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Supplementary Methods, Figures S1-S11, and Table Legends. Supplementary Methods: description of methods used in the Supplementary figures, expanded description of ELISA assays, and description of the computational model of multiple EGFR ligand antagonists. Supplementary Figure S1: Monoclonal EGFR antibodies poorly block signal amplification by high-affinity EGFR ligands. Supplementary Figure S2: Hierarchical clustering of EGFR ligand expression in primary colon, head and neck, and lung tumors. Supplementary Figure S3: Trends observed in EGFR ligand expression in TCGA data set are similarly apparent in primary colorectal samples analyzed by RT-qPCR. Supplementary Figure S4: Relative expression of EGFR ligands varies across cancer indications. Supplementary Figure S5: Simulated activity of a combination of two ligand antagonists versus single antagonists as a function of ligand affinity. Supplementary Figure S6: MM-151 antibodies have non-overlapping epitopes and can simultaneously engage the same EGF receptor molecule. Supplementary Figure S7: MM-151 elicits superior ligand antagonism. Supplementary Figure S8: MM-151 promotes potent down-regulation of EGFR that is dependent upon the oligoclonal combination of bivalent antibodies. Supplementary Figure S9: MM-151 promotes both antibody-dependent cell-mediated (ADCC) and complement-dependent (CDC) cytotoxicity. Supplementary Figure S10: Cell proliferation stimulated by high-affinity ligands is partially or completely resistant to cetuximab treatment but still sensitive to MM-151 treatment. Supplementary Figure S11: MM-151 overcomes signal amplification driven by high-affinity ligand. Legends for Supplementary Tables S1-S8.

Supplementary Tables S2, S3, S4. Structure of the computational model of multiple ligand antagonists: species (Table S2), parameters (Table S3), and Reactions (Table S4).

Supplementary Table S1. Cell lines and cell culture conditions

Supplementary Tables S5, S6, S7, S8. mRNA expression data for EGFR ligands as measured by RNAseq from The Cancer Genome Atlas (TCGA) across 14 cancer indications or by RT-qPCR of colorectal cancer FFPE samples. Table S5: Summary of TCGA datasets included in the analysis. Table S6: Raw and normalized expression data from TCGA. Table S7: EGFR ligand expression measured by RT-qPCR on commercially-obtained colorectal cancer FFPE samples. Table S8: Pairwise correlations of EGFR ligands across four TCGA indications.

Supplementary Methods, Figures 1-8, Table 1 from An ErbB3 Antibody, MM-121, Is Active in Cancers with Ligand-Dependent Activation

Extracellular vesicles (EVs) are an important intercellular communication system facilitating the transfer of macromolecules between cells. Delivery of exogenous cargo tethered to the EV surface or packaged inside the lumen are key strategies for generating therapeutic EVs. We identified two "scaffold" proteins, PTGFRN and BASP1, that are preferentially sorted into EVs and enable high-density surface display and luminal loading of a wide range of molecules, including cytokines, antibody fragments, RNA binding proteins, vaccine antigens, Cas9, and members of the TNF superfamily. Molecules were loaded into EVs at high density and exhibited potent in vitro activity when fused to full-length or truncated forms of PTGFRN or BASP1. Furthermore, these engineered EVs retained pharmacodynamic activity in a variety of animal models. This engineering platform provides a simple approach to functionalize EVs with topologically diverse macromolecules and represents a significant advance toward unlocking the therapeutic potential of EVs.

BackgroundTumor associated macrophages (TAMs) play a critical role in tumor immunosuppression and resistance to immune checkpoint blockade. Reprogramming ‘M2’ TAMs to a proinflammatory ‘M1’ phenotype by selectively silencing M2 phenotype-driving transcription factors, such as STAT6, is a promising strategy to relieve TAM-induced immunosuppression. We have developed exoASO-STAT6™, an investigational therapeutic candidate consisting of exosomes loaded with antisense oligonucleotides (ASOs) targeting STAT6. By leveraging the TAM tropism of exosomes, exoASO-STAT6™ is the first systemically administered exosome designed to selectively silence STAT6 in TAMs. Preclinical biodistribution studies demonstrated that the liver is the main organ targeted by exoASO after intravenous (IV) dosing.MethodsWe evaluated the translational potential of exoASO-STAT6 to treat hepatocellular carcinoma through pharmacokinetics (PK), pharmacodynamic (PD) and anti-tumoral efficacy studies in preclinical models, as well as computational analysis of human HCC datasets.ResultsPK/PD were evaluated in naïve mice dosed IV with a single dose of exoASO-STAT6. PK analysis showed that the STAT6-ASO is rapidly cleared from plasma. Retention of the ASO in liver was dose-dependent and observed for at least 21 days. exoASO-STAT6 induced significant Stat6 mRNA knockdown (KD) in liver tissue with maximum KD at day 7 (70% KD at the 30 ug dose). IV administration of exoASO-STAT6 in an orthotopic, CPI resistant HCC model attenuated tumor growth and induced complete remission of tumor lesions in 50% of mice, while combination therapy with anti-PD1 antibodies further enhanced anti-tumor activity (75% complete remissions). Gene expression and histological analysis of the liver showed effective remodeling of the tumor microenvironment including a significant increase in interferon and cytotoxic T-cell gene signatures and iNOS expression. PD studies were also performed in cynomolgus monkeys that demonstrated a dose-dependent and durable silencing of STAT6 mRNA (50% and 31% at 1- and 3- weeks post-dose, respectively). STAT6 knockdown correlated with a reduction in STAT6 target genes, IL4R and EGR2, confirming modulation of the STAT6 pathway. Finally, we identified a STAT6 macrophage transcriptional signature and show high expression in human HCC tumors, both in immune cell-rich and TAM-rich/CD8 T-cell low tumors that correlates with worse survival.ConclusionsIn summary, we demonstrate that exoASO-STAT6 has a durable PK/PD profile in the liver of several species and potent antitumoral efficacy in a preclinical model of HCC. Furthermore, we identify an inverse correlation between the STAT6 macrophage signature and survival in human HCC tumors. Altogether our data support the systemic administration of exoASO-STAT6 as a promising therapy for liver cancer.Ethics ApprovalFor MiceMice were maintained and treated at the animal care facility of Codiak Biosciences in accordance with the regulations and guidelines of the Institutional Animal Care and Use Committee (CB2017-001).Animal housing and experimental procedures (mice) were conducted according to the French and European Regulations and the National Research Council Guide for the Care and Use of Laboratory Animals and Institutional Animal Care and Use Committee of Oncodesign (Oncomet) approved by French authorities (CNREEA agreement N° 91).For cynomolgus monkeys:All animals were maintained and treated at the animal care facility of Altasciences in compliance with the Animal Welfare Act and recommendations set forth in the Guide for the Care and Use of Laboratory Animals (National Research Council 2011).

