NK Cell Engager Research Articles

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

Abstract 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<0.001) compared to BiKE. IL-15 alone did not enhance lytic degranulation. When PBMC were exposed to TriKE, only NK-cells, but not T-cells showed a proliferation specific pattern. Direct comparison of the BiKE and the TriKE showed that the TriKE had the ability to induce proliferation and expansion, the BiKE did not. Only with TriKE or IL-15 exposure was there a significantly enhanced expansion index. IFN-γ production from the same CD56+/CD3- NK-cell population was enhanced by 1615EpCAM, but levels did not approach levels seen with the IL12/IL18 combination that is known to stimulate cytokine production at supraphysiologic levels. We believe that this is a platform technology since anti-CD133 also can be substituted for anti-EpCAM to create functional anti-cancer stem cell TriKEs. These results indicate that we have successfully developed an immune engager that simultaneously mediates ADCC and also provides a self-sustaining costimulatory signal inducing NK effector cell expansion. 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.

Trastuzumab upregulates expression of HLA-ABC and T cell costimulatory molecules through engagement of natural killer cells and stimulation of IFNγ secretion

ABSTRACTIt is increasingly recognized that trastuzumab, an antibody approved for treating human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer, exerts some of its antitumor effects through enhanced T cell responses. Full activation of CD8+ T cells requires both expression of major histocompatibility complex class I molecules (HLA-ABC) and expression of the T cell costimulatory molecule CD80 or CD86; however, the impact of trastuzumab treatment on the expression of HLA-ABC and CD80 and CD86 has not been investigated in HER2-overexpressing breast cancer cells. In this study, we found that, while there is no direct correlation between the expression of HER2 and HLA-ABC in breast cancer, knockdown of HER2 or inhibition of HER2 kinase by lapatinib downregulated HLA-ABC expression. Trastuzumab had no meaningful effects on HLA-ABC expression in HER2-overexpressing breast cancer cells in monoculture; however, treatment of such cells with trastuzumab in co-culture with human peripheral blood mononuclear cells (PBMC) significantly upregulated not only HLA-ABC expression but also CD86 expression. We showed that this upregulation was mediated by interferon gamma (IFNγ) secreted from the natural killer (NK) cells in PBMC as a result of engagement of NK cells by trastuzumab. We further confirmed this effect of trastuzumab in vivo using a mouse mammary tumor model transduced to overexpress human HER2. Together, our data provide evidence that trastuzumab upregulates expression of HLA-ABC and T cell costimulatory molecules in HER2-overexpressing breast cancer cells in the presence of PBMC, which supports the view that T-cell-mediated immune responses are involved in trastuzumab-mediated antitumor effects.

Differential Expressions of Selected Activating and Inhibitory Receptors on K562-Stimulated Natural Killer (NK) Cells in HIV-1 and HIV-2 Infections

Context: The functional activity of NK cells depends on the balance between the engagement of activating and inhibitory receptors on the cell surface with their ligands, which enables them to kill infected cells. Objectives: The aim of this study was to evaluate and compare expressions of selected activating and inhibitory receptors on stimulated NK cells in HIV-1 and HIV-2 infections. Methods: PBMCs were analysed for activating (NKp30, NKp44, NKp46) and inhibitory (CD158a, CD158b, p70) receptor expressions in 30 HIV-1, 30 HIV-2 and 30 HIV uninfected healthy control (HC) subjects by flow cytometry after stimulating with K562 cells. Results: There was an expression of other receptors following an already in vitro engagement of NK cells with K562 cells. Higher expression of the activating receptors, NKp44 (p = 0.029) and NKp46 (p = 0.032) on NK cells from HIV-2 compared to HIV-1 infected individuals but similar NKp30 expression (p = 0.980). The levels of expression of inhibitory receptor CD158a were similar between HIV-1 and HIV-2 infected subjects (p = 0.309) but there was significant up-regulation of inhibitory receptors p70 (p = 0.010) and CD158b (p = 0.05) in HIV-1 compared to HIV-2 subjects. Conclusion: Despite the in vitro engagement of NK cells with stimulating K562 cells, our data showed differential expressions of other selected activating and inhibitory receptors in HIV-1 and HIV-2 infected subjects.

