BackgroundNatural killer group 2D (NKG2D) is an activating receptor of natural killer (NK) cells and other lymphocytes that mediates lysis of malignant cells through recognition of stress-induced ligands such as MICA and MICB. Such ligands are broadly expressed by cancer cells of various origins and serve as targets for adoptive immunotherapy with effector cells endogenously expressing NKG2D or carrying an NKG2D-based chimeric antigen receptor (CAR). However, shedding or downregulation of NKG2D ligands (NKG2DL) can prevent NKG2D activation, resulting in escape of cancer cells from NKG2D-dependent immune surveillance.MethodsTo enable tumor-specific targeting of NKG2D-expressing effector cells independent of membrane-anchored NKG2DLs, we generated a homodimeric recombinant antibody which harbors an N-terminal single-chain fragment variable (scFv) antibody domain for binding to NKG2D, linked via a human IgG4 Fc region to a second C-terminal scFv antibody domain for recognition of the tumor-associated antigen ErbB2 (HER2). The ability of this molecule, termed NKAB-ErbB2, to redirect NKG2D-expressing effector cells to ErbB2-positive tumor cells of different origins was investigated using peripheral blood mononuclear cells, ex vivo expanded NK cells, and NK and T cells engineered with an NKG2D-based chimeric receptor.ResultsOn its own, bispecific NKAB-ErbB2 increased lysis of ErbB2-positive breast carcinoma cells by peripheral blood-derived NK cells endogenously expressing NKG2D more effectively than an ErbB2-specific IgG1 mini-antibody able to induce antibody-dependent cell-mediated cytotoxicity via activation of CD16. Furthermore, NKAB-ErbB2 synergized with NK-92 cells or primary T cells engineered to express an NKG2D-CD3ζ chimeric antigen receptor (NKAR), leading to targeted cell killing and greatly enhanced antitumor activity, which remained unaffected by soluble MICA known as an inhibitor of NKG2D-mediated natural cytotoxicity. In an immunocompetent mouse glioblastoma model mimicking low or absent NKG2DL expression, the combination of NKAR-NK-92 cells and NKAB-ErbB2 effectively suppressed outgrowth of ErbB2-positive tumors, resulting in treatment-induced endogenous antitumor immunity and cures in the majority of animals.ConclusionsOur results demonstrate that combining an NKAB antibody with effector cells expressing an activating NKAR receptor represents a powerful and versatile approach to simultaneously enhance tumor antigen-specific as well as NKG2D-CAR and natural NKG2D-mediated cytotoxicity, which may be particularly useful to target tumors with heterogeneous target antigen expression.

C hloroethylnitrosureas (CNUs) are powerful DNA-reactive alkylating agents used in cancer therapy. Here, we analyzed cyto-and genotoxicity of nimustine (ACNU), a representative of CNUs, in synchronized cells and in cells deficient in repair proteins involved in homologous recombination (HR) or nonhomologous end-joining (NHEJ). We show that HR mutants are extremely sensitive to ACNU, as measured by colony formation, induction of apoptosis and chromosomal aberrations. The NHEJ mutants differed in their sensitivity, with Ku80 mutants being moderately sensitive and DNA-PKcs mutated cells being resistant. HR mutated cells displayed a sustained high level of γH2AX foci , which co-stained with Rad51 and 53BP1, indicating DNA double-strand breaks (DSB) to be formed. Using synchronized cells, we analyzed whether DSB formation after ACNU treatment was replication-dependent. We show that γH2AX foci were not induced in G 1 but increased significantly in S phase and remained at a high level in G 2 , where a fraction of cells became arrested and underwent, with a delay of > 12 h, cell death by apoptosis and necrosis. Rad51, ATM, MDC-1 and RPA-2 foci were also formed and shown to co-localize with γH2AX foci induced in S phase, indicating that the DNA damage response was activated. All effects observed were abrogated by MGMT, which repairs O 6 -chloroethylguanine that is converted into DNA cross-links. We deduce that the major genotoxic and killing lesion induced by CNUs are O 6 -chloroethylguanine-triggered