Background: Tumor-associated macrophages (TAMs) and myeloid derived suppressor cells (MDSCs) promote an immunosuppressive milieu by inhibiting T-cell activation and recruitment, leading to resistance to immune checkpoint therapies. Interleukin 10 (IL10) and prostaglandin E2 (PGE2) promote tumor initiation and spread, in part, through TAM polarization and MDSC recruitment. C/EBPβ is a critical transcription factor that regulates the immunosuppressive state of TAMs and MDSCs and is activated by IL10 and PGE2 signaling. We developed a proprietary engineered exosome loaded with antisense oligonucleotides (ASOs) targeting C/EBPβ (exoASO-C/EBPβ), that selectively delivers ASO to TAMs and MDSCs, thereby inhibiting C/EBPβ expression and inducing reprogramming from an immunosuppressive (M2) to a pro-inflammatory (M1) phenotype. Results: exoASO-C/EBPβ demonstrated dose-dependent target gene knockdown (KD) in primary human M2 macrophages in vitro with a 2-fold higher potency (IC50) compared to free ASO. C/EBPβ KD resulted in profound changes in gene expression and cytokine secretion profile consistent with reprogramming to a M1 phenotype. exoASO-C/EBPβ induced a significant induction of pro-inflammatory cytokines, in M2 macrophages generated with IL10 or PGE2, both of which show prominent activation of the C/EBPβ pathway. Biodistribution in tumor-bearing mice (IT and IV) demonstrate selective uptake by MDSCs and TAMs. Intra-tumoral (IT) administration of exoASO-C/EBPβ in CT26 tumors resulted in a significantly higher KD of C/EBPβ in tumor-associated CD11b+ cells, as compared to CD11b- cells. Additionally, exoASO-C/EBPβ induced changes in Nos2, CD206 and Csf1r mRNA expression in CD11b+ cells, demonstrating in vivo M2 to M1 reprogramming. Furthermore, IT microinjections of exoASO-C/EBPβ in YUMM1.7 tumor-bearing mice resulted in a significant induction of pro-inflammatory M1 markers TNFα and iNOS, in contrast, free C/EBPβ ASO showed limited increase in M1 markers. exoASO-C/EBPβ administered IT as a single agent demonstrated significant anti-tumor activity in multiple tumor models including: the CT26-colorectal cancer (60% complete responses (CRs)), MB49-bladder cancer (70% CRs) and B16F10-melanoma (30% CRs). Combination treatment with anti-PD1 in all these models further increased the anti-tumor efficacy. Finally, we used a myeloid cell specific C/EBPβ/PGE2/IL10 gene signature to identify cancer indications where exoASO-C/EBPβ therapy might have the most therapeutic significance. Conclusion: exoASO-C/EBPβ is a novel therapeutic candidate that selectively targets and attenuates a critical transcription factor in immunosuppressive myeloid cells, resulting in reprogramming of TAMs and potent anti-tumor activity across multiple TAM-rich mouse models as a monotherapy. Citation Format: Sushrut Kamerkar, Charan Leng, Olga Burenkova, SuChul Jang, Kelvin Zhang, Yanyan Liu, William Dahlberg, Kyriakos Economides, Timothy Soos, Dalia Burzyn, Sriram Sathyanarayanan. Exosome-mediated delivery of antisense oligonucleotides targeting C/EBPβ reprograms tumor-associated macrophages and induces potent single agent anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3508.

Prodrugs engineered for preferential activation in diseased versus normal tissues offer immense potential to improve the therapeutic index of preclinical and clinical-stage active pharmaceutical ingredients that either cannot be developed otherwise or whose efficacy or tolerability it is highly desirable to improve. Such approaches, however, often suffer from trial-and-error design, precluding predictive design and optimization. Here, using BET bromodomain inhibitors (BETi)—a class of epigenetic regulators with proven anti-cancer activity but clinical development hindered by systemic adverse effects–– we introduce a platform that overcomes these challenges. Through tuning of traceless linkers appended to a “brush prodrug” scaffold, we demonstrate that it is possible to correlate in vitro prodrug activation kinetics with in vivo tumor pharmacokinetics, leading to novel BETi prodrugs with enhanced anti-tumor efficacy and devoid of dose-limiting toxicities. This work has immediate clinical implications, introducing principles for the predictive design of prodrugs and potentially overcoming hurdles in drug development.

