A Diagnostic Strategy for Gauging Individual Humoral Ex Vivo Immune Responsiveness Following COVID-19 Vaccination.

Purpose: We describe a diagnostic procedure suitable for scheduling (re-)vaccination against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) according to individual state of humoral immunization. Methods: To clarify the relation between quantitative antibody measurements and humoral ex vivo immune responsiveness, we monitored 124 individuals before, during and six months after vaccination with Spikevax (Moderna, Cambridge, MA, USA). Antibodies against SARS-CoV-2 spike (S1) protein receptor-binding domain (S1-AB) and against nucleocapsid antigens were measured by chemiluminescent immunoassay (Roche). Virus-neutralizing activities were determined by surrogate assays (NeutraLISA, Euroimmune; cPass, GenScript). Neutralization of SARS-CoV-2 in cell culture (full virus NT) served as an ex vivo correlate for humoral immune responsiveness. Results: Vaccination responses varied considerably. Six months after the second vaccination, participants still positive for the full virus NT were safely determined by S1-AB levels ≥1000 U/mL. The full virus NT-positive fraction of participants with S1-AB levels <1000 U/mL was identified by virus-neutralizing activities >70% as determined by surrogate assays (NeutraLISA or cPas). Participants that were full virus NT-negative and presumably insufficiently protected could thus be identified by a sensitivity of >83% and a specificity of >95%. Conclusion: The described diagnostic strategy possibly supports individualized (re-)vaccination schedules based on simple and rapid measurement of serum-based SARS-CoV-2 antibody levels. Our data apply only to WUHAN-type SARS-CoV-2 virus and the current version of the mRNA vaccine from Moderna (Cambridge, MA, USA). Adaptation to other vaccines and more recent SARS-CoV-2 strains will require modification of cut-offs and re-evaluation of sensitivity/specificity.

CD16xCD33 Bispecific Killer Cell Engager (BiKE) as potential immunotherapeutic in pediatric patients with AML and biphenotypic ALL.

Similar to pediatric acute myeloid leukemia (AML) the subgroup of biphenotypic acute lymphoblastic leukemia (ALL) is a rare complex entity with adverse outcome, characterized by the surface expression of CD33. Despite novel and promising anti-CD19 targeted immunotherapies such as chimeric antigen receptor T cells and bispecific anti-CD19/CD3 antibodies, relapse and resistance remain a major challenge in about 30% to 60% of patients. To investigate the potential role of the fully humanized bispecific antibody CD16 × CD33 (BiKE) in children with CD33+ acute leukemia, we tested whether the reagent was able to boost NK cell effector functions against CD33+ AML and biphenotypic ALL blasts. Stimulation of primary NK cells from healthy volunteers with 16 × 33 BiKE led to increased cytotoxicity, degranulation and cytokine production against CD33+ cell lines. Moreover, BiKE treatment significantly increased degranulation, IFN-γ and TNF-α production against primary ALL and AML targets. Importantly, also NK cells from leukemic patients profited from restoration of effector functions by BiKE treatment, albeit to a lesser extent than NK cells from healthy donors. In particular, those patients with low perforin and granzyme expression showed compromised cytotoxic function even in the presence of BiKE. In patients with intrinsic NK cell deficiency, combination therapy of CD16xCD33 BiKE and allogeneic NK cells might thus be a promising therapeutic approach. Taken together, CD16xCD33 BiKE successfully increased NK cell effector functions against pediatric AML and biphenotypic ALL blasts and constitutes a promising new option for supporting maintenance therapy or "bridging" consolidation chemotherapy before hematopoietic stem cell transplantation.

CD33 Delineates Two Functionally Distinct NK Cell Populations Divergent in Cytokine Production and Antibody-Mediated Cellular Cytotoxicity.

The generation and expansion of functionally competent NK cells in vitro is of great interest for their application in immunotherapy of cancer. Since CD33 constitutes a promising target for immunotherapy of myeloid malignancies, NK cells expressing a CD33-specific chimeric antigen receptor (CAR) were generated. Unexpectedly, we noted that CD33-CAR NK cells could not be efficiently expanded in vitro due to a fratricide-like process in which CD33-CAR NK cells killed other CD33-CAR NK cells that had upregulated CD33 in culture. This upregulation was dependent on the stimulation protocol and encompassed up to 50% of NK cells including CD56dim NK cells that do generally not express CD33 in vivo. RNAseq analysis revealed that upregulation of CD33+ NK cells was accompanied by a unique transcriptional signature combining features of canonical CD56bright (CD117high, CD16low) and CD56dim NK cells (high expression of granzyme B and perforin). CD33+ NK cells exhibited significantly higher mobilization of cytotoxic granula and comparable levels of cytotoxicity against different leukemic target cells compared to the CD33- subset. Moreover, CD33+ NK cells showed superior production of IFNγ and TNFα, whereas CD33- NK cells exerted increased antibody-dependent cellular cytotoxicity (ADCC). In summary, the study delineates a novel functional divergence between NK cell subsets upon in vitro stimulation that is marked by CD33 expression. By choosing suitable stimulation protocols, it is possible to preferentially generate CD33+ NK cells combining efficient target cell killing and cytokine production, or alternatively CD33- NK cells, which produce less cytokines but are more efficient in antibody-dependent applications.