Abstract 654: CD28 shedding is a novel resistance mechanism to anti PD-1 therapy
Authors: Hakim Motti , Dror Anna Fridman , Gabay Tal Shahar , Alishekevitz Dror , Meilin Edna , Mandel Ilana , Sapir Yair , Shulman Avidor , Moshe Tehila Ben 
Published: June 2022

Abstract
Background: Resistance mechanisms to anti-PD1 therapy remain poorly understood. Studies have implicated that efficient anti-PD1 therapy relies specifically on intact CD28/B7 signaling. We have previously shown that membranal CD28 is actively shed by MMP-2 and MMP-13 upon T cell stimulation in humans, and that soluble CD28 can counteract the efficacy of anti-PD1 blocking antibodies in-vitro. Here we describe that CD28-shedding process is a potential novel resistance mechanism to PD-1 therapies in cancer patients, by using several ex-vivo and in-vivo methodologies. The prominent unfavorable effect of CD28-shedding on PD-1 blockade prompted the generation of BND-67, an agent that can selectively and efficiently block CD28 shedding.
Methods: Soluble CD28 detection in plasma of cancer patients and in the culture media of stimulated T cells was achieved using a proprietary in-house ELISA. Cancer patient processed tumors and PBMC were used ex-vivo to identify membrane/soluble CD28 by ELISA and flow-cytometry. MC-38 challenged human CD28 transgenic mice (CD28-shedding permissive model), and WT mice (non-permissive CD28-shedding model) were compared for efficacy to anti-PD1 blocking antibody. BND-67 was isolated by screening a VHH library for binding to the CD28-shedding prone region and blocking of CD28 shedding. The lead clone was further affinity-matured and reformatted into a half-life extended construct.
Results: CD28 receptor density was found to be lower on tumor-resident T cells then their matched counterparts in the periphery. Evaluation of plasma samples from patients undergoing anti-PD-1 therapy demonstrated an association of diseases relapse with elevated plasma levels of soluble CD28. Correspondingly, relapses from response to anti-PD-1 therapy were significantly higher in CD28-shedding permissive mice vs non-permissive mice. To overcome this novel regulatory mechanism, we generated BND-67, which is an affinity-matured VHH that can effectively bind CD28, block CD28 shedding and has no agonistic activity. In addition, an optimal approach for increasing the half-life of the agent was identified by fusing the VHH to human Fc.
Conclusions: We report here the discovery of a potential novel resistance mechanism to anti-PD1 blockade involving the shedding of the CD28 receptor. This process occurs in the setting of cancer and was further reinforced by results observed in transgenic mice. Biond has generated BND-67, a therapeutic drug candidate for the inhibition of this novel regulatory mechanism.
Citation Format: Motti Hakim, Anna Fridman Dror, Tal Shahar Gabay, Dror Alishekevitz, Edna Meilin, Ilana Mandel, Yair Sapir, Avidor Shulman, Tehila Ben Moshe. CD28 shedding is a novel resistance mechanism to anti PD-1 therapy [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 654.