytes Suppl. Fig. S2, T-DM1 does not directly induce platelet aggregation in washed platelets Suppl. Fig. S3, At high concentrations, DM1 inhibits agonist-induced platelet aggregation in platelet-rich plasma Suppl. Fig. S4, DM1 conjugates alter the morphology of megakaryocytes Suppl. Fig. S5, Uptake of T-[3H]DM1 varies by differentiation stage Suppl. Fig. S6, HER2 is not expressed on megakaryocytes or platelets Suppl. Fig. S7, Prolonged exposure to T-DM1 results in disruption of cytoskeletal structure in maturing megakaryocytes

PDF file - 507K, Supplemental Table 1A: PCR and Sanger Sequencing Primers Supplemental Table 1B: PCR and Sequenom Genotyping Primers Supplemental Figure 1. Kaplan-Meier estimates of disease free survival for BCIRG 006 study, entire population, third planned analysis. (N=3222) Supplemental Table 2. Patient Characteristics: Genotyped vs. Non-genotyped Supplemental Figure 2. Kaplan-Meier estimates of disease free survival in subset of patients enrolled in BCIRG006 (third planned analysis) who were genotyped for this study (N=1,286) Supplemental Figure 3. Kaplan-Meier estimates of disease free survival in subset of patients enrolled in BCIRG006 (third planned analysis) who were genotyped for this study with stratification for major prognostic factors Supplemental Table 3. Patients Characteristics by Genotype (Advanced Disease Cohort) Supplemental Table 4. Genotype and Allele Frequencies of the FCGR2A and FCGR3A Polymorphisms Supplemental Figure 4. Kaplan-Meier estimates of disease free survival in trastuzumab treated patients within the adjuvant cohort according to FcGammaR polymorphisms, broken down by treatment arm Supplemental Figure 5. Kaplan-Meier estimates of disease free survival in patients treated with chemotherapy alone (no trastuzumab) in the adjuvant cohort according to FcGammaR polymorphisms, broken down by treatment arm. Supplemental Figure 6. Kaplan-Meier estimates of overall survival (OS) in trastuzumab treated patients within the adjuvant cohort according to FcGammaR polymorphisms. Supplemental Figure 7. Kaplan-Meier estimates of progression free survival (PFS) in trastuzumab treated patients within the advanced disease cohort according to FcGammaR polymorphisms.

Purpose: Targeting HER2 with multiple HER2-directed therapies represents a promising area of treatment for HER2-positive cancers. We investigated combining the HER2-directed antibody–drug conjugate trastuzumab emtansine (T-DM1) with the HER2 dimerization inhibitor pertuzumab (Perjeta).Experimental Design: Drug combination studies with T-DM1 and pertuzumab were performed on cultured tumor cells and in mouse xenograft models of HER2-amplified cancer. In patients with HER2-positive locally advanced or metastatic breast cancer (mBC), T-DM1 was dose-escalated with a fixed standard pertuzumab dose in a 3+3 phase Ib/II study design.Results: Treatment of HER2-overexpressing tumor cells in vitro with T-DM1 plus pertuzumab resulted in synergistic inhibition of cell proliferation and induction of apoptotic cell death. The presence of the HER3 ligand, heregulin (NRG-1β), reduced the cytotoxic activity of T-DM1 in a subset of breast cancer lines; this effect was reversed by the addition of pertuzumab. Results from mouse xenograft models showed enhanced antitumor efficacy with T-DM1 and pertuzumab resulting from the unique antitumor activities of each agent. In patients with mBC previously treated with trastuzumab, lapatinib, and chemotherapy, T-DM1 could be dosed at the maximum tolerated dose (MTD; 3.6 mg/kg every 3 weeks) with standard dose pertuzumab. Adverse events were mostly grade 1 and 2, with indications of clinical activity.Conclusions: Dual targeting of HER2 with the combination of T-DM1 and pertuzumab in cell culture and mouse xenograft models resulted in enhanced antitumor activity. In patients, this combination showed an encouraging safety and tolerability profile with preliminary evidence of efficacy. Clin Cancer Res; 20(2); 456–68. ©2013 AACR.

PDF file 86K, Supplementary Methods and figure legends

PDF file 64K, NRG protects ZR-75-30, but not HCC1954, breast cancer cells from T-DM1 induced apoptosis

PDF file 158K, NRG protects BT-474, but not KPL-4, breast cancer cells from cytotoxicity induced by anti-mitotic agents DM1, docetaxel and vinorelbine

PDF file - 51KB, MEHD7945A increases cell fraction in apoptotic sub-G0 phase.

Supplementary Figure 6 from A Central Role for HER3 in HER2-Amplified Breast Cancer: Implications for Targeted Therapy

Supplementary Legends 1-6 from A Central Role for HER3 in HER2-Amplified Breast Cancer: Implications for Targeted Therapy