Jason Gary Sagert

Tumor-specific activation of an EGFR-targeting probody enhances therapeutic index.

A proteolytically activatable EGFR Probody demonstrates antitumor efficacy while alleviating toxicity. Seek and Destroy One of the main problems with current cancer therapies is lack of specificity: Traditional chemotherapeutics target all dividing cells, and even more restricted drugs, like monoclonal antibodies, may have on-target but off-tumor side effects. But what if you had a drug that was only turned on in the presence of the tumor? Desnoyers et al. now report the development of a Probody that targets epidermal growth factor receptor (EGFR) only in the presence of tumor. Cetuximab is a Food and Drug Administration–approved EGFR-targeting antibody used to treat metastatic colorectal cancer and head and neck cancer, but therapy often results in dose-limiting skin rash. The authors modified cetuximab to form a Probody (PB1)—where the antigen-binding sites are masked until the antibody is activated by proteases commonly found in the tumor microenvironment. The authors found that PB1 was largely inert while in circulation in mice, but that it had comparable efficacy to cetuximab in the presence of tumor. In nonhuman primates, PB1 demonstrated safety and decreased toxicity at higher doses than cetuximab. What’s more, ex vivo human primary tumor samples were sufficient to activate PB1. If these data hold true in human trials and for other antibodies, Probodies could be used to target cancer while minimizing treatment side effects. Target-mediated toxicity constitutes a major limitation for the development of therapeutic antibodies. To redirect the activity of antibodies recognizing widely distributed targets to the site of disease, we have applied a prodrug strategy to create an epidermal growth factor receptor (EGFR)–directed Probody therapeutic—an antibody that remains masked against antigen binding until activated locally by proteases commonly active in the tumor microenvironment. In vitro, the masked Probody showed diminished antigen binding and cell-based activities, but when activated by appropriate proteases, it regained full activity compared to the parental anti-EGFR antibody cetuximab. In vivo, the Probody was largely inert in the systemic circulation of mice, but was activated within tumor tissue and showed antitumor efficacy that was similar to that of cetuximab. The Probody demonstrated markedly improved safety and increased half-life in nonhuman primates, enabling it to be dosed safely at much higher levels than cetuximab. In addition, we found that both Probody-responsive xenograft tumors and primary tumor samples from patients were capable of activating the Probody ex vivo. Probodies may therefore improve the safety profile of therapeutic antibodies without compromising efficacy of the parental antibody and may enable the wider use of empowered antibody formats such as antibody-drug conjugates and bispecifics.

Abstract C158: Tumor-specific inhibition of jagged-dependent notch signaling using a Probody™ therapeutic.

Probodies represent a unique class of antibody-based therapeutics that specifically target activity to diseased tissues including cancer. Probodies are designed to be inactive in circulation and in healthy tissue and activated only within the tumor microenvironment by dysregulated protease activity. The Probody approach enables promising therapeutic targets not druggable with conventional antibodies due to toxicity. Inhibition of Notch signaling has revealed the therapeutic potential of targeting this pathway in cancer; however, systemic inhibition results in severe toxicities (e.g. gastrointestinal and cardiac) in preclinical and clinical studies limiting the development of Notch-targeting therapeutics. We have developed a fully human IgG1 monoclonal antibody that binds and inhibits the activity of both human and mouse Jagged1 and Jagged2 (Jagged1/2). In vitro cell based assays and in vivo ectopic xenograft mouse tumor models (BxPC3 and H292) show dose-dependent inhibition of Notch signaling and anti-tumor activity, respectively. However, the antibody also shows profound toxicities in mice, including weight loss, alopecia, hyperkeratosis, and athymia, due to on-target inhibition of Jagged1/2 in healthy tissue. To address these toxicities, we developed a fully recombinant anti-Jagged1/2 Probody, CTX-033, comprised of the same antibody but with a unique peptide mask that blocks the antigen binding site linked to the light chain of the antibody by a linker containing a substrate that is cleavable by one or more proteases upregulated in cancer. CTX-033 had similar anti-tumor activity in the mouse xenograft models as compared to the antibody. However, despite a two-fold higher systemic exposure to CTX-033 as compared to the antibody, Probody treated mice did not exhibit the toxicities associated with the antibody treatment. Furthermore, CTX-033 shows additive anti-tumor efficacy when combined with gemcitabine in the BxPC3 model. Notably, the Probody plus gemcitabine combination lacks the significant toxicity associated with the antibody plus gemcitabine combination treatment. To validate both Jagged expression and protease activity in patient tumor samples, we used a novel in situ assay, immunofluorescent Probody zymography. The results reveal broad expression of the Jagged ligands and specific activation and binding of CTX-033 in patient pancreatic adenocarcinoma tissue samples. The data described here with the anti-Jagged Probody, CTX-033, demonstrates (1) the potential of the Probody platform to enable the safe targeting of two key ligands in the Notch signaling pathway not possible with a traditional antibody format and (2) the presence of both Jagged1/2 and proteases capable of activating CTX-033 in patient tumor samples. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C158. Citation Format: Jason Sagert, James West, Olga Vasiljeva, Jennifer Richardson, Luc Desnoyers, Shouchun Liu, Annie Yang, Chihunt Wong, Elizabeth Menendez, Krishna Polu, Henry Lowman. Tumor-specific inhibition of jagged-dependent notch signaling using a Probody™ therapeutic. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C158.