Background: Tumor-associated macrophages (TAMs) promote tumor progression and resistance to immune checkpoint inhibitors and are thus attractive targets for cancer immunotherapy. The STAT6 transcriptional network is an important driver of the immune-suppressive M2 macrophage program in the tumor microenvironment (TME). Previous attempts to therapeutically target these transcriptional networks have not been successful. Exosomes serve as an efficient, natural, intercellular communication system that can deliver nucleic acids and other macromolecules. Leveraging the potential of exosomes, we have developed a novel, engineered exosome therapeutic candidate loaded with antisense oligonucleotides (ASO) targeting STAT6 (exoASO-STAT6), that effectively silences STAT6 expression in TAMs. Results: In vitro and in vivo studies demonstrate an enhanced delivery of ASO to M2 macrophages. exoASO showed a 2x improvement in uptake vs free ASO in human M2 macrophages in vitro. Following IV administration, exoASO demonstrated up to 11x increase in uptake in monocytes and MDSCs in the blood, Kupffer cells in liver and TAMs and MDSCs in the tumor. In vitro treatment with exoASO-STAT6 resulted in 90% target gene KD in human, mouse and cynomolgus monkey M2 macrophages, which was persistent for up to 10 days. Additionally, exoASO-STAT6 demonstrated greater potency than free ASO. STAT6 KD resulted in a 7x decrease in M2 marker CD163 and a 25x increase in pro-inflammatory cytokines such as IL-12 or TNFα, demonstrating effective macrophage reprogramming. In vivo efficacy studies in CT26 showed potent dose-dependent single agent activity of exoASO-STAT6, with a cumulative dose of 36 μg of ASO resulting in 94% TGI and 80% complete responses. CD8 T-cell depletion abrogated anti-tumor activity and the complete responders were resistant to tumor cell re-challenge, demonstrating a CD8-T cell mediated immunological memory response. In an orthotopic model of HCC that is resistant to anti-PD-1 or anti-CSF1R therapy, IV administration of exoASO-STAT6 significantly attenuated tumor growth, as observed by a 61% reduction in tumor mass and complete elimination of tumor lesions in 50% of treated mice. exoASO-STAT6 therapy resulted in a decrease in M2 markers such as Tgfb1 and Ccl17 and an increase in M1 markers such as IL1b. A significant increase in interferon and cytotoxic T-cell gene signatures was also observed, demonstrating effective reprogramming of the TME. Conclusion: exoASO-STAT6 is a novel therapeutic that selectively targets STAT6, a key transcription factor in TAMs. This therapy results in effective macrophage reprogramming to a pro-inflammatory M1 phenotype and potent single agent anti-tumor activity in multiple checkpoint refractory tumor models. In sum, exoASO-STAT6 represents a first-in-class strategy to target TAMs in a highly selective manner. Citation Format: Sushrut Kamerkar, Charan Leng, Olga Burenkova, Su Chul Jang, Christine McCoy, Kelvin Zhang, William Dahlberg, Marie Leblanc, Hugo Quillery, Sylvie Maubant, Olivier Duchamp, Kyriakos Economides, Timothy Soos, Dalia Burzyn, Sriram Sathyanarayanan. Engineered exosome- mediated STAT6 knockdown in tumor associated macrophages (TAMs) results in potent single agent activity in a hepatocellular carcinoma (HCC) model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1792.

Background: Tumor-associated macrophages (TAMs) are potent drivers of an immunosuppressive tumor microenvironment by reducing T cell recruitment and activation, promoting resistance to immune checkpoint inhibition. Experimental therapies blocking monocyte differentiation or eliminating TAMs show minimal anti-tumor activity. STAT6 and C/EBPβ have key roles in regulating the immunosuppressive state of TAMs however previous attempts to selectively target these transcription factors were unsuccessful. We have developed proprietary engineered exosome therapeutic candidates loaded with antisense oligos (ASOs) targeting STAT6 and C/EBPβ (exoASO). exoASO is designed to selectively deliver ASOs to TAMs and decrease STAT6 and C/EBPβ expression, leading to reprogramming of M2-like TAMs to a M1-like immune stimulatory phenotype. Results: exoASO-STAT6 and exoASO-C/EBPβ induced dose-dependent knockdown (KD) of target genes in primary human M2 macrophages with greater potency (IC50) than free ASO. Gene expression analysis and cytokine assays showed that exoASO-mediated KD resulted in a marked reprogramming to a proinflammatory (M1) phenotype. IV administration of exoASO in a TAM-rich model (CT-26) demonstrates selective uptake by myeloid derived suppressor cells and macrophages. We evaluated efficacy of exoASO-STAT6 or exoASO-C/EBPβ alone or in combination with anti-PD1 therapy. Both exoASO-STAT6 and exoASO-C/EBPβ were efficacious as monotherapies (50% and 60% complete responses (CR) respectively), whereas anti-PD1 or anti-CSFR1 mAb were not effective (0% CR). exoASO-C/EBPβ in combination with anti-PD1 mAb resulted in greater efficacy (80% CR), and enhanced survival (70% at day 55) compared to exoASO-C/EBPβ monotherapy (50% survival at day 55). In contrast, combination of anti-PD1 with exoASO-STAT6 did not show improved efficacy as compared to monotherapy. Durable anti-tumor responses were observed with both exoASO therapies as mice achieving CR against the primary tumor had no tumor growth upon re-challenge. We evaluated anti-tumor efficacy using an orthotopic hepatocellular carcinoma (HCC) model. Mice were dosed IV using: exoASO-C/EBPβ; free C/EBPβ ASO; anti-PD1 or; anti-CSF1R mAbs. exoASO-C/EBPβ treatment attenuated HCC-mediated increase in liver to body weight ratios (≤ 10%) and showed little to no observable tumor lesions in 50% of treated mice. In contrast, increased liver to body weight ratios (≥ 10%) and observable tumor lesions occurred in all mice (100%) treated with either free C/EBPβ ASO or anti-CSF1R mAb. Conclusion: exoASO-STAT6 and exoASO-C/EBPβ are efficacious as monotherapies in TAM-rich tumors when administered locally or systemically. exoASO-C/EBPβ has increased potency when administered in combination with checkpoint antibodies. Collectively, exoASOs against STAT6 and C/EBPβ are a first-in-class strategy to target TAMs in a highly selective manner. Citation Format: Sushrut Kamerkar, Dalia Burzyn, Olga Burenkova, Charan Leng, Raymond Yang, Katherin Kirwin, Tong Zi, Su Chul Jang, William Dahlberg, Eric Zhang, Scott Estes, Sylvie Maubant, Olivier Duchamp, Kyriakos Economides, Timothy Soos, Sriram Sathyanarayanan. Genetic reprogramming of TAMs by engineered exosomes results in potent single agent anti-tumor activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5696.

