Background Interferon alpha 2b (IFN) is an approved immunotherapy for treatment of multiple tumors; however, the toxicity of IFNa has limited its clinical use. Using CytomX proprietary Probody® Therapeutics (Pb-Tx) technology, a conditionally active, mouse cross reactive IFN-a (Pb-IFNa-A/D) with minimal activity in its prodrug form, was generated to enable the study of cancer immunobiology in the mouse. Pb-IFNa-A/D is activated by the elevated protease activity associated with the tumor microenvironment (TME), leading to preferential IFN activity in the TME but not in healthy tissues. Pb-IFNa-A/D has displayed robust activity in a range of tumors, including checkpoint non-responsive models. Methods The Pb-Tx platform technology attenuates activity of a molecule by blocking its active regions through affinity or steric interference. Such blockade, termed masking, is reversed upon proteolytic cleavage of a substrate-containing linker between the molecule and the mask by tumor-associated proteases. To investigate the pharmacodynamic activity and evaluate biomarkers related to response to Pb-IFNa-A/D, we screened 25 syngeneic murine tumor models with both monotherapy and PD-1 checkpoint blockade combination. In addition to monitoring efficacy outcomes, tumor tissue and peripheral blood were collected 48 hours post administration for pharmacodynamic response measurement. Results Pb-IFNa-A/D monotherapy demonstrated anti-tumor activity in a range of syngeneic tumor models, including some refractory to PD-1 blockade. Combination of Pb-IFNa-A/D and PD-1 blockade demonstrated enhanced antitumor activity in comparison to PD-1 alone. We assayed peripheral blood cytokine levels 48hrs post administration of Pb-IFNa-A/D and observed a significant increase in chemokines including CXCL10, and cytokines including CCL2/3, while PD-1 blockade showed no significant increase. The combination treatment significantly increased CXCL10 in comparison to both monotherapies. To analyze changes in lymphocyte activation we performed peripheral blood immunophenotyping. Pb-IFNa-A/D and combination treatment demonstrated a significant increase in peripheral blood lymphocyte activation by CD69 and Granzyme B staining and were correlated with response. To evaluate the on-tumor changes in response to Pb-IFNa-A/D, we performed RNA-seq on tumor tissue. We observed an increase in interferon-stimulated genes with Pb-IFNa-A/D and combination treatment. In agreement with peripheral observations, Pb-IFNa-A/D and combination treatment similarly increased Granzyme-B and CXCL10 expression. Conclusions Pb-IFNa-A/D demonstrated robust anti-tumor activity in a range of syngeneic tumor models. Pharmacodynamic activity in the periphery and tumor demonstrates an IFN-stimulated immune response. These data support Pb-IFNa-A/D as a promising clinical candidate as both a single agent and in combination with checkpoint blockade, potentially expanding their benefits to patients with unresponsive tumors.

BackgroundDespite its potential, the toxicity of interferon alpha has limited its clinical use. CytomX proprietary Probody® Therapeutics (Pb-Tx) technology allowed us to create a conditionally active IFN-a2b (Pb-IFN-a2b) with minimal activity in its prodrug form. The prodrug is activated in the tumor microenvironment (TME), leading to preferential activity in the TME but not in healthy tissues. Pb-IFN-a2b demonstrated a substantially enhanced tolerability profile compared to standard IFN therapy without compromising its antitumor effects.MethodsThe Pb-Tx platform technology attenuates activity of a molecule by blocking its active regions through affinity or steric interference. Such blockade, termed masking, is reversed upon proteolytic cleavage of a linker between the molecule and the mask by tumor associated proteases.ResultsPb-IFN-a2b demonstrated considerable reduction (1000-fold or more) of interferon signaling in vitro and significantly reduced immune cell activation. Exposure to viable tumor tissues or tumor-associated proteases in vitro fully restored its bioactivity, including the ability of Pb-IFN to stimulate tumor-infiltrating immune cells. Antitumor activity of the Pb-IFN-a2b in xenograft studies is equal to or greater than Peg-IFN-α2b. In syngeneic mouse tumor models, Pb-IFN demonstrated significant antitumor activity that was further enhanced by PD-(L)1 blockade. Activation of lymphocytes by the molecule was observed in tumors but not in secondary lymphoid organs. Toxicology studies performed in hamsters demonstrated enhanced tolerability of the molecule compared to its unmasked control. In addition, Pb-IFN-a2b suppressed growth of hamster melanoma tumor model RPMI1846 at dose levels that were above the tolerated dose of the unmasked control. Biomarkers of IFN signaling were greatly attenuated in non-human primates compared to the unmasked control. In cynomolgus monkey, Pb-IFN-a2b demonstrated linear pharmacokinetics, extended half-life, and was well tolerated at weekly doses up to 60 mg/kg.ConclusionsPb-IFN-a2b shows improved tolerability and antitumor activity in preclinical studies compared to traditional IFN treatment. These data support Probody cytokine therapeutics as a promising addition to current immunotherapy regimens, potentially expanding their benefits to patients with typically unresponsive tumors.Ethics ApprovalAll animal experiments were reviewed and approved by CytomX’s Institutional Animal Care and Use Committee (IACUC Protocol AP303).