Abstract 2665: Transforming Notch ligands into tumor-antigen targets: A Probody-Drug Conjugate (PDC) targeting Jagged 1 and Jagged 2

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The development of antibody drug conjugates (ADC) holds significant promise for improving outcomes in patients with cancer. However, toxicity can limit the number of accessible targets for these highly potent and empowered antibody formats due to expression in healthy tissue. Probody formatted ADCs enable opening up the therapeutic window for high potential but previously inaccessible targets, such as the Jagged ligands in the Notch pathway. Probodies are fully recombinant biotherapeutics comprised of a monoclonal antibody whose binding to target antigen is blocked by a masking peptide. Upon cleavage of a specific substrate-linker by tumor-specific proteases, the activated Probody binds its target, resulting in tumor-localized activity. Jagged expression is observed in a wide variety of patient tumors including multiple myeloma, pancreatic cancer, breast cancer, and prostate cancer. We previously described a novel anti-Jagged 1/2 antibody that is efficacious in slowing tumor growth in mouse in-vivo tumor models but results in systemic toxicity. A corresponding Probody mitigates systemic toxicities associated with inhibition of Jagged-induced Notch signaling while maintaining anti-tumor efficacy. Here we show that the Notch ligands Jagged 1 and Jagged 2 have properties that could also enable an antibody-drug conjugate (ADC) approach because the ligands are both expressed on the cell surface and can internalize an anti-Jagged antibody. Using FACS we have shown that Jagged 1/2 are expressed on several human cancer cell lines and by fluorescent IHC staining, the expression of Jagged 1/2 is maintained in the corresponding xenograft tumors. To further explore the potential of Jagged as an ADC target, we engineered a Probody Drug Conjugate (PDC) conjugated to the microtubule inhibitor MMAE. This PDC is efficacious in a pancreatic xenograft tumor model BxPC3. Importantly, in the BxPC3 model the PDC shows equivalent in vivo efficacy to the corresponding ADC without causing the systemic toxicity associated with ADC treatment. Supportive of the potential clinical benefit of an anti-Jagged PDC, more than 75% of lung, pancreatic, and breast cancer patient tumor samples evaluated show moderate to high Jagged 1/2 expression as measured by IHC staining. These data demonstrate that the ProbodyTM platform has potential to enable the use of drug conjugates to target Jagged ligands in the Notch pathway. Citation Format: Jason Sagert, Jim West, Chihunt Wong, Luc Desnoyers, Olga Vasiljeva, Jennifer Richardson, Krishna Polu, Henry Lowman. Transforming Notch ligands into tumor-antigen targets: A Probody-Drug Conjugate (PDC) targeting Jagged 1 and Jagged 2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2665. doi:10.1158/1538-7445.AM2014-2665

Abstract 4570: Development of a proteolytically activatable EGFR Probody for cancer therapy.

Antibodies directed to specific disease-related antigens have proven to be very successful therapeutics for a variety of disease indications. In spite of their high affinity and specificity for target antigen, target-mediated toxicity constitutes a major limitation for the development of antibodies to certain targets. We have addressed this type of on-target toxicity by developing a new class of targeting antibodies (Probody™ therapeutics) that remain in an inert, masked form until proteolytically activated at the site of disease. As a proof-of-concept for the construction of a Probody, we used cetuximab as a starting point. Cetuximab is an EGFR-targeted antibody approved for the treatment of colorectal and head-and-neck cancers that produces an on-target toxicity in the form of a skin rash that afflicts 88% of patients treated with the antibody. We engineered an EGFR Probody by incorporating an inhibitory masking peptide fused to the antibody light chain. Masking of the Probody is achieved through a linker that also incorporates a substrate that is cleaved by one or more proteases up-regulated in cancer. In vitro, EGFR binding and cell-based activities of the masked Probody were diminished compared to those of cetuximab, but treatment with exogenous target proteases activated the Probody and restored activity comparable to cetuximab. Using tumor xenograft models in mice, we demonstrated that the Probody remained masked in systemic circulation but was activated and accumulated in the tumor microenvironment. Tumor activation of the Probody translated to efficacy similar to that seen with cetuximab. Consistent with our results in mice, the Probody remained efficiently masked in non-human primates and did not cause skin toxicity such as that observed in animals treated with cetuximab. Together, these results demonstrate that antibody activity can be specifically targeted to diseased tissue by utilizing locally overexpressed proteases as activating agents, suggesting that a variety of antigens not previously amenable to an antibody therapeutic approach may be successfully addressed with Probodies. Citation Format: Luc R. Desnoyers, Annie Yang, Tony W. Liang, Stephen Moore, Jason Sagert, Daniel R. Hostetter, Elizabeth Menendez, Fei Han, Michael Krimm, Ken Wong, Jennifer Richardson, Jim W. West, Shouchun Liu, Olga Vasiljeva, Henry B. Lowman. Development of a proteolytically activatable EGFR Probody for cancer therapy. [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 4570. doi:10.1158/1538-7445.AM2013-4570

