Abstract 2846A: CD28 shedding is a novel immune-regulatory mechanism found in cancer patients which directly inhibits anti PD-1 effect
Authors: Hakim Motti , Dror Anna Fridman , Alishekevitz Dror , Meilin Edna , Shilovizky Orit , Weber Jeffrey S. , Sapir Yair , Shulman Avidor , Moshe Tehila Ben 
Published: September 2020

Abstract
Background
The interaction between the CD28 receptor and its B7 ligands has a crucial role in the induction of a potent and persistent anti-tumor T cell response. Indeed, the most effective immune oncology treatments target CTLA-4 and PD-1, proteins that directly downregulate CD28 activity, further emphasizing the need for better understanding of the regulation of CD28.
Shedding of immune receptors is one mechanism that the immune system employs to modulate signalling cascades. Here, we describe, for the first time, the finding that membranal CD28 is actively shed by specific proteases upon T cell stimulation in humans, and its implications on immune oncology.
Methods
The detection of soluble CD28 in plasma of cancer patients and in the culture media of stimulated T cells was achieved using a proprietary in-house ELISA. Cytokine levels were measured by ELISA. The effect of recombinant human CD28 on immune cells was studied using superantigen stimulations, MLR, flow cytometry and time-lapse live-cell-microscopy. MOG immunized splenocytes were used in ex-vivo recall assay.
Results
Elevated levels of soluble CD28 were found in 15% of plasma samples from cancer patients across various tumor types. The origin of soluble CD28 can be ascribed to proteolytic cleavage of the receptor, as treatments with protease inhibitors diminished the amount of soluble CD28 in a concentration-dependent manner. A variety of T cell stimulations in vitro resulted in release and accumulation of soluble CD28. Moreover, the shedding of membranal CD28 is found to be a continuous process even after removal of the stimuli. The proteolytic site was mapped to the stalk region of human CD28. This stalk region is not conserved in mice, and indeed soluble CD28 was not detected in MOG stimulated mice splenocytes, suggesting that the shedding mechanism may be unique to humans. In various MLR assays, the presence of soluble CD28 resulted in inhibition of effector cytokine secretion and T cells clustering together with elevation in tumor supportive cytokines, such as IL-6, IL-10 and IDO. Importantly, soluble CD28 counteracted anti PD-1 activity and inhibited the secretion of inflammatory cytokines, such as IL-2 and IFNγ. Evaluation of plasma samples from patients undergoing anti-PD1 therapy demonstrated an association of clinical response with decreased plasma levels of soluble CD28.
Conclusions
We report here the discovery of a novel regulatory mechanism which blocks the CD28/B7 activation pathway by generating a decoy human CD28 receptor. This regulatory mechanism occurs in the setting of cancer as aberrant levels of soluble CD28 were found in the plasma of cancer patients, where its presence was associated with reduced response to anti-PD1 therapy.
The development of therapeutic drug candidates for the inhibition of this regulatory mechanism is in progress.
Citation Format: Motti Hakim, Anna Fridman Dror, Dror Alishekevitz, Edna Meilin, Orit Shilovizky, Jeffrey S. Weber, Yair Sapir, Avidor Shulman, Tehila Ben Moshe. CD28 shedding is a novel immune-regulatory mechanism found in cancer patients which directly inhibits anti PD-1 effect [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2846A.