James William West

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 SY09-01: Next-generation anti-CTLA-4 antibodies

The activity of ipilimumab as a single agent and in combination with nivolumab (anti-PD-1) in melanoma, as well as the use of ipilimumab/nivolumab combinations in other malignancies, has confirmed the importance of CTLA-4 blockade in immunotherapy. The antitumor effect of this treatment also results in significant immune-related adverse events that limit dosing and result in patient discontinuation. We have taken two approaches to alter the activity of ipilimumab so as to improve its potency and its safety profile. One approach is to enhance the antibody-dependent cellular cytotoxicity (ADCC) activity of ipilimumab in order to increase the potential for Treg depletion at the tumor site; this would be expected to increase the activity of the antibody. The second approach is to produce a prodrug form of ipilimumab (an anti-CTLA-4 Probody therapeutic) that will have reduced activity systemically, but will become proteolytically cleaved at the tumor site to produce the fully functional antibody; the goal of this approach is to reduce the adverse event profile while retaining the antitumor activity of ipilimumab. It has previously been shown that antitumor activity of anti-CTLA-4 antibodies in mouse models of cancer is dependent on the ability of the antibody to bind activating FcγRs and mediate ADCC against Tregs at the tumor site (1, 2). Although human IgG1 Abs have been shown to be effective mediators of ADCC in patients with hematologic malignancies, it is still unclear whether ipilimumab mediates Treg depletion in solid tumors. Using in vitro ADCC assays, we have found that a nonfucosylated (NF) version of ipilimumab (ipilimumab-NF) has increased activity compared to ipilimumab. Ipilimumab-NF also demonstrates increased IL-2 secretion in peripheral mononuclear cells treated with the superantigen SEB as compared to ipilimumab. Transgenic mice that express human FcγRs in the place of mouse FcγRs were used to investigate the activity of anti-mouse surrogate CTLA-4 antibody engineered with either a human IgG1 or human IgG1-NF Fc region in a mouse tumor model. In these mice, the IgG1-NF version of anti-mouse CTLA-4 was found to significantly increase antitumor activity and Treg depletion at the tumor site compared to the IgG1. These data suggest that the clinical activity of ipilimumab could be enhanced by use of the nonfucosylated version of the Ab. In addition, ipilimumab-NF was tested for its ability to enhance a vaccine response in Mauritian cynomolgus macaques. Ipilimumab-NF was shown to result in increased vaccine-induced T-cell responses compared to ipilimumab using two replication-incompetent adenovirus serotype 5 viral vectors encoding SIV antigens as assessed by MHC-I tetramers and IFN-gamma ELISPOT in Mauritian cynomolgus macaques expressing the common allele, Mafa-A1*063. In a second approach, using Probody platform technology developed by CytomX, we have developed an anti-CTLA-4 Probody therapeutic (Probody Tx) based on ipilimumab. Probody Txs utilize a masking peptide that binds to the antigen-binding site of the Ab to reduce target binding. The mask extends from the light chain of the Ab via a linker sequence that contains cleavage sites for proteases preferentially active at the tumor site relative to healthy tissue. The ipilimumab Probody Tx binds to CTLA-4 with significantly lower affinity than the parental antibody and has reduced activity in in vitro assays. When tested in a mouse tumor model using human CTLA-4 KI mice, the ipilimumab-Probody Tx has comparable antitumor activity and Treg depletion at the tumor compared to ipilimumab. In contrast, ipilimumab-Probody Tx-treated mice show reduced levels of activated peripheral Tregs compared to ipilimumab-treated mice, even at doses 8-fold higher than are required for antitumor efficacy, consistent with reduced activity of the Probody Tx outside the tumor microenvironment. The development of next-generation anti-CTLA-4 antibodies holds promise for improving the utility of ipilimumab for single-agent or combination therapy. The two improvements to ipilimumab outlined above could each lead to a superior therapeutic outcome and merit further investigation. References 1. Selby MJ, Engelhardt JJ, Quigley M, Henning KA, Chen T, Srinivasan M, et al. Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells. Cancer Immunol Res 2013;1:32-42. 2. Simpson TR, Li F, Montalvo-Ortiz W, Sepulveda MA, Bergerhoff K, Arce F, et al. Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma. J Exp Med 2013;210:1695-710. 1. Citation Format: Alan J. Korman, John Engelhardt, John Loffredo, Jose Valle, Rahima Akter, Raja Vuyyuru, Natalie Bezman, Paula So, Robert Graziano, Kimberly Tipton, James West, Bryan Irving, Mark Selby. Next-generation anti-CTLA-4 antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr SY09-01. doi:10.1158/1538-7445.AM2017-SY09-01

Abstract A081: A PD-L1-targeted Probody provides antitumor efficacy while minimizing induction of systemic autoimmunity