Abstract C165: Development of a probody drug conjugate (PDC) against CD166 for the treatment of multiple cancers

Antibody drug conjugates (ADCs) have shown their greatest clinical utility when targeting antigens expressed at very high levels on cancer cells that have coincidentally lower expression in normal tissues. This is exemplified by the approvals of trastuzumab emtansine for her2neu 3+ breast cancer and brentuximab vedotin for Hodgkins Disease and Anaplastic large-cell lymphoma. Both drugs are approved for subsets of specific cancer types where target antigen expression is particularly high relative to expression in normal tissues. There are other cell surface antigens that are highly expressed on cancer cells and normal tissues, but the utility of such antigens as ADC targets is restricted by their corresponding expression in normal tissues. One such target is CD166 (ALCAM), which shows 3+ expression by IHC in most donors of multiple cancer types, e.g., ca. 70% prevalence in breast, prostate, and lung cancers but also expression in multiple normal tissues including lung, GI tissues, and liver. Thus CD166 has not been progressed as a target for ADCs. Probody™ therapeutics are fully recombinant antibody prodrugs that are converted to active antibodies by tumor-associated proteases. Preclinical in vivo studies show that Probody therapeutics remain substantially inactive in normal tissues and in circulation. As such, Probody drug conjugates (PDCs), unlike ADCs, enable targeting of high expression tumor targets that are also expressed in normal tissues. We have developed an anti-CD166 Probody therapeutic selected for specific binding, internalization, and cross reactivity to cynomolgus macaque as a species for toxicology assessments. This therapeutic has been conjugated to spdb-DM4 and tested in preclinical models of efficacy and safety. Treatment with the PDC has led to complete regressions in models of lung and breast cancer at therapeutically relevant doses. These same doses were assessed for safety in cynomolgus monkeys. The safety and efficacy profiles for the anti-CD166 PDC are supportive of progression to clinical development of this anti-CD166 Probody drug conjugate. Citation Format: Annie Yang Weaver, Shweta Singh, Amy DuPage, Jason Sagert, Jeanne Flandez, Elizabeth Menendez, Judi Ford, Michael Krimm, Stephen Moore, Margaret Nguyen, Andrew Jang, Eric Brecht, Yuanhui Huang, Linnea Diep, Nicole Lapuyade, Tereza Sputova, James West, Olga Vasiljeva, Shouchun Liu, Jennifer Richardson, W. Michael Kavanaugh, Jonathan A. Terrett, Luc R. Desnoyers. Development of a probody drug conjugate (PDC) against CD166 for the treatment of multiple cancers. [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 C165.

Abstract 2664: An anti-Jagged-1/-2 Probody demonstrates inhibition of Jagged-dependent Notch signaling and is activated in multiple types of tumors

