Early Reconstitution of CD6+ T Cells after Hematopoietic Cell Transplantation Identifies a Suitable Target for Acute Graft Versus Host Disease Treatment Using Anti-CD6 Monoclonal Antibody Itolizumab
Authors: Rambaldi Benedetta , Kim Haesook T. , Reynolds Carol , Chamling Rai Sharmila , Asano Takeru , Arihara Yohei , Gooptu Mahasweta , Koreth John , Cutler Corey , Nikiforow Sarah , Ho Vincent T. , Antin Joseph H. , Romee Rizwan , Soiffer Robert J. , Ampudia Jeanette , Ng Cherie Tracy , Connelly Stephen , Ritz Jerome 
Published: November 2020

CD6 is a co-stimulatory receptor expressed on T cells that binds activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen-presenting cells and various epithelial and endothelial tissues. The CD6-ALCAM pathway modulates T cell activation, proliferation and trafficking playing a central role in inflammation. Early studies by Soiffer and colleagues demonstrated that ex-vivo depletion of CD6+ donor T cells prior to hematopoietic cell transplantation (HCT) decreased the incidence of acute graft versus host disease (aGVHD), highlighting the importance of CD6+ cells in GVHD pathogenesis. Itolizumab, a humanized IgG1 anti-CD6 monoclonal antibody, has been shown to block CD6 signaling, leading to a reduction in T cell activation and proliferation. The aim of this study was to characterize expression of CD6 early after HCT and to examine the effects of Itolizumab on T cell responses in the setting of aGVHD.
We analyzed immune reconstitution in 95 adult patients who underwent allogeneic HCT for hematological malignancies. Patients received myeloablative (50.5%) or reduced intensity (49.5%) conditioning. Almost all patients received peripheral blood stem cell grafts from HLA-matched related (MRD; 27.4%), matched unrelated (MUD; 66.3%) or mismatched unrelated donors (MMUD; 6.3%). Tacrolimus and methotrexate were used for GVHD prophylaxis. Forty-two of 95 patients developed grade I-IV aGVHD at a median of 50 days (range 20-294) after HCT and systemic immunosuppression was started in 81% of these cases. aGVHD grade severity was 38.1%, 40.5%, 7.1% and 14.3% of grade I, II, III and IV, respectively. Patient samples were collected at 1, 2, 3 and 6 months after HCT and analyzed using multi-color flow cytometry. Nine healthy donors (HD) were analyzed as controls. Suppressive activity of Itolizumab was tested in vitro using peripheral blood mononuclear cells (PBMCs) obtained from HD and patients before (preGVHD) and after aGvHD onset (postGVHD) after 72 hour stimulation with antiCD3/CD2/CD28 coated beads. T cell proliferation was measured by CFSE dilution, while T cell activation and maturation were monitored by expression of CD25 and CD45RO, respectively. To test if Itolizumab activity was dependent on the presence of the CD6 cognate ligand, ALCAM, HD CD3+ T cells were stimulated with anti-CD3 antibody with or without ALCAM-Fc for 96 hours.
CD6+ T cells reconstituted early after transplant, accounting for 77.8% (range, 69.9-93.7) of CD4Treg, 98.1% (range, 96.5-99.5) of CD4Tcon, 84% (range, 77.1-92.1) of CD8 T cells at 1 month. This pattern remained unchanged at 2, 3 and 6 months and in HD. CD6 expression (MFI) was the highest in Tcon and lowest in Treg in patients as well as in HD. Compared to HD, CD6 MFI in CD4 Treg was significantly lower in patients at 1,2,3 and 6 months after HCT (p&lt;0.002 at 1,2,3 months and 0.01 at 6 months) (Fig 1A and 1B). CD6 MFI was significantly lower in post-GVHD Tcon (P=0.0004) and Treg (P=0.04) compared to patients without GVHD (Fig 1C).
Prior to GVHD onset, Itolizumab inhibited CD4 and CD8 T cell proliferation in a similar fashion to HD control. This effect was less prominent in T cells collected after GVHD onset, when patients were already receiving immunosuppressive medications, especially for the CD8 T cells (Fig 1D). Similar results were observed for CD25 and CD45RO expression (Fig 1D). Addition of ALCAM-Fc to anti-CD3 antibody enhanced T cell proliferation, activation and maturation. Itolizumab efficiently inhibited T cell proliferation, activation and maturation in the presence of ALCAM-Fc and anti-CD3 antibody, while no effect was observed in the presence of anti-CD3 antibody alone (Fig 2A). Finally, Itolizumab alone did not induce significant CDC, ADC or ADCC in vitro (Fig 2B).
In conclusion, we demonstrate that CD6 positive T cells reconstitute rapidly after HCT with higher levels of expression in Tcon compared to Treg and CD8 T cells. Percentages of CD6+ T cells do not change during aGVHD, but levels of CD6 expression decrease in Tcon and Treg after aGVHD. Itolizumab efficiently inhibits T cell proliferation and activation after in vitro TCR stimulation of PBMCs from patients with aGvHD. Functional inhibition of the CD6-ALCAM pathway without T cell depletion may be a novel therapeutic strategy for treating aGvHD. A phase I/II study using Itolizumab as first line treatment in combination with steroids for patients with aGVHD is currently ongoing (NCT03763318).

Disclosures
Rambaldi: Equillium: Research Funding. Koreth:EMD Serono: Consultancy; Biolojic Design Inc: Consultancy; Cugene: Membership on an entity's Board of Directors or advisory committees; Regeneron: Other: Research Support; BMS: Other: Research Support; Miltenyi: Other: Research Support; Clinigen: Other; Therakos: Membership on an entity's Board of Directors or advisory committees; Equillium: Consultancy; Moderna Therapeutics: Consultancy; Amgen: Consultancy. Cutler:Generon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Mesoblast: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kadmon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Medsenic: Consultancy, Membership on an entity's Board of Directors or advisory committees. Nikiforow:Kite: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Nkarta: Membership on an entity's Board of Directors or advisory committees. Soiffer:Juno: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees; Be the Match/ National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Cugene: Consultancy; alexion: Consultancy; Kiadis: Membership on an entity's Board of Directors or advisory committees; Precision Bioscience: Consultancy; Mana Therapeutics: Consultancy; Rheos Therapeutics: Consultancy; Novartis: Consultancy; VOR Biopharma: Consultancy. Ampudia:Equillium: Current Employment, Current equity holder in publicly-traded company. Ng:Equillium: Current Employment, Current equity holder in publicly-traded company. Connelly:Equillium: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Ritz:Rheos Medicines: Consultancy; Amgen: Research Funding; Avrobio: Consultancy; Talaris Therapeutics: Consultancy; LifeVault Bio: Consultancy; Infinity Pharmaceuticals: Consultancy; Equillium: Research Funding; Falcon Therapeutics: Consultancy; Kite Pharma: Research Funding; TScan Therapeutics: Consultancy.