Antibodies to T cell checkpoint molecules like PD-1, PD-L1 and CTLA-4 can unleash potent and durable anti-tumor immunity in many different cancer types. These drugs induce anti-tumor immune responses by activating T cells in tumors, but because similar mechanisms control anti-tumor immunity and self-tolerance, they can also induce systemic autoimmunity by activating autoreactive T cells in normal tissues. Combinations of checkpoint inhibitors greatly increase clinical responses, but similarly increase these toxicities, thereby limiting their clinical utility. New methods are therefore needed that provide anti-tumor activity without deregulating systemic immunity. Probody™ therapeutics are recombinant, proteolytically-activated antibody prodrugs, designed to widen therapeutic index by minimizing interaction with normal tissue and maximizing interaction with tumor. Probodies exploit the fundamental dysregulation of extracellular protease activity that exists in tumors relative to healthy tissue. The amino terminus of the antibody light chain is extended with a protease-cleavable linker and a masking peptide designed to block antigen binding to the antibody. Cleavage of the linker by specific tumor-associated proteases leads to dissociation of the mask and release of an antibody competent to bind to antigen in the tumor. Probodies bind only minimally to antigen in healthy tissue because there are insufficient active proteases present to remove the mask. The feasibility of the Probody approach to checkpoint targets is supported by preclinical studies in which intratumoral delivery of low dose immune modulators enables durable, systemic anti-tumor responses despite negligible systemic exposure. Here we demonstrate the ability of a Probody targeting PD-L1 to provide equivalent anti-tumor efficacy in mice to that of its antibody parent, while minimizing induction of systemic autoimmunity. A mouse/human cross-reactive anti-PD-L1 antibody was generated by phage display, and engineered into a Probody. Binding to mouse PD-L1 by the Probody in its masked state was significantly reduced relative to its binding following proteolytic removal of the mask. The presence of active proteases capable of releasing the Probody9s mask was demonstrated in frozen sections of MC38 and CT26 murine syngeneic tumors. Systemic administration of the PD-L1 Probody to mice bearing established MC38 syngeneic tumors induced anti-tumor efficacy comparable to the unmasked parental antibody at the same dose. In contrast, the PD-L1 Probody provided protection from induction of autoimmune diabetes in NOD mice at doses sufficient to induce maximal diabetes with the parental antibody. Finally, PD-L1 occupancy by the Probody on peripheral blood and splenic T cells was significantly reduced compared to that of the parental antibody at the same dose. This finding is consistent with the reduced ability of the Probody in its masked form to bind to PD-L1 and to induce autoimmunity outside of the tumor. By demonstrating equivalent efficacy with an improved safety profile, these results provide validation of the Probody concept for a T cell checkpoint target. Probodies should expand opportunities for immunotherapies, particularly those that are limited by enhanced toxicities when used in combination. Citation Format: Chihunt Wong, Li Mei, Kenneth R. Wong, Elizabeth E.M. Menendez, Olga Vasiljeva, Jennifer H. Richardson, James W. West, Michael Kavanaugh, Bryan A. Irving. A PD-L1-targeted Probody provides antitumor efficacy while minimizing induction of systemic autoimmunity. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A081.

Abstract 3211: PD-1-targeted Probody therapeutics provide anti-tumor efficacy and a 10-fold dose protection against systemic autoimmunity in preclinical studies

Immuno-oncology approaches have transformed the treatment of advanced melanoma, NSCLC and an increasing number of other cancers. Antibodies targeting the T cell checkpoint molecules CTLA-4 and PD-1 can reverse tumor suppression of T cell activation that prevents effective anti-tumor immunity. However, blockade of checkpoint molecules, particularly in combination, in non-tumor tissues can result in autoimmune side effects that can limit therapeutic utility. Combination of PD-1 and CTLA-4 blockade provides increased response rates but with concomitant increases in immune-related adverse events. Here we demonstrate that Probody™ therapeutics targeting PD-1 synergize with a CTLA-4 checkpoint inhibitor to eradicate established tumors in a mouse model while protecting against PD1-mediated systemic autoimmunity. Probody therapeutics are recombinant, proteolytically-activated antibody prodrugs that have the potential to meaningfully widen therapeutic index by minimizing interactions with normal tissue while retaining anti-tumor activity. Probody therapeutics take advantage of protease activities that are abundant in tumors but suppressed in normal tissues. An extension of the antibody light chain with a masking peptide blocks binding of the antibody to antigen in normal tissue. Tumor-associated proteases cleave and release the mask, enabling the active antibody to bind antigen preferentially in the tumor. The feasibility of the Probody approach to checkpoint targets is supported by preclinical and clinical studies in which intratumoral delivery of low dose immune modulators enables anti-tumor responses despite negligible systemic exposure. For preclinical assessment of PD-1 as a Probody target, a family of Probody therapeutics derived from the anti-mouse PD-1 antibody J43 was developed. The intact Probody therapeutics demonstrated reduced binding to mouse PD-1 relative to the parental antibody that was completely restored following proteolytic activation. The anti-tumor efficacy of the J43 antibody and Probody therapeutics was assessed following systemic administration as single agents or in combination with an anti-CTLA-4 antibody to mice bearing established MC38 syngeneic tumors. As single agents, the PD-1 antibody and Probody therapeutics reduced tumor growth relative to an isotype antibody control and synergized with CTLA-4 blockade. Consistent with their reduced ability to bind target in the absence of proteolytic activation, the PD-1 Probody therapeutics were up to 10 times less potent than the parental anti-PD-1 antibody in inducing autoimmune diabetes in NOD mice. Our preclinical findings demonstrate that PD-1 Probody therapeutics retain anti-tumor efficacy with improved safety profiles and therefore have the promise to enable better tolerated PD-1 combination immunotherapies. PROBODY is a trademark of CytomX Therapeutics, Inc. Citation Format: Kimberly A. Tipton, Chanty Chan, Kenneth R. Wong, Victoria Singson, Jennifer H. Richardson, W Michael Kavanaugh, Bryan A. Irving, James W. West. PD-1-targeted Probody therapeutics provide anti-tumor efficacy and a 10-fold dose protection against systemic autoimmunity in preclinical studies. [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 3211.