Probodies represent a unique class of antibody therapeutics that specifically target therapeutic activity to diseased tissues, such as cancer. A Probody is a fully recombinant protein that contains a masking peptide that blocks the antibody9s antigen-binding site and is linked to the light chain of the antibody by a protease-cleavable peptide substrate. Probodies are designed to be inactive in circulation and in healthy tissues but activated within the tumor microenvironment. The Probody™ approach is designed to widen the therapeutic index of antibodies to promising therapeutic targets that may be limited by significant toxicity. One such promising set of targets are components of the Notch pathway, which is involved in both oncogenic and healthy tissue signaling. We have developed a fully human IgG1 monoclonal antibody that binds and inhibits the activity of both human and mouse Jagged-1 and Jagged-2, two key ligands in the Notch signaling pathway. A Probody produced from this antibody induced dose-dependent inhibition of Notch signaling, resulting in anti-tumor activity in in vivo tumor models without significant toxicity. To further explore the clinical relevance of the anti-Jagged Probody for the treatment of cancer, we evaluated (i) the heterogeneity of Jagged expression in multiple tumor types, (ii) the activation of the Jagged Probody in tumor biopsies, and (iii) the blockade of Notch signaling using an intracellular pharmacodynamic biomarker. Patient tumor tissues were screened for Jagged-1/-2 IHC across lung, pancreatic and breast cancer_ indications where activation of the Notch pathway is implicated. Overall, more than 75% of patient tumors demonstrated moderate to high Jagged-1/-2 expression. We also developed and validated a technique termed IHZ™ analysis that enables the measurement of proteolytic Probody activation and binding to Jagged ligands ex vivo in human tumor sections. We evaluated 120 patients across the three tumor types and observed ∼99% of samples that were positive for antibody binding were also positive by IHZ analysis, signifying sufficient proteolytic activity to activate the Probody. Finally, we evaluated the accumulation of the Notch Intracellular Domain (NICD), a biomarker of Notch signaling, in the nucleus of tumor cells in animals treated with antibody. Notably, our data showed that Notch signaling blockade as indicated by a decrease in the accumulation of NICD correlated with dose-response in in vivo models. Taken together, our data suggest that the anti-Jagged-1/-2 Probody may have wide therapeutic utility as a tumor-specific inhibitor of Jagged-dependent Notch signaling in oncology. Citation Format: Olga Vasiljeva, Elizabeth Menendez, Jason Sagert, James W. West, Jennifer Richardson, Luc Desnoyers, Shouchun Liu, Judi Ford, Krishna Plou, Henry Lowman. An anti-Jagged-1/-2 Probody demonstrates inhibition of Jagged-dependent Notch signaling and is activated in multiple types of tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2664. doi:10.1158/1538-7445.AM2014-2664

Abstract 2975: Development of a probody drug conjugate (PDC) targeting CD71 for the treatment of solid tumors and lymphomas

The targets of Antibody Drug Conjugates (ADCs) have typically been selected by identifying transmembrane antigens that are highly expressed in tumors but are low or absent in normal tissues. The number of potential ADC targets meeting these requirements is limited, either because expression in tumors is not high enough for optimal efficacy, or because expression in normal tissues is too high, leading to toxicity. Probody (TM) therapeutics are antibody prodrugs designed to remain inactive until proteolytically activated in the tumor microenvironment. Probody technology therefore has the potential to enable targeting of more desirable tumor antigens with higher, more persistent and more homogeneous expression in tumors, while limiting toxicity due to interaction with these antigens in normal tissues. CD71 (transferrin receptor) is an example of a highly desirable ADC target, because of its well-characterized ability to efficiently internalize and deliver an ADC payload intracellularly. Further, CD71 is expressed at homogeneously high levels (3+ expression by IHC) in almost all tumor types, including in metastatic disease. However, because CD71 is also expressed on multiple normal cell types, including many progenitor hematological cells, we reasoned that a CD71-targeted ADC would be challenging to develop. To enable targeting of CD71, we have developed an anti-CD71 Probody Drug Conjugate (PDC) CX-2005, which can be activated by multiple proteases in the tumor microenvironment, but which remains in a relatively inactive form while in circulation and in normal tissues. CX-2005 produces complete tumor regressions at therapeutic doses in mouse models of lymphoma, breast cancer and lung cancer. Consistent with our hypothesis that it would be difficult to develop an anti-CD71 ADC, treatment of cynomolgus monkeys with an anti-CD71 ADC at doses that were efficacious in mouse tumor models caused life-threatening depletion of CD71-expressing hematopoietic cells, including neutrophils, lymphocytes and RBCs. In contrast, these toxicities were not observed in monkeys treated with the same dose of the anti-CD71 PDC, consistent with the Probody therapeutic avoiding interaction with these normal cells. Our data demonstrate that, in preclinical studies, Probody drug conjugates can safely and effectively target attractive tumor antigens like CD71 which have been difficult to effectively approach with traditional ADCs due to their expression on critical normal tissues. Further, our data support the development of Probody therapeutics directed against CD71 in multiple different cancers. PROBODY is a trademark of CytomX Therapeutics, Inc. Citation Format: Shweta Singh, Amy DuPage, Annie Yang Weaver, Jason Sagert, Clayton White, Michael Krimm, Yuanhui Huang, Linnea Diep, Kim Tipton, Shouchun Liu, Jennifer Richardson, W. Michael Kavanaugh, Jonathan A. Terrett, Luc R. Desnoyers. Development of a probody drug conjugate (PDC) targeting CD71 for the treatment of solid tumors and lymphomas. [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 2975.