Impact of Itolizumab on the Expression of CD6 and the Function of Effector T Cells
Authors: Ampudia Jeanette , Chu Dalena , Badrani Jana , Doherty Taylor , Connelly Stephen , Ng Cherie Tracy 
Published: November 2020

Introduction: CD6 is a T-cell costimulatory receptor that has been implicated in the pathogenesis of multiple autoimmune and inflammatory (AI) diseases. In GVHD, CD6 is expressed on reconstituting T cells soon after transplant (Rambaldi et al., 2019), including Th1 and Th17 cells which are both implicated in the induction and pathogenesis of acute GVHD (aGVHD). CD6 is highly expressed on these cells and promotes immune synapse formation, T-cell activation, and T-cell migration via interaction with its ligand activated leukocyte cell adhesion molecule (ALCAM). Furthermore, studies have demonstrated that ex-vivo depletion of CD6+ donor cells prior to hematopoietic cell transplantation (HCT) decreases the incidence of aGVHD (Soiffer et al., 1992; Soiffer et al., 1998), highlighting the importance of CD6. While the contribution of CD6 to T cell activation has been well described, less is known regarding the expression levels and role of CD6 on effector and memory T cells (Teff) which are prominent in all diseases including aGVHD. Consequently, the aim of this study was to determine the role of CD6 specifically on effector T cells, and further illuminate the mechanism of itolizumab, an anti-CD6 monoclonal antibody.
Methods: Naïve T cells were enriched from frozen PBMCs via a naïve T cell magnetic separation kit (Stemcell). Naïve T cells were polarized towards a Th1 phenotype for 6 days with a Th1 differentiation cocktail (Stemcell) and CD3/CD28 T cell activator (Stemcell) and rested overnight prior to entering restimulation conditions. Naïve T cells were polarized towards a Th17 phenotype for 8 days with IL-6, IL-1β, TGF-β, IL-23, anti-IL-4 and IFN-γ, the CD3/CD28 T cell activator was used to activate the T cells. To re-stimulate differentiated T cells, anti-CD3 mAb and ALCAM-Fc or anti-CD3 mAb alone were coated on 48-well plates overnight at 4oC. Th1 or Th17 T cells were labeled with CFSE and seeded with isotype control or itolizumab for 72hrs. Cells were collected for flow cytometry analysis and supernatant collected for relevant cytokine detection. To assess surface levels of CD6, cryopreserved PBMCs were thawed and incubated with itolizumab or isotype at 37oC for specific timepoints. Following incubation, cells were washed and stored at 4oC for subsequent staining. All samples were stained at the same time and surface levels of CD6 was detected using a monoclonal anti-CD6 antibody that does not compete with itolizumab.
Results: Blockade of the CD6 pathway, using itolizumab during restimulation of differentiated Teff cells in the presence of ALCAM, inhibited multiple effector functions including proliferation and changes in cell size. An average of a 40% decrease in CFSE proliferation was observed across multiple donors. Furthermore, treatment of Teff cells with itolizumab resulted in a significant decrease in expression level of T cell markers of activation and exhaustion such as CD25, PD-1 and Tim3. This effect was exclusively in the presence of ALCAM, indicating that the effect was specific to blockade of the CD6-ALCAM pathway. When levels of CD6 were assessed, CD45RO+ (Teff/mem) cells expressed higher levels than CD45RA+CD45RO- (Tnaive). Itolizumab treatment of in vitro generated CD45RO+ T cells inhibited this stimulation-induced increase in CD6 in a dose-dependent manner, suggesting that the drug may modulate surface CD6 expression as a mechanism separate from physical blockade of CD6.
Conclusions: These findings are the first to characterize the CD6-ALCAM pathway as a key regulator of differentiated effector T-cell function. Modulation of activation markers and CD6 itself by itolizumab, suggest modulation of Teff activity by both direct and indirect inhibition of CD6 signaling. These data further support targeting the CD6-ALCAM pathway to inhibit both naïve and effector T cell populations in aGVHD.

Disclosures
Ampudia: Equillium: Current Employment, Current equity holder in publicly-traded company. Chu:Equillium: Current Employment, Current equity holder in publicly-traded company. Doherty:Equillium Inc.: Research Funding. Connelly:Equillium: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Ng:Equillium: Current Employment, Current equity holder in publicly-traded company.