Joerg U. Schmohl

IL15 Trispecific Killer Engagers (TriKE) Make Natural Killer Cells Specific to CD33+ Targets While Also Inducing Persistence, In Vivo Expansion, and Enhanced Function

Purpose: The effectiveness of NK cell infusions to induce leukemic remission is limited by lack of both antigen specificity and in vivo expansion. To address the first issue, we previously generated a bispecific killer engager (BiKE) containing single-chain scFv against CD16 and CD33 to create an immunologic synapse between NK cells and CD33+ myeloid targets. We have now incorporated a novel modified human IL15 crosslinker, producing a 161533 trispecific killer engager (TriKE) to induce expansion, priming, and survival, which we hypothesize will enhance clinical efficacy. Experimental Design: Reagents were tested in proliferation and functional assays and in an in vivo xenograft model of AML. Results: When compared with the 1633 BiKE, the 161533 TriKE induced superior NK cell cytotoxicity, degranulation, and cytokine production against CD33+ HL-60 targets and increased NK survival and proliferation. Specificity was shown by the ability of a 1615EpCAM TriKE to kill CD33-EpCAM+ targets. Using NK cells from patients after allogeneic stem cell transplantation when NK cell function is defective, the 161533 TriKE restored potent NK function against primary AML targets and induced specific NK cell proliferation. These results were confirmed in an immunodeficient mouse HL-60-Luc tumor model where the 161533 TriKE exhibited superior antitumor activity and induced in vivo persistence and survival of human NK cells for at least 3 weeks. Conclusions: Off-the-shelf 161533 TriKE imparts antigen specificity and promotes in vivo persistence, activation, and survival of NK cells. These qualities are ideal for NK cell therapy of myeloid malignancies or targeting antigens of solid tumors. Clin Cancer Res; 22(14); 3440–50. ©2016 AACR. See related commentary by Talmadge, p. 3419

Enhanced ADCC and NK Cell Activation of an Anticarcinoma Bispecific Antibody by Genetic Insertion of a Modified IL-15 Cross-linker

Previously, we constructed a bispecific NK-cell-engager (BiKE) bearing single-chain variable fragments (scFv) against CD16 on NK cells and EpCAM on tumor cells. This BiKE facilitated antigen-specific antibody-dependent cell-mediated cytotoxicity (ADCC) but did not induce NK cell expansion. We incorporated a modified interleukin-15 cross-linker to create a trispecific construct (TriKE) in order to improve activation, proliferation, and survival of NK cells. Synthesis and assembly of hybrid genes encoding the TriKE was accomplished using DNA-shuffling and DNA-ligation techniques. The TriKE was tested for specificity, efficacy, proliferative capability, and cytokine profile using functional assays. The molecular modifications improved yield without compromising binding to EpCAM(+) HT-29 colorectal carcinoma cells. (51)Chromium-release and degranulation assays showed better killing rates with TriKE compared to BiKE. TriKE was more active in a variety of different carcinoma cell lines. TriKE showed the ability to stimulate expansion of CD56(+)CD3(-) NK cells. BiKE and TriKE showed enhanced but not supraphysiologic levels of cytokine secretion. 1615EpCAM TriKE drives enhanced ADCC while significantly improving proliferation, activation, and survival of NK cell effectors. The TriKE provides a selectively delivered self-sustaining signal at the NK/tumor cell synapse. Targeted cytokine stimulation, rather than systemic cytokine administration, may impact toxicity in patients rendering the TriKE a promising new off-the-shelf carcinoma therapy.

Mutagenic Deimmunization of Diphtheria Toxin for Use in Biologic Drug Development

Background: Targeted toxins require multiple treatments and therefore must be deimmunized. We report a method of protein deimmunization based on the point mutation of highly hydrophilic R, K, D, E, and Q amino acids on the molecular surface of truncated diphtheria-toxin (DT390). Methods: Based on their surface position derived from an X-ray-crystallographic model, residues were chosen for point mutation that were located in prominent positions on the molecular surface and away from the catalytic site. Mice were immunized with a targeted toxin containing either a mutated DT390 containing seven critical point mutations or the non-mutated parental toxin form. Results: Serum analysis revealed a significant 90% reduction in anti-toxin antibodies in mice immunized with the mutant, but not the parental drug form despite multiple immunizations. The experiment was repeated in a second strain of mice with a different MHC-haplotype to address whether point mutation removed T or B cell epitopes. Findings were identical indicating that B cell epitopes were eliminated from DT. The mutant drug form lost only minimal activity in vitro as well as in vivo. Conclusion: These findings indicate that this method may be effective for deimmunizing of other proteins and that discovery of a deimmunized form of DT may lead to the development of more effective targeted toxin.