Tumor-associated macrophages (TAMs) are potent drivers of an immunosuppressive tumor microenvironment and may be an attractive therapeutic target. Experimental therapies tested thus far block myeloid cell differentiation, leading to ineffective antigen presentation and minimal antitumor activity. Exosomes serve as an efficient, natural, intercellular communication system that can deliver nucleic acids and other macromolecules. We have developed novel, engineered exosome therapeutic candidates that selectively deliver antisense oligos (ASOs) to TAMs, targeting key transcription factors that control the immunosuppressive program, such as STAT6 and C/EBPβ. These transcription factors, which are otherwise “undruggable” with current therapeutic strategies, are activated through various signaling molecules such as IL-4 and IL-10, which results in a protumorigenic macrophage phenotype (M2). Exosomes loaded with STAT6 or C/EBPβ ASOs (exoASO) induced dose-dependent knockdown (KD) of target genes in primary human M2 macrophages with greater potency than the free ASO. Gene expression analysis and cytokine assays showed that exoASO-mediated KD resulted in a marked reprogramming to a proinflammatory (M1) phenotype, as measured by a significant decrease in CD163 and TGF-β1 expression, and a concomitant induction of TNFα and IL-12p40 production. Intratumorally dosed exoASO preferentially associated with tumor myeloid cells, resulting in a 50% KD of STAT6 and C/EBPβ mRNA transcripts in tumor-associated CD11b+ cells, compared to a 20% KD with an equivalent dose of free ASO. Modulation of mRNAs for CD206, Csf1r, and iNos was also observed, and is consistent with M2 to M1 conversion. ExoASO-STAT6 treatment of mice with CT26 tumors showed significant antitumor activity as a monotherapy (80% tumor growth inhibition, 30% complete responses). Similar monotherapy activity was also observed in vivo with exoASO C/EBPβ therapy. In contrast, equivalent doses of free ASO or anti-CSF1R monotherapy did not have any significant effects on tumor growth. Thus, exoASO therapeutics can specifically target and effectively reduce the expression of transcription factors in TAMs, inducing effective reprogramming and resulting in potent antitumor activity. Collectively, exoASOs against STAT6 and C/EBPβ represent a first-in-class strategy to target tumor-associated myeloid cells in a highly selective manner. Citation Format: Suhrut Kamerkar, Dalia Burzyn, Charan Leng, Olga Burenkova, Su Chul Jang, Raymond Yang, Adam Boutin, Katherine Kirwin, Tong Zi, William Dahlberg, Eric Zhang, Kelvin Zhang, Scott Estes, Kyriakos Economides, Timothy Soos, Sriram Sathyanarayanan. Reprogramming of tumor-associated M2 macrophages with antisense oligonucleotide-loaded exosomes results in potent single-agent antitumor activity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A50.

Bone Marrow Transplant (BMT) is a potentially curative treatment for malignant and non-malignant blood disorders. Current regimens for patient preparation, or conditioning, prior to BMT limit the use of this curative procedure due to regimen-related mortality and morbidities, including risks of organ toxicity, infertility and secondary malignancies. Targeted preparation using antibody drug conjugates (ADCs) to mouse CD45 has previously been shown to be sufficient to enable bone marrow transplant (BMT) in syngeneic immune competent mice (Palchaudhuri et al. Nature Biotech 2016 34:738–745), and this approach to preparation has the potential to expand the utility of BMT if it can be successfully translated to patients. To further investigate the utility of this tool ADC in murine transplant models, we explored anti-CD45-saporin (CD45-SAP) in an allogeneic minor mismatch transplant model (Balb/c donor into DBA/2 recipients). The goal of the work was to identify the level of immune suppression, if any, that needs to be used in combination with CD45-SAP to enable high donor chimerism in the allogeneic setting. CD45-SAP (1.9 mg/kg, iv) was evaluated alone or in combination with additional immune modulating agents: clone 30F11 (25 mg/kg, IP), a naked anti-CD45 antibody that mimics ATG by relying on effector function to enable potent peripheral B- and T -cell depletion; pre-transplant Cytoxan (PreTCy, 200 mg/kg, IP), 2 Gy total body irradiation (TBI), and post-transplant Cytoxan (PTCy, 200 mg/kg, IP) to prevent graft versus host disease as well as block host versus graft rejection. 9 Gy TBI was used as the conventional conditioning positive control. Conditioned mice were transplanted with 2 × 107 whole bone marrow cells, and chimerism assessed over 12 weeks. CD45-SAP in combination with PTCy achieved comparable peripheral myeloid chimerism, a readout of stem cell engraftment, to 9 Gy TBI (>90%) at 8 weeks post-transplantation (Fig 1). The addition of 30F11 to the CD45-SAP/PTCy protocol had no effect on peripheral donor chimerism (59% vs 61%), suggesting additional lymphodepletion is not required. In contrast, the single agents alone, 2 Gy TBI in combination with 30F11 and PTCy resulted in These results indicate CD45-SAP in combination with PTCy is sufficient to enable high levels of donor chimerism in the minor mismatched setting without the need for additional immune suppression. CD45-SAP was more effective at conditioning than 2Gy TBI or PreTCy. Future experiments will investigate the CD45-SAP/PTCy protocol in haploidentical and fully mismatched allogeneic mouse models.

