Cryopreserved CAR-like NK Cells Pre-Complexed with the CD30/CD16A Bispecific Innate Cell Engager (ICE®) AFM13 for the Treatment of CD30 + Malignancies
Authors: Reusch Uwe , Ellwanger Kristina , Fucek Ivica , Müller Thomas , Schniegler-Mattox Ute , Pahl Jens , Tesar Michael , Koch Joachim 
Published: November 2021

Abstract
AFM13 is a bispecific tetravalent innate cell engager (ICE®) with two binding sites for CD16A and CD30 on immune cells and tumor cells, respectively. AFM13 is designed to potently activate anti-tumoral responses of natural killer (NK) cells, through antibody-dependent cellular cytotoxicity (ADCC) and macrophages via antibody-dependent cellular phagocytosis (ADCP) towards CD30 + tumor cells. It is widely known that the number of NK cells and their anti-tumor activity are often compromised in patients with cancer, including CD30 + malignancies such as Hodgkin lymphoma (HL) (Reiners, K.S. et al., Mol Ther 2013;21:895-903). Accumulating evidence suggests that the selective, high affinity binding of ICE® to CD16A enables stable pre-complexing with NK cells to enhance ADCC.
Early clinical studies with AFM13 have demonstrated safety and efficacy, both as mono- and combination therapy with an anti-PD-1 checkpoint inhibitor (pembrolizumab) (Bartlett, N.L. et al., Blood 2020;136(21):2401-2409), in patients with relapsed or refractory (R/R) HL or peripheral T cell lymphoma (PTCL). Currently, AFM13 monotherapy is being evaluated in a registration-directed Phase 2 study to treat patients with PTCL (NCT04101331). Additionally, a Phase 1b/2a study in patients with PTCL (NCT03192202) previously demonstrated that NK cell numbers pre- and post-dosing of AFM13 may positively correlate with anti-tumoral response (Sawas, A. et al., Blood 2020;136(1):25-26). Therefore, the safety and efficacy of freshly prepared cord blood-derived allogeneic NK cells pre-complexed with AFM13, followed by three weekly infusions of AFM13 monotherapy, is currently being evaluated in patients with R/R CD30 + lymphomas in a Phase 1 dose escalation study (NCT04074746). The first four enrolled patients, all with HL, have shown a response rate of 100% without any safety concerns (Rezvani, K. et al., presentation AACR 2021).
To further develop this CAR-like NK cell immunotherapeutic towards an off-the-shelf product, we evaluated the feasibility of cryopreservation of pre-complexed NK cells and assessed the biological activity of such cryopreserved and pre-complexed NK cells after thawing. In this study, the cell surface retention, suitability for NK cell preloading, and cytotoxic activity of AFM13 and other CD30-specific antibody formats was assessed. Furthermore, the compatibility of these formats with cryopreservation of the resulting CAR-like NK cell product was investigated. Cell surface retention assays on primary NK cells confirm substantially slower dissociation kinetics of the tetravalent bispecific ICE® compared to corresponding IgG1 or Fc-enhanced IgG1, enabling efficient and durable pre-complexing of NK cells with ICE®. Surface plasmon resonance analyses confirm long retention of ICE® independent of the CD16A polymorphism. More importantly, these assays demonstrate that the high ADCC potency and efficacy of NK cells, pre-complexed with CD16A-specific tetravalent ICE®, is maintained after one freeze-thaw cycle.
These data suggest that high-affinity pre-complexing of adoptive NK cells with bispecific, CD16A-selective ICE® could be a novel cryopreserved off-the-shelf NK cell product for the effective depletion of tumor cells without the limitations and potential risks associated with the CAR-NK cell technology.

Disclosures
Reusch: Affimed: Current Employment, Current holder of stock options in a privately-held company. Ellwanger: Affimed: Current Employment, Current holder of stock options in a privately-held company. Fucek: Affimed: Current Employment, Current holder of stock options in a privately-held company. Müller: Affimed GmbH: Current Employment, Current holder of stock options in a privately-held company. Schniegler-Mattox: Affimed: Current Employment, Current holder of stock options in a privately-held company. Pahl: Affimed: Current Employment, Current holder of stock options in a privately-held company. Tesar: Affimed: Current Employment, Current holder of stock options in a privately-held company. Koch: Affimed: Current Employment, Current holder of stock options in a privately-held company.