Effective Treatment of Resistant Hematological Malignancies by IL-2 Activated NK Cells Using Haploidentically Mismatched Donors by Induction of Graft-Versus-Tumor (GVT) Effect While Avoiding GVHD.
Authors: Slavin Shimon , Or Reuven , Ackerstein Aliza , Samuel Simcha , Shapira Michael Y. , Resnick Igor , Bitan Menachem , Aker Memet , Gelfand Yael , Eizik Osnat , Morecki Shoshana 
Published: April 2020

Abstract
Whereas some patients with chemotherapy-resistant hematological malignancies may be successfully treated by allogeneic stem cell transplantation (SCT) through induction of graft-versus-tumor (GVT) effects, others may fail to respond or succumb to uncontrolled acute and/or chronic GVHD. We have previously documented that GVT effects induced by intentionally mismatched lymphocytes are much more potent and occur much faster, however, control of GVHD is mandatory. A pilot clinical trial was conducted in 19 patients (age 4–63): 6 AML; 2 ALL; 2 biphenotypic leukemia; 3 RAEB-t; 4 NHL; 2 Hodgkin’s; all fully resistant to chemotherapy (4 failing prior autologous and 1 prior allogeneic SCT). A total of 15 received haploidentical related transplant; 4 received matched sibling and 1 received matched unrelated donor. Prevention of rejection of mismatched allografts was accomplished by activation induced clonal deletion following infusion of donor with buffy coat 24 hours before administration of Cytoxan 120–180mg/kg to eliminate host anti-donor activated T lymphocytes. Additional conditioning included Fludarabine 30mg/m2 x 6; Busulfex 3.2mg/kg x 2 or low dose TBI 200–400 cGy ± Melphalan 70–140mg/m2 and ATG 40mg/kg or Alemtuzumab (60mg). Positively selected CD34+ cells mobilized with G-CSF were infused on day 0. Purified CD34+ cells were isolated by Miltenyi’s cliniMACS following mobilization with G-CSF. Based on our preclinical work suggesting that fully mismatched isolated NK cells cause no GVHD, 22 procedures were done involving administration of purified NK cells 1–15 months post SCT. Donor mononuclear cells were activated for 4 days at 37oC in 5% CO2 in air with rIL-2 (6,000 I.U./ml) and administered on day 4. NK cells were positively selected with Miltenyi’s anti-CD56 (n=15) or by anti-CD3 immunomagnetic beads (n=3). In 4 cases, CD56+ cells were separated before rIL-2 culturing. All 4 patients with matched donors received treatment of overt relapse despite GVHD. One, receiving the treatment prophylactically after transplant, is alive and well &gt;29 months. Of 15 recipients of haploidentically mismatched allografts, 5 entered CR. One is alive and well after failing 2 prior transplants &gt;27 months with no GVHD at all. Three patients died with no evidence of disease: 1 due to hepatitis B; 1 due to aspergillosis that was present before transplant and 1 committed suicide for unrelated causes. One patient died of CNS disease. Overall, GVHD developed in 4/19 cases (1 grade 2; 1 grade 4; 1 limited chronic; 1 extensive chronic), however, these patients had excessive T cell contamination with NK cells. In conclusion, carefully purified IL-2 activated intentionally mismatched NK cells can be most effective against resistant tumor cells and can be administered with minimal or no risk of GVHD. Immunotherapy with mismatched NK cells can be a practical approach to induce GVT effects with no GVHD.