Although EGFR is a validated therapeutic target across multiple cancer indications, the often modest clinical responses to current anti-EGFR agents suggest the need for improved therapeutics. Here, we demonstrate that signal amplification driven by high-affinity EGFR ligands limits the capacity of monoclonal anti-EGFR antibodies to block pathway signaling and cell proliferation and that these ligands are commonly coexpressed with low-affinity EGFR ligands in epithelial tumors. To develop an improved antibody therapeutic capable of overcoming high-affinity ligand-mediated signal amplification, we used a network biology approach comprised of signaling studies and computational modeling of receptor–antagonist interactions. Model simulations suggested that an oligoclonal antibody combination may overcome signal amplification within the EGFR:ERK pathway driven by all EGFR ligands. Based on this, we designed MM-151, a combination of three fully human IgG1 monoclonal antibodies that can simultaneously engage distinct, nonoverlapping epitopes on EGFR with subnanomolar affinities. In signaling studies, MM-151 antagonized high-affinity EGFR ligands more effectively than cetuximab, leading to an approximately 65-fold greater decrease in signal amplification to ERK. In cell viability studies, MM-151 demonstrated antiproliferative activity against high-affinity EGFR ligands, either singly or in combination, while cetuximab activity was largely abrogated under these conditions. We confirmed this finding both in vitro and in vivo in a cell line model of autocrine high-affinity ligand expression. Together, these preclinical studies provide rationale for the clinical study of MM-151 and suggest that high-affinity EGFR ligand expression may be a predictive response marker that distinguishes MM-151 from other anti-EGFR therapeutics. Mol Cancer Ther; 14(7); 1625–36. ©2015 AACR.

The ErbB family of receptor tyrosine kinases comprises four members: epidermal growth factor receptor (EGFR/ErbB1), human EGFR 2 (HER2/ErbB2), ErbB3/HER3, and ErbB4/HER4. The first two members of this family, EGFR and HER2, have been implicated in tumorigenesis and cancer progression for several decades, and numerous drugs have now been approved that target these two proteins. Less attention, however, has been paid to the role of this family in mediating cancer cell survival and drug tolerance. To better understand the complex signal transduction network triggered by the ErbB receptor family, we built a computational model that quantitatively captures the dynamics of ErbB signaling. Sensitivity analysis identified ErbB3 as the most critical activator of phosphoinositide 3-kinase (PI3K) and Akt signaling, a key pro-survival pathway in cancer cells. Based on this insight, we designed a fully human monoclonal antibody, seribantumab (MM-121), that binds to ErbB3 and blocks signaling induced by the extracellular growth factors heregulin (HRG) and betacellulin (BTC). In this article, we present some of the key preclinical simulations and experimental data that formed the scientific foundation for three Phase 2 clinical trials in metastatic cancer. These trials were designed to determine if patients with advanced malignancies would derive benefit from the addition of seribantumab to standard-of-care drugs in platinum-resistant/refractory ovarian cancer, hormone receptor-positive HER2-negative breast cancer, and EGFR wild-type non-small cell lung cancer (NSCLC). From preclinical studies we learned that basal levels of ErbB3 phosphorylation correlate with response to seribantumab monotherapy in mouse xenograft models. As ErbB3 is rapidly dephosphorylated and hence difficult to measure clinically, we used the computational model to identify a set of five surrogate biomarkers that most directly affect the levels of p-ErbB3: HRG, BTC, EGFR, HER2, and ErbB3. Preclinically, the combined information from these five markers was sufficient to accurately predict which xenograft models would respond to seribantumab, and the single-most accurate predictor was HRG. When tested clinically in ovarian, breast and lung cancer, HRG mRNA expression was found to be both potentially prognostic of insensitivity to standard therapy and potentially predictive of benefit from the addition of seribantumab to standard of care therapy in all three indications. In addition, it was found that seribantumab was most active in cancers with low levels of HER2, consistent with preclinical predictions. Overall, our clinical studies and studies of others suggest that HRG expression defines a drug-tolerant cancer cell phenotype that persists in most solid tumor indications and may contribute to rapid clinical progression. To our knowledge, this is the first example of a drug designed and clinically tested using the principles of Systems Biology.

In this paper we consider the question of how quality assurance can have a real, positive impact on the quality of teaching and learning at universities, considering the realities of different systems—the system of control and the system of quality culture—in using the example of two universities: the KNITU-KAI in Russia and the TU Dresden in Germany. The study involved 40 lecturers of technical courses and 120 third-year students of technical courses from both universities. The authors put forward a working hypothesis that quality assurance has a positive effect on the quality of teaching and learning if it carries out its informational, motivational and administrative functions. The results have shown that the aforementioned functions are not fulfilled by both universities and a special organizational culture is not the only condition that must be met for effective evaluation efforts. In order to fulfill motivational, informational and administrative functions, it is important as well that the quality assurance system is transparent and that all participants see that the surveyed data and results lead to measures for quality development. It is equally important that all university members participate in the process of quality assurance.

Mutations in the extracellular domain (ECD) of EGFR have been reported in colorectal (CRC) patients as a mechanism of acquired resistance to the EGFR-directed antibodies cetuximab and panitumumab. Similar to EGFR intracellular domain mutations observed in patients treated with EGFR kinase inhibitors, these EGFR-ECD mutations inhibit the binding of the antibodies while maintaining EGFR signaling activity. MM-151, an investigational agent consisting of three anti-EGFR IgG1 antibodies, has been recently reported to overcome these mutations in over-expression and patient-derived cell line models. Here, we extend these studies to characterize the mechanisms of action that enable MM-151 to provide robust inhibition of EGFR-ECD mutations beyond that achievable by other monoclonal and oligoclonal EGFR antibodies. First, utilizing a cell-free binding assay with recombinant EGFR-ECD mutants, we found that MM-151, but not the Sym004 combination of two antibodies, maintained oligoclonal binding to all mutants. Second, we performed a panel of 2D and 3D in vitro experiments with over-expression and genome-edited (CRISPR-Cas9) cell lines harboring EGFR-ECD mutations to characterize target engagement, cell signaling, proliferation, and the impact of hetero- versus homozygous expression of EGFR wild type or mutant alleles. We observed that the three mechanisms of action for MM-151- antagonism of both low- and high-affinity EGFR ligands, EGFR down-regulation, and immune-effector activity— were preserved in these models. Third, we performed xenograft experiments to evaluate the behavior of MM-151 and found that MM-151 maintained inhibitory activity in an in vivo context. The results of our multiscale experiments identified the impact of combining three antibodies to overcome the high plasticity of the EGFR extracellular domain and thus reveal the potential for clinical evaluation of MM-151 in both EGFR-naïve and EGFR-refractory CRC populations. Citation Format: Hongfang Wang, Shawn P. Carey, Olga Burenkova, Jian Tang, Nastaran Gerami-Moayed, Sabrina Arena, Alberto Bardelli, Rachel Nering, Jeffrey D. Kearns. MM-151 elicits broad and unique inhibition of cells harboring EGFR extracellular domain mutations — results of multiscale experiments with genome-edited cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2148.

