The OXS-C3550 TriKE was developed following continued research with the TriKE platform at the Masonic Cancer Center, University of Minnesota. As demonstrated in non-clinical models, this targeted immunotherapy directs immune cells to kill cancer cells while diminishing drug-related toxicity.
These data suggest that OXS-C3550 has functional advantages over our first-generation TriKE and warrants clinical testing. These advantages include, better induction of NK cell function, better induction of proliferation, better stability of binding, and better in vivo tumor control.
In addition, these promising new findings will direct our future TriKE and TetraKE development towards consideration of using the modified CD16 component (camelid version), which we believe has great potential in the tumor immunotherapy setting.
OXS C3550 will focus on AML, the most common form of adult leukemia with 43,000 new cases each year. These patients will require frontline therapy, usually chemotherapy including cytarabine and an anthracycline, a therapy that has not changed in over 40 years. Also, about half will have relapses and require alternative therapies. In addition, about 20,000 new cases of myelodysplastic syndrome (MDS) are diagnosed each year and there are minimal treatment options (Siegel et al, 2014). At a minimum, OXS-C3550 can be expected to serve as a relatively safe, inexpensive, and easy to use therapy for resistant/relapsing AML. From a biologic standpoint, it could also be combined with chemotherapy as frontline therapy.
The NK cell cancer-killing activity is expected to be increased by bringing the NK cells in close proximity to the cancer cells. This can be achieved by “engagers” (linkers) that bind to CD16 on the surface of NK cells and bind specific proteins (such as CD33) on the surface of cancer cells, thus linking the NK cell to the cancer cell. The original anti-CD16-IL-15-anti-CD33 TriKE (OXS-3550) utilized the inclusion of a modified Interleukin-15 (IL-15), a peptide that activates NK cells, in the “engager” further increases NK cancer-cell killing capabilities and improves their function in the tumor microenvironment (Vallera et al,2016). This second-generation generation anti-CD16-IL-15-anti-CD33 TriKE (OXS-C3550) utilizes a modified anti-CD16 component while incorporating the wild-type IL-15.
It was thought that steric factors, possibly mediated by the two scFv arms containing a VH and a VL, may hinder maximal functionality in this molecule by mispairing at the time of refolding. To address this, we substituted the anti-CD16 scFv arm in the TriKE platform with a novel humanized camelid anti-CD16 single-domain antibody to create a second-generation molecule (OXS-C3550). Single-domain antibodies have several advantages, including better stability and solubility, more resistance to pH changes, can better recognize hidden antigenic sites, lack of a VL portion thus preventing VH/VL mispairing and they are suitable for construction of larger molecules. In our bacterial production system, the OXS-C3550 construct yielded better purities and quantities than the first-generation TriKE molecule. OXS-C3550 induced a potent increase in NK cell degranulation, measured by CD107a expression against HL-60 AML tumor targets when compared to the first-generation TriKE (70.75±3.65% vs. 30.75±5.05%). IFNg production was similarly enhanced (29.2±1.8% vs. 6.55±1.07%). OXS-C3550 also exhibited a robust increase in NK cell proliferation (57.65±6.05% vs. 20.75±2.55%).
OXS-C3550 also yielded better tumor control in a primary AML tumor model (NSG mice engrafted with HL-60-luc targets and human NK cells). OXS-C3550 treated mice had 130-fold less tumor burden than the first-generation TriKE treated mice (1.86×107±2.76×106 vs 2.45×109±4.26×108 p/sec/cm2/sr, P = 0.0005, n = 5), indicating a strong improvement in AML tumor control with OXS-C3550.