Final Results for the 1703 Phase 1b/2 Study of Elotuzumab in Combination with Lenalidomide and Dexamethasone in Patients with Relapsed/Refractory Multiple Myeloma

Final Results for the 1703 Phase 1b/2 Study of Elotuzumab in Combination with Lenalidomide and Dexamethasone in Patients with Relapsed/Refractory Multiple Myeloma

Pharmacokinetics and Safety of Elotuzumab in Combination with Lenalidomide and Dexamethasone in Patients with Multiple Myeloma and Various Levels of Renal Function: Results of a Phase 1b Study

INTRODUCTIONRenal impairment affects more than a third of patients with relapsed multiple myeloma (MM). Renal impairment predicts poor outcome with conventional treatments, while reversal of renal function is associated with improved outcome.1,2 Better therapies for patients with MM and renal impairment are needed. Elotuzumab (Elo) is a humanized monoclonal antibody targeted against Signaling Lymphocytic Activation Molecule F7 (SLAMF7; also called CS1). Through both direct activation and engagement of natural killer cells, Elo selectively kills SLAMF7-expressing myeloma cells and, in combination with lenalidomide (Len) and dexamethasone (dex), has shown objective responses in clinical trials.3 This study evaluated the pharmacokinetics (PK) and safety of Elo in combination with Len/dex in renally impaired patients with MM.METHODSThis multicenter, open-label, Phase Ib trial (NCT01393964) enrolled patients with symptomatic newly diagnosed or relapsed/refractory MM and normal renal function (NRF; creatinine clearance [CrCl] ≥90 mL/min), severe renal impairment (SRI; CrCl <30 mL/min not requiring dialysis), or end-stage renal disease (ESRD; requiring dialysis). Elo was given in 28-day cycles until disease progression or unacceptable toxicity. Intravenous Elo (10 mg/kg) dosing occurred on Day 1 of the first cycle, weekly in cycles 2 and 3, and biweekly in subsequent cycles. Len (5–25 mg, depending on renal function) was given on Days 1–21 of each cycle and dex (40 mg) was given weekly. The primary endpoint was single-dose Elo PK. Other endpoints were safety and efficacy. Treatment responses were based on International Myeloma Working Group criteria.RESULTSA total of 26 patients were treated, 8 with NRF (median age 60 years), 9 with SRI (median age 75 years), and 9 with ESRD (median age 56 years). Median (range) baseline CrCl values were 105 (84–146) mL/min and 26 (15–33) mL/min for the NRF and SRI groups, respectively. One patient (CrCl 33 mL/min; SRI group) was excluded from the PK analysis. The majority (46%) had International Staging System stage III disease. Twenty-three patients (89%) had received prior therapy (median 2 regimens, range 1–7). Prior bortezomib, thalidomide, or Len treatment occurred in 21 (81%), 11 (42%), and 9 (35%) patients, respectively. Differences in baseline characteristics for race were seen between the NRF (8/8 white) and ESRD groups (1/9 white). Median Elo treatment duration for the NRF, SRI, and ESRD groups was 5, 15, and 8 months, respectively. Treatment discontinuation occurred in 6 (75%) NRF, 4 (44%) SRI, and 5 (56%) ESRD patients, primarily due to disease progression. Preliminary mean Elo PK values were calculated in 5 patients (1 NRF, 2 SRI, 2 ESRD). Maximum serum concentrations were 219, 214, and 203 µg/mL in the NRF, SRI, and ESRD groups, respectively. AUC0-Twas 46762, 52806, and 40357 h.µg/mL in the NRF, SRI, and ESRD groups, respectively. Half-life was 19, 13, and 8 days in the NRF, SRI, and ESRD groups, respectively. Total body clearance was 0.14, 0.15, and 0.29 mL/h/kg in the NRF, SRI, and ESRD groups, respectively. Adverse events are summarized in the table. No deaths or second primary malignancies were reported. Infusion reactions (Grade 2) were seen in 3 patients. Objective response rates (ORRs) were 75% (NRF, 6/8), 67% (SRI, 6/9), and 56% (ESRD, 5/9). Thirty-eight percent (3/8) of NRF, 56% (5/9) of SRI, and 11% (1/9) of ESRD patients had a very good partial response or better.Table:Adverse eventsAdverse eventNRF(n=8)SRI(n=9)ESRD(n=9)Any AE (any grade)899Most common AEs (occurred in >25% of patients)Fatigue646Diarrhea254Back pain334Anemia253Constipation631Pyrexia414Peripheral edema144Hyperglycemia332Thrombocytopenia143Rash241Hypokalemia133Cough421Dyspnea322Any Grade 3–4 AE787CONCLUSIONSElo, in combination with Len/dex, was well tolerated by patients with MM regardless of renal function. Updated PK assessments will be provided at the time of presentation. Responses were observed in all 3 renal function groups and ORR in patients with NRF was consistent with previous studies.1. San Miguel JF, et al. Leukemia. 2008;22:842-9.2. Knudsen LM et al. Eur J Haematol.2000;65:175-81.3. Lonial S, et al. J Clin Oncol. 2012;30:1953-9.This study is being funded by Bristol-Myers Squibb.Professional medical writing and editorial assistance was provided by Matthew Thomas at Caudex Medical and was funded by Bristol-Myers Squibb. DisclosuresOff Label Use: Elotuzumab is an investigational agent being studied for the treatment of multiple myeloma. Jagannath:Celgene; Bristol-Myers Squibb; Sanofi-Aventis: Honoraria. Zonder:Bristol-Myers Squibb: Advisory board Other. Kaufman:Millennium; Celgene; Novartis; Onyx; Janssen; Spectrum: Honoraria; Novartis; Onyx; Merck; Celgene: Research Funding; Millennium; Celgene; Novartis; Onyx; Janssen; Spectrum: Consultancy. Lynch:Bristol-Myers Squibb: Employment. Bleickardt:Bristol-Myers Squibb: Employment. Paliwal:Bristol-Myers Squibb: Employment. Vij:Array: Honoraria; Millennium: Honoraria; Novartis: Honoraria; Janssen: Honoraria; Sanofi: Honoraria; Onyx: Honoraria, Research Funding; Celgene: Honoraria, Research Funding.

