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BiKE and TriKE selectively bind antigen expressing targets

Supplemental Figure Legends

Supplementary Figure 2. Studies of effector cells from normal donors indicate that NK cells proliferate moreso than T cells in response to TriKE, but T cells can expand.

Blocking studies show specificity of BiKEs and TriKEs.

Supplementary Figure 1. To address the input of the IL-15 moiety on NK cell cytotoxicity, we compared the 161533 TriKE to equivalent molar concentrations of IL-15 or IL-15 plus 1633 BiKE. Activation markers were studied.

Purpose: The effectiveness of NK cell infusions to induce leukemic remission is limited by lack of both antigen specificity and in vivo expansion. To address the first issue, we previously generated a bispecific killer engager (BiKE) containing single-chain scFv against CD16 and CD33 to create an immunologic synapse between NK cells and CD33+ myeloid targets. We have now incorporated a novel modified human IL15 crosslinker, producing a 161533 trispecific killer engager (TriKE) to induce expansion, priming, and survival, which we hypothesize will enhance clinical efficacy.Experimental Design: Reagents were tested in proliferation and functional assays and in an in vivo xenograft model of AML.Results: When compared with the 1633 BiKE, the 161533 TriKE induced superior NK cell cytotoxicity, degranulation, and cytokine production against CD33+ HL-60 targets and increased NK survival and proliferation. Specificity was shown by the ability of a 1615EpCAM TriKE to kill CD33-EpCAM+ targets. Using NK cells from patients after allogeneic stem cell transplantation when NK cell function is defective, the 161533 TriKE restored potent NK function against primary AML targets and induced specific NK cell proliferation. These results were confirmed in an immunodeficient mouse HL-60-Luc tumor model where the 161533 TriKE exhibited superior antitumor activity and induced in vivo persistence and survival of human NK cells for at least 3 weeks.Conclusions: Off-the-shelf 161533 TriKE imparts antigen specificity and promotes in vivo persistence, activation, and survival of NK cells. These qualities are ideal for NK cell therapy of myeloid malignancies or targeting antigens of solid tumors. Clin Cancer Res; 22(14); 3440–50. ©2016 AACR.See related commentary by Talmadge, p. 3419

A human ovarian cancer cell line, which migrates to mouse ovaries and establishes peritoneal carcinomatosis, was used to evaluate the cooperative effect of an antiangiogenic gene therapy combined with chemotherapy. The ovarian carcinoma cell line MA148 was genetically modified by “Sleeping Beauty” transposon-mediated delivery of DsRed2 fluorescent protein. Stable, high-level expression of DsRed protein enabled in vivo imaging of peritoneal dissemination of ovarian cancer. Both external and internal imaging, along with histopathology, showed migration of i.p. injected human ovarian cancer cell line to mouse ovaries. Using this model, we evaluated the effect of adeno-associated virus (AAV)–mediated expression of a mutant endostatin either alone or in combination with carboplatin treatment. A single i.m. injection of recombinant AAV (rAAV)-mutant human endostatin with P125A substitution (P125A-endostatin) showed sustained expression of mutant endostatin. Antiangiogenic gene therapy inhibited orthotopic growth of ovarian cancer and resulted in 33% long-term tumor-free survival. A single cycle of carboplatin treatment combined with mutant endostatin gene therapy resulted in 60% of the animals remaining tumor free for >200 days, which was significantly better than rAAV-LacZ and/or carboplatin. Combination treatment delayed tumor appearance in 40% of the animals, wherein the residual tumors were smaller in size with limited or no peritoneal metastasis. These studies suggest that AAV-mediated gene therapy of P125A-endostatin in combination with carboplatin is a useful method to inhibit peritoneal dissemination of ovarian carcinoma. (Cancer Res 2006; 66(8): 4319-28)

Introduction: Pediatric germ cell tumors (GCTs) are rare and heterogeneous tumors that are grouped together due to their presumed common cell of origin, the primordial germ cell (PGC). GCTs typically occur in the testes or ovaries; however, abnormal prenatal PGC migration along with lack of apopotosis can result in tumors in extragonadal locations, including the central nervous system (intracranial and intraspinal GCTs; iGCTs). Little is known about the etiology of iGCTs, although international incidence data suggest that the highest incidence occurs in Asian countries. Autoimmune encephalitis (AE) is a paraneoplastic event characterized by abnormal behavior, seizures, and movement disorders; a number of case reports find AE in conjunction with GCT. We note with interest that a recent registry based study in California reported the highest incidence of AE in Asians. In this analysis, we used 1992-2009 data from the National Cancer Institute's Surveillance, Epidemiology and End Results (SEER) Program to determine whether the increased rates of iGCT observed in Asian countries was also seen in Asian/Pacific Islanders living in the United States. Methods: Frequencies and incidence rates were evaluated for the entire cohort and for demographic subgroups based on sex, age category (0-9 and 10-29 years), race (white, black, American Indian/Alaskan Native and Asian/Pacific Islander), and tumor location (pineal gland vs. other) as sample size permitted. Analyses were conducted using SEER*Stat 7.1.0. Results: Two peaks in incidence were observed, the first before age 1 year in both sexes and the second at age 10 years for females and age 18 years for males. We observed a significantly higher incidence rate of iGCT in Asian/Pacific Islanders compared with whites (RR=1.98, 95% CI 1.51-2.56 for males and RR=3.21, 95% CI 1.99-5.05 for females) in the 10-29 year age group. This difference was observed for tumors located both in the pineal gland and for tumors in other locations. Conclusions: The increased rates of iGCT we observed in individuals of Asian descent in the SEER registry are in agreement with data from the International Agency for Research on Cancer, where the highest incidence rates were observed in Japan, Singapore, and the Maori population of New Zealand. The explanation for these rate differences has not been investigated to date; however, the increased incidence in individuals of Asian ancestry in the United States suggests that genetic susceptibility may play a role in etiology. The higher frequency of AE in Asians and the co-occurrence with GCT in case reports is also intriguing. It is possible that AE could be triggered by PGCs abnormally present in the cranio-spatial axis prior to GCT diagnosis, and it will be of interest to determine whether a diagnosis of AE precedes iGCT. These differences in incidence patterns by race and the possible link with AE merit further investigation. Citation Format: Jenny N. Poynter, Rachel Fonstad, Jakub Tolar, Logan G. Spector, Julie A. Ross. Incidence of intracranial germ cell tumors by race in the United States, 1992-2009: A potential link with autoimmune encephalitis?. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2291. doi:10.1158/1538-7445.AM2013-2291