Background:Adoptive cellular therapy (ACT) with redirected T cells expressing a chimeric antigen receptor (CAR) or transgenic T‐cell‐receptor (tTCR) has revolutionized cellular immunotherapy to hematological neoplasia, in particular to acute lymphoid leukemia, and also shows great promise as therapy for solid tumors. While CARs can only detect fully cell surface expressed target structures, TCR‐mediated recognition is not limited to surface antigens, but covers processed tumor neoantigens derived from the whole proteome. However, mispairing of transgenic and endogenous TCRs and restriction to patient‐derived, autologous T lymphocytes with variable “fitness” and T cell subsets due to individual health conditions and age of the patient exemplify the current limitations encountered in TCR‐redirected ACT. The natural killer (NK) cell line NK92 elicits lytic activity comparable to T cells, has been approved by the FDA for ACT and shown not to cause graft‐vs‐host disease. Since NK cell redirection is currently limited to CARs, NK92 cells engineered to express a CD3/TCR or CD3/CD8/TCR complex might evolve as an attractive, standardized cellular source for off‐the‐shelf TCR‐based ACT.Aims:Thus, the goal of this study was to explore NK92‐CD3+ and NK92‐CD3+CD8+ variants redirected to acute myeloid leukemia (AML) by expression of different AML‐reactive TCRs for antileukemic immunity in vitro and in vivo.Methods:NK92 cells expressing the γδεζ‐chains of the CD3 complex (NK92CD3+) were kindly provided by Dr. C. Wölfel (III. Dept. of Medicine) and further genetically engineered to express different human CD8 isoforms. Upon retroviral transfer (RT) of 3 different codon‐optimized and murinized TCRs obtained from AML‐specific CD8+CTL clones into NK92‐CD3+cells TCR expression was examined by flow cytometry (FC). NK92CD3+TCR+as well as NK92CD3+CD8+TCR+cells (together referred to as NK92TCR+) were enriched using antibiotic selection and immunomagnetic cell sorting (MACS®). Biological reactivity of redirected NK92TCR+cells to patient‐derived (primary) AML blasts was tested in vitro by IFN‐γ ELISPOT and 51Cr‐release assays. Comparable expression‐profiling of checkpoint molecules on NK92 and NK92TCR+cells such as e.g. CD80/86, PD1/PDL1, NKG2A, TIM3, TIGIT and HLA‐E was performed by FC. A patient‐derived AML‐xenograft (PDX) model in NSG mice has been already established.Results:Following RT and enrichment >80% of NK92‐CD3+cells expressed a given TCR, and >90% of NK92CD3+CD8+TCR+cells could be obtained. Upon coculture with different primary AML samples and corresponding EBV‐LCL blasts as controls NK92CD3+TCR+ elicited TCR dependent IFN–γ release and exhibited cytolytic activity to some but not all AML samples. CD8 coexpression slightly increased reactivity as measured by IFN‐γ release. Interestingly, as TCR‐redirected NK92 antileukemic responses appeared to be inhibited to some but not all AML samples we found high expression of HLA‐E on these AML blasts and of NGK2A on NK92TCR+ cells. Current blocking studies to examine whether a HLA‐E/NKG2A interaction might impair NK92TCR+ mediated AML reactivity are ongoing and will be presented. Moreover, first preliminary in vivo experiments of adoptively transferred NK92TCR+ cells into a NSG‐AML PDX model indicated biological activity but further studies are in progress to evaluate this point in more detail.Summary/Conclusion:These studies demonstrate that the established and FDA approved NK92 cell line can be redirected to elicit TCR‐mediated antitumoral immunity to AML. NK92TCR+ cells might thus represent a promising universal tool for an ‘off‐the‐shelf’ ACT product