Increased Aurora A expression occurs in a variety of human cancers and induces chromosomal abnormalities during mitosis associated with tumor initiation and progression. MLN8054 is a selective small-molecule Aurora A kinase inhibitor that has entered Phase I clinical trials for advanced solid tumors. MLN8054 inhibits recombinant Aurora A kinase activity in vitro and is selective for Aurora A over the family member Aurora B in cultured cells. MLN8054 treatment results in G 2 /M accumulation and spindle defects and inhibits proliferation in multiple cultured human tumor cells lines. Growth of human tumor xenografts in nude mice was dramatically inhibited after oral administration of MLN8054 at well tolerated doses. Moreover, the tumor growth inhibition was sustained after discontinuing MLN8054 treatment. In human tumor xenografts, MLN8054 induced mitotic accumulation and apoptosis, phenotypes consistent with inhibition of Aurora A. MLN8054 is a selective inhibitor of Aurora A kinase that robustly inhibits growth of human tumor xenografts and represents an attractive modality for therapeutic intervention of human cancers.

Introduction: The Epidermal Growth Factor Receptor (EGFR) pathway is a key driver of late-stage colorectal cancer (CRC) and EGFR inhibitors, such as cetuximab and panitumumab, prolong survival in a large subset of patients. Not all patients respond to EGFR inhibitor therapy, however, and resistance develops in those who respond. Accumulating evidence suggests that alternative signaling pathways circumvent EGFR blockade. Several studies have shown that tumors with high heregulin, IGF-1 or HGF respond poorly to EGFR-targeted therapies. Further, Phase 2 studies suggest that combined inhibition of EGFR and HGF with panitumumab and rilotumumab significantly improves response rates compared to panitumumab alone. These data support the notion that more than one pathway may be simultaneously active in late-stage CRC and that EGFR-directed therapy may be improved through combination strategies. Merrimack has built a portfolio of biologic agents targeting key oncogenic receptors that block ligand binding and pathway activation, providing a unique opportunity to develop combination therapies in a biomarker-driven fashion. MM-151 targets EGFR; MM-121 (seribantumab) targets ERBB3; MM-141 (istiratumab) targets both IGF-1R and ERBB3; and MM-131 targets Met and EpCAM. Here we investigate responsiveness to different resistance ligands and the ability of drug combinations to block ligand-mediated cell growth more effectively than monotherapy. Experimental procedures: We evaluated the prevalence of resistance ligands in colorectal adenocarcinoma patient samples by RNA in situ hybridization. To assess the effect of ligands and drugs on cell growth we assembled a panel of 29 genetically diverse CRC cell lines. Using a carefully optimized in vitro 3D culture system, we measured cell viability in response to MM-151, MM-121, MM-141, MM-131 or a combination of the drugs using MM-151 as the backbone. The screen was performed in the presence or absence of exogenously added ligands: EGF, heregulin, IGF-1 or HGF. Using these data, we selected representative xenograft models for in vivo analysis of drug combination efficacy. Results: We show that colorectal cancer tumors co-express resistance ligands and that ligand prevalence appears to increase following treatment. Results from our in vitro screen show that CRC cell lines fall into two groups: those that are unresponsive to ligand stimulation and those that are responsive. Interestingly, cell lines that are responsive to one ligand tend to respond to other ligands as well, suggesting that blocking more than one pathway may prove more effective than blocking only a single pathway. In pre-clinical models of cell lines and xenografts, where ligands are co-expressed, combinations of targeted antibodies, using MM-151 as the backbone, have greater activity than monotherapy. Conclusions: These data suggest that subsets of patients with high expression of individual or multiple ligands could benefit from different combinations of targeted therapies. A Phase 1 study evaluating the safety, pharmacology and preliminary activity of the co-administration of MM-151 and MM-121 (seribantumab) in heregulin positive cancer patients is currently enrolling patients with advanced colorectal cancer, non-small cell lung cancer, and head and neck cancer. Citation Format: Marisa J. Wainszelbaum, Jessica Fessler, Johanna Lahdenranta, Olga Burenkova, Nat Gerami-Moayed, Yasmin Hashambhoy-Ramsay, Victoria Rimkunas, Gavin MacBeath. Inhibition of ERBB3 with MM-121, IGF1-R with MM-141 or Met with MM-131 increases the activity of EGFR inhibitor MM-151 in colorectal cancer models expressing multiple resistance ligands. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C25.

Purpose: Heregulin-mediated activation of the human epidermal growth factor receptor 3 (HER3/ErbB3) is required for the growth and survival of many epithelial cancers. This signaling pathway is also emerging as a mechanism of resistance to targeted agents and chemotherapies. MM-121 (seribantumab) is an investigational human monoclonal anti-ErbB3 antibody that has previously been shown to effectively block ligand-dependent activation of ErbB3 in a range of tumors, and has demonstrated clinical activity in biomarker positive patients in several Phase II trials. The purpose of this study was to examine in three indications of interest, the ability of heregulin to induce resistance to standard chemotherapies and the reversal of this effect by MM-121. Such systematic evaluation of different combinations can serve as a guide for the future clinical development of MM-121. Methods: To assess the effect of heregulin and MM-121 on chemotherapies in cancer cells, we conducted a high throughput proliferation screen in 3D cultures. A panel of 60 cell lines of relevant clinical indications (ovarian, breast and lung cancer) was selected and tested for the sensitivity to respective standard-of-care chemotherapies in the absence or presence of exogenously added heregulin. Using these data, we analyzed the rescuing capacity of heregulin and the MM-121 combination's sensitivity, and selected representative combinations for in vivo models. Results: We show that in a large panel of cancer cell lines the presence of heregulin can induce resistance to multiple chemotherapies with very different mechanisms of action. The combination of MM-121 with any one of these chemotherapies can reverse the heregulin-meditated rescue and provide an additive treatment effect at therapeutically relevant doses achieved in the clinic. These results were further validated in xenograft mouse models of all three indications, using representative chemotherapies and doses. In addition, biomarker analysis revealed that ErbB3 receptor levels largely determine responsiveness to heregulin and MM-121. Conclusions: MM-121 is an anti-ErbB3 antibody designed to block ligand-mediated signaling, and currently in clinical development. The results presented here demonstrate the role of heregulin in reducing the sensitivity of tumors to standard-of-care chemotherapies, and the effect of ErbB3 pathway inhibition across indications. Citation Format: Kristina Masson, Viara Grantcharova, Olga Burenkova, Marisa Wainszelbaum, Sergio Iadevaia, Sharlene Adams, Andreas Raue, Akos Czibere, Birgit Schoeberl, Gavin MacBeath. The ErbB3-targeting antibody MM-121 (seribantumab) reverses heregulin-driven resistance to multiple chemotherapies on tumor cell growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-243. doi:10.1158/1538-7445.AM2015-LB-243