Tetraspecific scFv construct provides NK cell mediated ADCC and self-sustaining stimuli via insertion of IL-15 as a cross-linker

Background The design of a highly effective anti-cancer immune-engager would include targeting of highly drug refractory cancer stem cells (CSC). The design would promote effective antibody-dependent cell-mediated cytotoxicity (ADCC) and simultaneously promote costimulation to expand and self-sustain the effector NK cell population. Based on our bispecific NK cell engager platform we constructed a tetraspecific killer engager (TetraKE) comprising single-chain variable fragments (scFvs) binding FcγRIII (CD16) on NK cells, EpCAM on carcinoma cells and CD133 on cancer stem cells in order to promote ADCC. Furthermore, an Interleukin (IL)-15-crosslinker enhanced NK cell related proliferation resulting in a highly active drug termed 1615EpCAM133. Results Proliferation assays showed TetraKE promoted proliferation and enhanced NK cell survival. Drug-target binding, NK cell related degranulation, and IFN-γ production was specific for both tumor related antigens in EpCAM and CD133 bearing cancer cell lines. The TetraKE showed higher killing activity and superior dose dependent degranulation. Cytokine profiling showed a moderately enhanced IFN-γ production, enhanced GM-CSF production, but no evidence of induction of excessive cytokine release. Methods Assembly and synthesis of hybrid genes encoding the TetraKE were performed using DNA shuffling and ligation. The TetraKE was tested for efficacy, specificity, proliferation, survival, and cytokine production using carcinoma cell lines and functional assays measuring NK cell activity. Conclusion 1615EpCAM133 combines improved induction of ADCC with enhanced proliferation, limited cytokine response, and prolonged survival and proliferation of NK cells. By linking scFv-related targeting of carcinoma and CSCs with a sustaining IL-15 signal, our new construct shows great promise to target cancer and CSCs.

Expression of surface-associated 82kDa-proMMP-9 in primary acute leukemia blast cells inversely correlates with patients' risk

With its ability to degrade extracellular matrix proteins and activate growth factors and cytokines, matrix metalloproteinase (MMP)-9 is an important regulator of cell function. Previously, we reported that myeloid leukemic cells express a unique 82kDa-proMMP-9 variant on their cell surface that is not affected by its natural inhibitor. In this study, we generated monoclonal antibodies that specifically recognize 82kDa-proMMP-9. Flow cytometry analysis using these antibodies revealed significant surface expression of 82kDa-proMMP-9 in monocytes, but minimal amounts in T and B cells isolated from peripheral blood of nine healthy donors and 22 patients with acute myeloid leukemia (AML). In all AML patients, blasts expressed 82kDa-proMMP-9 at levels of 4%-46%, with significantly higher levels in patients with a better risk defined according to National Comprehensive Cancer Network (NCCN) guidelines (ρ = -0.748, p < 0.001) and favorable phenotype according to the French-American-British classification (p = 0.02) compared with patients with adverse prognoses. Receiver operating characteristic curve analysis confirmed the diagnostic accuracy of 82kDa-proMMP-9 measurement in AML blasts (area under the curve: 0.893 [0.739-1.000], p = 0.019). It led us to define a cutoff value of 11.5% for identifying patients with lower NCCN risk (p = 0.005) and with a tendency toward a higher probability of response to anthracycline-based therapy (p = 0.109) and increased event-free survival (p = 0.24). Thus, 82kDa-proMMP-9 expression on blasts may represent a novel independent marker of prognosis in patients with AML.

Expression of RANK-L and in part of PD-1 on blasts in patients with acute myeloid leukemia correlates with prognosis.

Co‐stimulatory receptor (COR) and ligand (COL) expression on immune effectors are known to be relevant for immunological interactions and might be of prognostic relevance if expressed on acute myeloid leukemia (AML) blasts as reported for receptors of the tumor necrosis factor receptor family.

Development of a Deimmunized Bispecific Immunotoxin dDT2219 against B-Cell Malignancies