Type I interferons can exert direct antitumor effects, modulate tumor stroma, and induce de novo antitumor immune responses. They have demonstrated combination activity with PD-(L)1 blockade to potentially expand the benefit to patients with unresponsive tumors. Despite its potential, the toxicity of interferon alpha has limited its clinical use. Here we applied CytomX proprietary Probody® Therapeutics (Pb-Tx) technology to create a conditionally active IFN-α2b (Pb-IFN-α2b) with minimal activity in its prodrug form. The prodrug is activated in the tumor microenvironment (TME), leading to preferential activity in the TME but not in healthy tissues. Pb-IFN-α2b demonstrated an enhanced tolerability profile compared to standard IFN therapy without compromising its antitumor effects. The Pb-Tx platform technology attenuates activity of a molecule by blocking its active regions through affinity or steric interference. Such blockade, termed masking, is reversed upon proteolytic cleavage of a substrate-containing linker between the molecule and the mask by tumor associated proteases. Pb-IFN molecules were engineered with a dual masking approach combining the effects of steric inhibition by Fc fusion and affinity interference by a peptide mask. Pb-IFN-α2b demonstrated significant reduction (1000-fold or more) of its specific activity in vitro, including antiproliferative effects and immune cell activation. Treatment with tumor-associated proteases or exposure to viable tumor tissues fully restored its activity. Activated but not masked Pb-IFN-α2b induced a gene expression profile consistent with interferon signaling in primary human immune cells. In vitro studies with dissociated human tumors demonstrated the ability of Pb-IFN to activate tumor immune infiltrate, which could be further enhanced by concomitant PD-L1 blockade. Antitumor activity of the Pb-IFN-α2b in xenograft studies is equal to or greater than Peg-IFN-α2b. Pb-IFN-α2b demonstrated significant antitumor activity in syngeneic mouse tumor models without evidence of toxicity. Consistent with in vitro observations, this anti-tumor activity was further enhanced by PD-(L)1 blockade. Toxicology studies performed in hamsters demonstrated enhanced tolerability of the molecule compared to its unmasked control. Pb-IFN-α2b did not cause hematological changes or body weight loss associated with unmasked interferon. In cynomolgus monkey Pb-IFN-α2b demonstrated linear pharmacokinetics, extended half-life, and was well tolerated at doses up to 15 mg/kg. Pb-IFN-α2b shows improved tolerability and antitumor activity in preclinical studies compared to traditional IFN treatment. These data support Probody cytokine therapeutics as a promising addition to current immunotherapy regimens, potentially expanding their benefits to patients with typically unresponsive tumors. Citation Format: Alexey Berezhnoy, Hsin Wang, Nicole Lapuyade, Na Cai, Carol LePage, Michael B. Winter, Ivan Ye, Hong Lu, Michael Krimm, Ken Wong, Robert T. Dunn, Leila Boustany, Madan Paidhungat, Marcia Belvin, Erwan Le Scolan, Dylan Daniel. Probody-interferon-alpha 2b combines antitumor activity with improved tolerability [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 2071.

Agonist-induced Rap1 GTP loading results in integrin activation involved in T cell trafficking and functions. MRL proteins Rap1-interacting adapter molecule (RIAM) and lamellipodin (LPD) are Rap1 effectors that can recruit talin1 to integrins, resulting in integrin activation. Recent work also implicates direct Rap1-talin1 interaction in integrin activation. Here, we analyze in mice the connections between Rap1 and talin1 that support integrin activation in conventional CD4(+) T (Tconv) and CD25(Hi)Foxp3(+)CD4(+) regulatory T (Treg) cells. Talin1(R35E, R118E) mutation that disrupts both Rap1 binding sites results in a partial defect in alpha(L)beta(2), alpha(4)beta(1), and a alpha(4)beta(7) integrin activation in both Tconv and Treg cells with resulting defects in T cell homing. Talin1(R35E,R118E) Tconv manifested reduced capacity to induce colitis in an adoptive transfer mouse model. Loss of RIAM exacerbates the defects in Treg cell function caused by the talin1(R35E,R118E) mutation, and deleting both MRL proteins in combination with talin1(R35E,R118E) phenocopy the complete lack of integrin activation observed in Rap1a/b-null Treg cells. In sum, these data reveal the functionally significant connections between Rap1 and talin1 that enable alpha(L)beta(2), alpha(4)beta(1), and alpha(4)beta(7) integrin activation in CD4(+) T cells.