NK Cells: Key to Success of DC-Based Cancer Vaccines?

The cytotoxic and regulatory antitumor functions of natural killer (NK) cells have become attractive targets for immunotherapy. Manipulation of specific NK cell functions and their reciprocal interactions with dendritic cells (DCs) might hold therapeutic promise. In this review, we focus on the engagement of NK cells in DC-based cancer vaccination strategies, providing a comprehensive overview of current in vivo experimental and clinical DC vaccination studies encompassing the monitoring of NK cells. From these studies, it is clear that NK cells play a key regulatory role in the generation of DC-induced antitumor immunity, favoring the concept that targeting both innate and adaptive immune mechanisms may synergistically promote clinical outcome. However, to date, DC vaccination trials are only infrequently accompanied by NK cell monitoring. Here, we discuss different strategies to improve DC vaccine preparations via exploitation of NK cells and provide a summary of relevant NK cell parameters for immune monitoring. We underscore that the design of DC-based cancer vaccines should include the evaluation of their NK cell stimulating potency both in the preclinical phase and in clinical trials.

The Human Herpesvirus-7 (HHV-7) U21 Immunoevasin Subverts NK-Mediated Cytoxicity through Modulation of MICA and MICB

Herpesviruses have evolved numerous immune evasion strategies to facilitate establishment of lifelong persistent infections. Many herpesviruses encode gene products devoted to preventing viral antigen presentation as a means of escaping detection by cytotoxic T lymphocytes. The human herpesvirus-7 (HHV-7) U21 gene product, for example, is an immunoevasin that binds to class I major histocompatibility complex molecules and redirects them to the lysosomal compartment. Virus infection can also induce the upregulation of surface ligands that activate NK cells. Accordingly, the herpesviruses have evolved a diverse array of mechanisms to prevent NK cell engagement of NK-activating ligands on virus-infected cells. Here we demonstrate that the HHV-7 U21 gene product interferes with NK recognition. U21 can bind to the NK activating ligand ULBP1 and reroute it to the lysosomal compartment. In addition, U21 downregulates the surface expression of the NK activating ligands MICA and MICB, resulting in a reduction in NK-mediated cytotoxicity. These results suggest that this single viral protein may interfere both with CTL-mediated recognition through the downregulation of class I MHC molecules as well as NK-mediated recognition through downregulation of NK activating ligands.