MM-121/SAR 256212 is a fully human anti ErbB3/HER3 antibody that blocks ligand-induced receptor activation. Formation of EGFR/ErbB3 (ErbB1-3) heterodimers has been implicated as a major driver of tumor growth and survival in non-small cell lung cancer (NSCLC). Although erlotinib remains the standard-of-care treatment for patients with EGFR-wild-type NSCLC who have failed platinum combination chemotherapy, clinical benefit is typically modest. To address this unmet medical need, we investigated the combination of erlotinib with MM-121 in pre-clinical models of NSCLC. We initially assembled a panel of 25 EGFR-WT NSCLC cell lines spanning the most common histological subtypes (adenocarcinoma, squamous and large-cell carcinoma). Clinical studies show that tumors harboring activating Ras mutations less commonly respond to ErbB-directed therapies. To explore the effect of Ras mutations on responsiveness to MM-121, we also selected our cell lines to include a variety of H-/K-/N-Ras genotypes. Using a carefully optimized in-vitro 3D culture system, we then measured cell viability in response to MM-121, erlotinib or a combination of the drugs in the presence or absence of exogenously added epidermal growth factor (EGF) and/or heregulin-β1 (HRG). Our results indicate that MM-121 inhibits HRG-driven cell proliferation in the studied cell lines. In the five cell lines exhibiting dual-EGF-HRG-driven cell proliferation, the combination of MM-121 with erlotinib demonstrated superior inhibition of cell viability over erlotinib alone.To assess the ability of MM-121 to inhibit tumor cell growth independent of exogenously supplied HRG, we established mouse xenografts from three of the cell lines and treated mice with erlotinib, MM-121, or a combination of the two. All three in-vivo models showed greatly enhanced inhibition of tumor cell growth compared to erlotinib alone in a manner consistent with our in-vitro results. Analyzing our in vitro data we found that mutations in Ras genes (HRAS, KRAS, NRAS) do not preclude response to MM-121 or incremental benefit from the combination of MM-121 with erlotinib over erlotinib alone. Non-adenocarcinoma origin likewise did not preclude response to MM-121, although a weak trend towards diminished activity in the ten non-adenocarcinoma NSCLC cell lines was apparent. Merrimack Pharmaceuticals and Sanofi are co-developing MM-121 and a Phase 1-2 study of MM-121 in combination with erlotinib is currently enrolling patients with EGFR-wild-type NSCLC. Citation Format: Marisa J. Wainszelbaum, Mark S. Sevecka, Olga Burenkova, Gabriela Garcia, William Kubasek, Gavin MacBeath. In-vitro studies of MM-121/SAR 256212, an anti-ErbB-3 antibody, in combination with erlotinib in EGFR-wild-type NSCLC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5464. doi:10.1158/1538-7445.AM2013-5464

Approximately 75% of breast cancers are estrogen receptor (ER) positive. Although endocrine therapies such as tamoxifen, fulvestrant, and letrozole have demonstrated significant efficacy in treating ER+ breast cancer patients, intrinsic or acquired resistance has limited their success. Recent studies suggest that crosstalk between ErbB receptor signaling and ER signaling may contribute to resistance to endocrine therapy. Overexpression of human epidermal growth factor receptor 2 (HER2, synonymous with ErbB2) and upregulation of the ErbB3 ligand heregulin are associated with poor prognosis and reduced overall survival. MM-111 is a novel bispecific antibody fusion protein that specifically targets the ErbB2/ErbB3 heterodimer and blocks heregulin binding to ErbB3. MM-111 inhibits ligand-induced ErbB3 phosphorylation, tumor cell cycle progression, and tumor growth when ErbB2 is overexpressed. We hypothesized that combination of endocrine therapies with MM-111 may improve anti-tumor efficacy. In an estrogen-stimulated BT474-M3 ER-positive breast cancer cell three-dimensional spheroid assay, MM-111, when used as a single agent, showed growth inhibitory effects similar to the anti-estrogen drugs tamoxifen and fulvestrant. Combination of MM-111 with anti-estrogen therapy showed superior activity to either drug alone. In the presence of heregulin, MM-111 maintained its growth inhibitory activity, whereas the inhibitory effect of tamoxifen and fulvestrant was diminished. This suggests that activation of ErbB3 confers tumor cell resistance to anti-estrogen therapies. When both estrogen and heregulin were present, the combination of MM-111 and the anti-estrogen drugs demonstrated a significantly greater inhibitory effect than either drug alone. Western blot analysis showed that treatment of BT-474-M3 cells with the combination of MM-111 and the anti-estrogen drugs significantly increased apoptosis markers such as cytochrome C and BAX. Furthermore, an in vivo BT474-M3 xenograft model showed resistance to tamoxifen treatment (5 mg/pellet, 60-day release). In this xenograft model MM-111 sensitized tumor response to tamoxifen and the combination treatment dramatically inhibited tumor growth. In conclusion, the combination of MM-111 and endocrine therapies may provide a potent regimen that overcomes acquired resistance to endocrine therapies in ER+, ErbB2-overexpressing breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 655. doi:10.1158/1538-7445.AM2011-655