Diphtheria toxin (DT) related targeted toxins are effective in cancer treatment, but efficacy diminishes in time because of their immunogenic potential and/or former vaccinations. In order to overcome this limitation for DT2219, a promising bispecific targeted toxin which targets CD19 and CD22, we deimmunized the DT moiety, and thereby developed an exciting improved drug (dDT2219) which still has the potential to sufficiently target B-cell malignancies but also limits clearance because of its reduced immunogenicity. The DT moiety was modified by inducing point mutations in prominent positions on the molecular surface. The new engineered dDT2219 was tested for activity, efficacy, and specificity using functional assays, proliferation assays, and flow cytometry. Furthermore, 12 samples of Chronic Lymphatic Leukemia (CLL) patients were used to assess binding. Immunogenicity was determined using a BALB/c mouse model. dDT2219 was efficient and specific against B-cell malignancies such as Bukitt-Lymphoma cell lines Daudi and Raji. dDT2219 showed specific binding on targets and on CLL samples. Intraperitoneal vaccination of immune competent mice showed that even after multiple administrations with increasing doses, induction of neutralizing antibodies was significantly lower in the dDT2219 treated animal group. The new dDT2219 combines potent anti-tumor cell activity with a reduced immunogenicity. With regard to the frequent development of neutralizing antibodies after multiple administrations with immunotoxins, dDT2219 shows promise to overcome this limitation and thus might maintain effectiveness even after multiple treatment cycles.

Expression of 4-1BB and its ligand on blasts correlates with prognosis of patients with AML

Costimulatory ligands (COLs) and their receptors (COR) regulate immune reactions and cellular survival and might be relevant in acute myeloid leukemia (AML). This study evaluated the clinical relevance of 4-1BBL, glucocorticoid-induced TNFR-related protein (GITR) and ligand (GITRL), CD80, and CD86 in case of expression on AML blasts. 98 patients were evaluated at initial diagnosis. Immunophenotypically evaluated specific fluorescence index (SFI) levels of COR and COL on blasts were correlated with morphological, cytogenetic, and several prognostic parameters. Significantly higher COR expression was seen in monocytic versus non-monocytic AML subtypes; GITR, p=0.05; GITRL, p=0.005; CD86, p=0.001). Cut-off values for two COR and their ligands were evaluated: cases presenting with 4-1BB values above cut-off 1.2 SFI levels correlated (tendentially) significantly with a higher probability for disease-free survival (DFS, p=0.06) and a favorable HR of 0.2; p=0.04 for relapse. HR for death was also significantly lower in this group (0.12; p=0.04). In contrast, a lower probability for DFS and overall survival was seen in cases with 4-1BBL expression above 2.2 SFI levels (p=0.08 and p=0.09). In addition, multivariate analysis showed a significantly higher probability of death in this group (HR 10.3, p=0.04). Expression of CD80 and CD86 did not show significant prognostic relevance. On initial diagnosis, 4-1BB and 4-1BBL qualify as markers for prediction of patients’ course and represent a valuable screening target for patients with AML at initial diagnosis.

Abstract 1493: Improvement of the bispecific antibody ADCC platform by genetic insertion of IL-15 as a cross-linker to create NK cell reactive TriKEs

Natural killer (NK)-cell related anti-tumor surveillance is limited by the ability of the tumor to escape killing. Previously, we constructed a bispecific NK-cell engager (BiKE) consisting of two scFV against CD16 (FcγRIII) on NK cells and EpCAM on tumor cells (EpCAM16). Epithelial cell adhesion molecule (EpCAM) is a transmembrane protein with prevalent expression on carcinomas making it a valuable marker for cancer targeting. This BiKE facilitated ADCC and tumor elimination, but did not account for the cellular expansion required for the success of T CARs. To improve this, we incorporated a modified interleukin (IL)-15 crosslinker to create a trispecific construct (TriKE) to enhance activation, proliferation, and to prolong survival of NK-cells. IL-15 was chosen since it is an established immunostimulatory cytokine with known effects on NK cells and is recognized as a promising cancer cure drug in NIH guided review. TriKE was assembled, expressed in E.coli, extracted, refolded, and purified to >90% with a molecular weight of 68,860 daltons. To determine the functional activity of 1615EpCAM, its killing ability was measured in standard Cr-51 release assays with EpCAM+ HT-29 colorectal cancer cells. The 1615EpCAM TriKE induced the highest level of killing compared to BiKE and other controls. To determine if the effect of IL-15 in the drug correlated with NK-cell levels, donors were selected with different naturally occurring NK cell levels. Freshly isolated PBMCs were added to HT-29 cells at E:T ratios of 20:1, 6.6:1, and 2:1. Donors showed increasingly higher levels of Cr-51 kill with TriKE, but not with BiKE indicating that greater the presence of NK-cells, the greater the TriKE effect. In order to study lytic degranulation as a function of NK-cell activity, CD107a expression was measured. Cells treated with TriKE showed significantly elevated degranulation when co-cultivated with targets (p Citation Format: Daniel A. Vallera, Joerg U. Schmohl, Martin Felices, Jeffrey S. Miller. Improvement of the bispecific antibody ADCC platform by genetic insertion of IL-15 as a cross-linker to create NK cell reactive TriKEs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1493.