BackgroundCytokines have been shown to elicit broad anti-tumor activity in preclinical models. These results have translated into the approval for clinical use of IFN-alpha and IL-2 before the checkpoint therapy era. However, to date, the clinical success of cytokines has been limited by systemic toxicity and poor exposure. CytomX Therapeutics has developed a new class of antibodies called Probody® therapeutics (Pb-Tx), designed to widen the therapeutic window by minimizing binding to targets in healthy tissue while being preferentially activated in the tumor microenvironment (TME) by tumor-associated proteases. CytomX has applied the Pb-Tx platform across multiple modalities including traditional antibodies, antibody-drug conjugates and T-cell engaging bispecifics and has advanced multiple programs into clinical studies. Here we have expanded the Pb-Tx platform with a conditionally activated cytokine version of IFN-α2b that has the potential to improve the therapeutic index of IFN-alpha therapy and allow systemic delivery.MethodsWe engineered an IFN-α2b with a dual masking strategy using a cleavable Fc domain at one end of IFN-a2b, and a cleavable affinity peptide mask at the other end. The construct was optimized to both maximize cleavability and minimize IFN-a2b toxicity. All animal experiments were reviewed and approved by CytomX's Institutional Animal Care and Use Committee (IACUC Protocol AP303).ResultsThe optimized IFN-a2b conditionally activated cytokine strongly reduced IFN-a2b activity in vitro (5,000X) in its dual-masked form. Its activity was fully restored upon protease activation. Transcriptional profiling of in vitro treated PBMC confirmed reduction of interferon-mediated activities of the masked molecule. In vitro studies with dissociated tumors indicated its ability to activate tumor immune infiltrate, that could be further enhanced by concomitant PD-L1 blockade. In mouse xenograft studies, conditionally activated IFN-a2b cytokines induced complete regression at doses as low as 0.1mpk (activity comparable to peginterferon). Surrogate conditionally activated IFN-a2b molecules were also highly potent in syngeneic mice in vivo efficacy studies. Finally, we established an in vivo safety model in hamster which has been shown to be sensitive to IFN-a-mediated toxicity in the liver and bone marrow. In hamster, we showed that conditionally activated IFN-a2b cytokines are well tolerated up to 15mpk and have reduced systemic IFN-a2b mediated toxicity as compared to the unmasked cytokine.ConclusionsTaken together these preclinical data further support the development of conditionally activated IFN-a2b with the potential to improve the therapeutic index of IFN-a therapy and to enable single agent and combination treatment in multiple clinical settings.Ethics ApprovalAll animal experiments were reviewed and approved by CytomX's Institutional Animal Care and Use Committee (IACUC Protocol AP303).

Integrin activation mediates lymphocyte trafficking and immune functions. Conventional T cell (Tconv cell) integrin activation requires Rap1-interacting adaptor molecule (RIAM). Here, we report that Apbb1ip−/− (RIAM-null) mice are protected from spontaneous colitis due to IL-10 deficiency, a model of inflammatory bowel disease (IBD). Protection is ascribable to reduced accumulation and homing of Tconv cells in gut-associated lymphoid tissue (GALT). Surprisingly, there are abundant RIAM-null regulatory T cells (T reg cells) in the GALT. RIAM-null T reg cells exhibit normal homing to GALT and lymph nodes due to preserved activation of integrins αLβ2, α4β1, and α4β7. Similar to Tconv cells, T reg cell integrin activation and immune function require Rap1; however, lamellipodin (Raph1), a RIAM paralogue, compensates for RIAM deficiency. Thus, in contrast to Tconv cells, RIAM is dispensable for T reg cell integrin activation and suppressive function. In consequence, inhibition of RIAM can inhibit spontaneous Tconv cell–mediated autoimmune colitis while preserving T reg cell trafficking and function.