In women, breast cancer is among the most common cancers and the fifth most common cause of cancer deaths. Due to the heterogeneity of the disease, 10-year progression free survival can vary widely with stage and type from 98% to 10%. We present data showing that MM-121, a fully human monoclonal anti-ErbB3 antibody, is efficacious in studies of both hormone dependent (ER+) and triple negative (ER-,PR-, Low Erb2) breast cancer lines that express the molecular profile consistent with MM-121 response (co-expression of ErbB3 and HRG). Using a combination of computational and experimental approaches, ErbB3 was identified as a critical transducer of oncogenic signaling leading to the development of MM-121, a first in class anti-ErbB3 antibody. We have previously demonstrated that MM-121, when used as a single agent, inhibits heregulin-induced signaling events in human cancer cell lines. Moreover, MM-121 caused dose-dependent inhibition of tumor growth in multiple xenograft models of human cancer, including ovarian, renal cell, pancreatic, lung, and prostate cancer. Estrogen dependent, or ER+ breast cancers, make up about 80% of breast cancers. A standard treatment for ER+ breast cancer includes hormone therapy; however a substantial number of ER+ breast cancers eventually develop resistance requiring additional treatments. Here we show that in ER+ breast cancer cells, MM-121 can block HRG induced ErbB3 activation and VEGF secretion as well as HGF induced pErbB3 in vitro. Additionally, in ER+ breast cancer xenograft models, MM121 is effective in combination with both chemotherapy agents and targeted therapies and may be effective in ER+ breast cancers that are refractory to hormone treatment. The triple negative breast cancers are characterized by poor prognosis, aggressiveness, and reduced responsiveness to standard treatments. MM-121 used as a single agent therapy was able to suppress tumor growth in triple negative primary human tumors, when grown as xenografts suggesting that MM-121 monotherapy may be clinically efficacious in triple negative breast cancers. Together, these data suggest that MM-121, when used as a single agent or in combination with other therapies, could offer significant clinical benefit to both ER+ and triple negative breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1806.

One of the challenges faced by targeted therapeutics currently in the clinic is the relatively small population of patients who derive significant benefit from their use. We report the development of a preclinical classifier which can correctly predict xenograft response to MM-121, an anti-ErbB3 antibody, based on the measurement of a few key biomarkers in cell lysates. Deregulation of the ErbB family receptors is common in many cancers. Using a combination of computational modeling and quantitative experiments we identified ErbB3 as a key mediator of mitogenic signaling downstream of the ErbB receptors. Based on these results, we developed MM-121, a first in class anti-ErbB3 monoclonal antibody that blocks heregulin-induced signaling and inhibits tumor growth in multiple xenograft models of human cancer. Here we present our efforts to derive a predictive biomarker signature that identifies tumors that are responsive to MM-121. Using our computational model of the ErbB signaling pathway we identified the five most critical proteins for predicting activation of phospho-AKT - a key mediator of cell survival and apoptosis. These proteins include MM-121's target, ErbB3, and its ligand, heregulin. We profiled these biomarkers in a large panel of cancer cell lines, and using the measured effect of MM-121 on inhibiting tumor growth in eight xenograft tumor models, we determined a classification rule for predicting xenograft response. We subsequently used this classification rule to correctly predict a priori MM-121 response in 11 xenograft models. These results suggest that our computationally-derived biomarker signature is sufficient for predicting response to MM-121 in xenografts, and could offer significant clinical benefit by helping select patients for MM-121 treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3756.

13059 Background: The mitotic kinase Aurora A is implicated in the development of multiple tumor types. MLN8054 is an oral, potent and selective small-molecule inhibitor of Aurora A with broad efficacy in preclinical models of cancer. Inhibition of Aurora A by MLN8054 induces accumulation of mitotic cells, followed by apoptosis. This study explores relationships between Aurora A inhibition, mitotic index, and tumor growth inhibition for xenograft models with different sensitivity to MLN8054. The marker response in mouse skin was also studied. Methods: Mice bearing subcutaneous xenografts were dosed orally qd or bid with MLN8054 for 21 days. Pharmacodynamic markers were studied after 1–2 doses. Formalin-fixed xenograft tissues were stained with the mitotic markers pHisH3 and MPM2, or with an antibody to the T288 autophosphorylation site on Aurora A. Tumor growth inhibition (TGI) was calculated using the formula 100 - [ΔT/ΔC * 100], where ΔT is the volume change for treated tumors, and ΔC is the volume change for control tumors. Results: HCT116 human colon xenografts were sensitive to MLN8054 on a qd or bid schedule (84% and 96% TGI respectively for 30mg/kg dose). The T288 autophosphorylation site was used to directly demonstrate inhibition of Aurora A, which resulted in dose-dependent duration of the elevation in mitotic index. Efficacy was similar for qd vs bid dosing of 30mg/kg MLN8054, and accordingly we found that a single dose was sufficient to elevate the mitotic index for about 20–24h in this model. SW480 human colon xenografts have MLN8054 sensitivity similar to that of HCT116, but more modest effects on mitotic index were observed. The mitotic index profile of SW480 is similar to that of MDA-MB-231 xenografts, the most insensitive model studied. Elevated mitotic index was also observed in mouse skin. Conclusions: We found that mitotic index measurements coupled with the T288 autophosphorylation site as a direct marker of Aurora A activity are useful for monitoring inhibition of Aurora A by MLN8054 in tumor and/or skin biopsies. In a sensitive model, greater duration of mitotic index elevation results in greater efficacy. Our continuing work aims to better understand the differences in marker and efficacy responses between xenograft lines, incorporating the pT288 antibody as a direct marker of Aurora A inhibition. [Table: see text]