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1064 Characterization of preclinical anti-tumor and pharmacodynamic activity in response to conditionally active IFNa with or without checkpoint blockade
  1. Benjamin Povinelli,
  2. Kamaljeet Kaur,
  3. Alexey Berezhnoy,
  4. Hsin Wang,
  5. Nicole Lapuyade,
  6. Carol LePage,
  7. Michael Winter,
  8. Olga Vasiljeva,
  9. Madan Paidhungat,
  10. Vangipuram Rangan,
  11. Erwan Le Scolan,
  12. Leila Boustany,
  13. Marcia Belvin and
  14. Dylan Daniel
  1. CytoX Therapeutics, South San Francisco, CA, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


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.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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