Development of Automated Separation, Expansion, and Quality Control Protocols for Clinical-Scale Manufacturing of Primary Human NK Cells and Alpharetroviral Chimeric Antigen Receptor Engineering.

In cellular immunotherapies, natural killer (NK) cells often demonstrate potent antitumor effects in high-risk cancer patients. But Good Manufacturing Practice (GMP)-compliant manufacturing of clinical-grade NK cells in high numbers for patient treatment is still a challenge. Therefore, new protocols for isolation and expansion of NK cells are required. In order to attack resistant tumor entities, NK cell killing can be improved by genetic engineering using alpharetroviral vectors that encode for chimeric antigen receptors (CARs). The aim of this work was to demonstrate GMP-grade manufacturing of NK cells using the CliniMACS® Prodigy device (Prodigy) with implemented applicable quality controls. Additionally, the study aimed to define the best time point to transduce expanding NK cells with alpharetroviral CAR vectors. Manufacturing and clinical-scale expansion of primary human NK cells were performed with the Prodigy starting with 8-15.0 × 109 leukocytes (including 1.1-2.3 × 109 NK cells) collected by small-scale lymphapheresis (n = 3). Positive fraction after immunoselection, in-process controls (IPCs), and end product were quantified by flow cytometric no-wash, single-platform assessment, and gating strategy using positive (CD56/CD16/CD45), negative (CD14/CD19/CD3), and dead cell (7-aminoactinomycine [7-AAD]) discriminators. The three runs on the fully integrated manufacturing platform included immunomagnetic separation (CD3 depletion/CD56 enrichment) followed by NK cell expansion over 14 days. This process led to high NK cell purities (median 99.1%) and adequate NK cell viabilities (median 86.9%) and achieved a median CD3+ cell depletion of log -3.6 after CD3 depletion and log -3.7 after immunomagnetic CD3 depletion and consecutive CD56 selection. Subsequent cultivation of separated NK cells in the CentriCult® chamber of Prodigy resulted in approximately 4.2-8.5-fold NK cell expansion rates by adding of NK MACS® basal medium containing NK MACS® supplement, interleukin (IL)-2/IL-15 and initial IL-21. NK cells expanded for 14 days revealed higher expression of natural cytotoxicity receptors (NKp30, NKp44, NKp46, and NKG2D) and degranulation/apoptotic markers and stronger cytolytic properties against K562 compared to non-activated NK cells before automated cultivation. Moreover, expanded NK cells had robust growth and killing activities even after cryopreservation. As a crucial result, it was possible to determine the appropriate time period for optimal CAR transduction of cultivated NK cells between days 8 and 14, with the highest anti-CD123 CAR expression levels on day 14. The anti-CD123 CAR NK cells showed retargeted killing and degranulation properties against CD123-expressing KG1a target cells, while basal cytotoxicity of non-transduced NK cells was determined using the CD123-negative cell line K562. Time-lapse imaging to monitor redirected effector-to-target contacts between anti-CD123 CAR NK and KG1a showed long-term effector-target interaction. In conclusion, the integration of the clinical-scale expansion procedure in the automated and closed Prodigy system, including IPC samples and quality controls and optimal time frames for NK cell transduction with CAR vectors, was established on 48-well plates and resulted in a standardized GMP-compliant overall process.

A phase 1 study of the bispecific anti-CD30/CD16A antibody construct AFM13 in patients with relapsed or refractory Hodgkin lymphoma.

AFM13 is a bispecific, tetravalent chimeric antibody construct (TandAb) designed for the treatment of CD30-expressing malignancies. AFM13 recruits natural killer (NK) cells via binding to CD16A as immune effector cells. In this phase 1 dose-escalation study, 28 patients with heavily pretreated relapsed or refractory Hodgkin lymphoma received AFM13 at doses of 0.01 to 7 mg/kg body weight. Primary objectives were safety and tolerability. Secondary objectives included pharmacokinetics, antitumor activity, and pharmacodynamics. Adverse events were generally mild to moderate. The maximum tolerated dose was not reached. Pharmacokinetics assessment revealed a half-life of up to 19 hours. Three of 26 evaluable patients achieved partial remission (11.5%) and 13 patients achieved stable disease (50%), with an overall disease control rate of 61.5%. AFM13 was also active in brentuximab vedotin-refractory patients. In 13 patients who received doses of ≥1.5 mg/kg AFM13, the overall response rate was 23% and the disease control rate was 77%. AFM13 treatment resulted in a significant NK-cell activation and a decrease of soluble CD30 in peripheral blood. In conclusion, AFM13 represents a well-tolerated, safe, and active targeted immunotherapy of Hodgkin lymphoma. A phase 2 study is currently planned to optimize the dosing schedule in order to further improve the therapeutic efficacy. This phase 1 study was registered at www.clinicaltrials.gov as #NCT01221571.