Cynomolgus Monkey Invariant NKT (iNKT) Cells Recover Following Depletion by Treatment with the Anti-Invariant T Cell Receptor Antibody NKTT120

Abstract

4836 Invariant Natural Killer T (iNKT) cells are a small subset of T lymphocytes (ranging from 0.01 – 0.1% of CD3+ T cells) that share surface markers and functional characteristics with T cells and natural killer (NK) cells. Unlike other T cells, they recognize glycolipid antigens presented by the MHC class-I-like protein CD1d rather than peptide antigens. In contrast to most T cells which express diverse T cell receptor (TCR) sequences, iNKT cells express a unique, highly conserved, semi-invariant TCR-α chain (Vα24-Jα18 in humans), which preferentially pairs with specific TCR-β chains (Vβ11 in humans). Like cells of the innate immune system, iNKT cells are rapid-onset cells with a universal receptor. They also share properties of T cells like requiring thymic positive selection and recognition of antigen presented on the MHC-I like molecule CD1d. As such, they serve as a bridge between the innate and adaptive immune systems. They can play either a pro-inflammatory role to enhance or an immuno-regulatory role to attenuate a developing immune response. iNKT cells have been shown to be involved in mediating tissue injury and inflammation in multiple organ systems. There is a growing recognition that chronic inflammation is associated with the pathophysiology of Sickle Cell Disease (SCD). Wallace et al. (Blood 114:667, 2009) found an increased ratio of activated iNKT cells in peripheral blood of patients with SCD compared to normal volunteers. Our study of peripheral blood iNKT cells from 5 SCD patients and 10 unaffected African American volunteers found that an average of 50.5% of the iNKT cells of the SCD patients were activated as indicated by up regulation of the lymphocyte activation marker CD69. In the healthy controls an average of only 8.9% of iNKT cells expressed CD69. A key role of iNKT cell activation in the pathology of SCD is supported by studies in a mouse model of SCD (Blood 114:667, 2009) that suggest iNKT cell depletion could reduce inflammation in the SCD patient. We have developed a humanized monoclonal antibody (NKTT120) that binds the CDR3 loop of the human and the old-world non-human primate invariant TCR with high specificity. NKTT120 depletes iNKT cells in human iTCR transgenic mice and cynomolgus monkeys (Macaca fascicularis). In the transgenic mice, iNKT cells reappear in the peripheral circulation within weeks after complete depletion. We conducted a study in cynomolgus monkeys to explore the relationship between dose and duration of iNKT cell depletion. Each group of animals (n=3) received a single dose of NKTT120. The dosing groups were 10, 30, 100 and 300 ug/kg, respectively. iNKT cells and other lymphocytes were monitored 24, 48, 72, 96 and 168 hours following dosing and weekly thereafter using flow cytometry. In all dose groups, iNKT cells were depleted within 24 hours with no significant changes in the other cells of the lymphocytic series. No adverse events have been noted to date. iNKT cells in the 10 ug/kg and the 30 ug/kg dose group were recovered by week 5 and week 7 respectively. As of week 12 post-dosing, iNKT cells in the 100 and the 300 ug/kg dose groups have not recovered. The kinetics of recovery in these higher dose animals will shed light on the impact of what is expected to be full tissue depletion on iNKT cell recovery. Overall, our study has shown that we can safely deplete iNKT cells in non-human primates and that iNKT cells can recover after depletion. Percent of Cells (Mean±SD) Estimated by FACS Analysis Before and Following NKTT120 IV Administration Shows Recovery Following Depletion Lymphocyte Profiles Pre Sample Day 1 Post Dose Week 5 Post Dose Week 7 Post Dose 10 ug/kg 30 ug/kg 100 ug/kg 300 ug/kg 10 ug/kg 30 ug/kg 100 ug/kg 300 ug/kg 30 ug/kg 100 ug/kg 300 ug/kg iNKT Cells* 0.29±0.17 0.02±0.03 0 0 0 0.14±0.12 0.01±0.005 0 0 0.05±0.01 0 0.01±0.01 NK Cells** 11.6±3.9 8.1±1.8 8.5±2.1 7.8±4 11.1±2.5 11.4±2.6 16.2±4.3 8.9±3.1 14.3±4.3 13.2±3.1 9.2±4.1 13.2±2.5 Mature T cells** 73.1±5.5 69.6±4 70±7.5 75.9±4.3 65.1±3.6 70.6±4.5 67.4±5.1 78.2±5.7 66.5±4.4 72.3±4.9 80.8±6.3 69.3±3.2 CD8 T Cells** 26.0±4.7 22.1±4.2 20.7±2.8 27.8±3.5 22.4±4.1 26.2±5.5 24.1±6.3 31.8±3.7 26.5±6.1 25.4±5.8 32.7±5.1 27.2±6.9 CD4 T Cells** 42.0±4 44.3±7.9 45.2±5.2 43.2±1.5 38.3±4.1 41.1±6.9 39.5±1.7 40.9±2.6 35.4±3.3 43.2±7.7 42.2±0.7 37.4±5.4 B Cells** 3.4±2.1 8.9±2.4 6.8±1.6 5.1±1 8.5±5.1 7±1.1 7.4±2.9 4.3±0.6 7.8±4.9 5.5±1.5 2.4±0.2 6.1±3.5 * % of CD3 ** % of Lymphocytes Disclosures: Scheuplein: NKT Therapeutics: Employment, Equity Ownership. Macdonald:NKT Therapeutics: Employment, Equity Ownership. Zeigler:MPI Research: Employment; NKT Therapeutics: Research Funding. LeBel:MPI Research: Employment; NKT Therapeutics: Research Funding. Thariath:NKT Therapeutics: Employment, Equity Ownership. Truneh:NKT Therapeutics: Consultancy, Equity Ownership. Mashal:NKT Therapeutics: Employment, Equity Ownership. Nathan:NKT Therapeutics: Consultancy. Schaub:NKT Therapeutics: Employment, Equity Ownership.