Tumor-restricted activation of Vγ9Vδ2 T cells via bispecific Evobodies: a novel strategy for safe and potent immunotherapy in ovarian cancer

Abstract

Introduction: Vγ9Vδ2 T cells have been clinically evaluated-both in vivo and ex vivo-for their efficacy against solid tumors over several decades. Although recent therapeutic approaches have renewed hope, significant and reproducible benefits for patients with solid tumors remains to be demonstrated.

Material & methods: We have developed bispecific biologics in an IgG-extended format that bind both to Folate Receptor alpha (FOLR1), which is highly expressed in the majority of ovarian cancers, and to the activating butyrophilin (BTN)3A. By reducing the affinity of the BTN3A agonist and leveraging the increased avidity of the tetravalent, bispecific antibody, activation of BTN3A is restricted to FOLR1-positive tumors, thereby avoiding off-target activation of non-tumor cells.

Results: Using RTCA co-culture assays with Vγ9Vδ2 T cells and tumor cell lines, we identified "Evobodies" that exhibit a strong therapeutic window and high potency against FOLR1-positive cells, while sparing healthy, FOLR1-negative tissue. Moreover, the lead molecule demonstrates high efficacy in a human autologous, patient-derived ex vivo tumor tissue model at unaltered/physiological effector-to-target (E:T) ratios. Importantly, we show that our tumor-engaging molecules avoid premature immune cell activation, degranulation, and cytokine release in the absence of FOLR1-positive tumor cells. They likely establish a cytokine gradient from the tumor site, harnessing the full potential of the natural local anti-infection response to target cancer cells.

Discussion: Thus, Evobodies represent a novel, first-in class of biologics for solid tumor treatment and are well-suited for further clinical development.

Keywords: BTN3A; FOLR1; Vγ9Vδ2 T cells; evobody; immunotherapy; ovarian cancer.

Figure 1

Generation and Optimization of bispecific Evobodies. (A) Homology model of the estimated structure of an Evobody. Dark blue: heavy chain of IgG1 agonist binding to BTN3A engineered with an Fc LALA mutation (L234A/L235A) to abrogate Fcγ receptor binding and effector functions, light blue: corresponding light chain, purple: scFvs binding to FOLR1, grey: linker sequences. (B) Chromatogram of the size exclusion chromatography (SEC) of recombinantly expressed Evobody. The red dashed line indicates the peak fractions that were collected and contained the correctly assembled Evobody. (C) SDS-PAGE of purified Evobody after SEC with two different volumes being loaded. For comparison, a mAb standard was loaded at decreasing amounts. 10% gel with reducing conditions. (D) Dose response curve of ETA-005 and the inverted format ETA-005 determined via an RTCA cytotoxicity assay with expanded Vγ9Vδ2 T cell lines from a healthy donor co-cultured in triplicates with OVCAR-3 cells. (E, F) Dose response curves for ETA-005 (before germline reset) and ETA-067 (after germline reset) determined via an RTCA cytotoxicity assay with expanded Vγ9Vδ2 T cells from a healthy donor co-cultured in triplicates either with OVCAR-3 wild-type cells (E) or with FOLR1 knock-out cells (F). (G) Comparison of ETA-005 and ETA-067 regarding their EC50 values from cytotoxicity assays with FOLR1-positive WT OVCAR-3 cells and FOLR1-negative cells (Top table) and their K_d-values as from surface plasmon resonance experiments to determine their affinity to BTN3A (bottom table).

Figure 2

Evobodies can induce the release of activating cytokines and proliferation of Vγ9Vδ2 T cells specifically at the tumor site. (A) Schematic representation of the comparison of tumor tissue and peripheral blood with regard to estimated Evobody availability and desired cytokine concentration. Created with Illustrations from NIAID NIH BIOART Source. (B) Expanded Vγ9Vδ2 T cells from healthy donors were co-cultured with OVCAR-3 cells for up to 24 h while donor-matched PBMCs were cultured for the same time period without OVCAR-3 cells. Evobody ETA-062 was added at concentrations of 1 nM to expanded Vγ9Vδ2 T cells and 10 nM to PBMCs. Supernatants were taken after 4, 8 and 24 h and the concentration of IFN-γ and TNF-α was determined via ELISA. Every line represents one donor (n=2 donors). (C/D) Vγ9Vδ2 T cell lines plus rIL-2 from healthy donors (n=4) were cultured in medium or with 300 nM BrHPP or 1 nM of the indicated Evobodies in the presence of anti-human CD107a mAb. (C) The percentage of CD107-positive Vδ2 T cells and (D) of dead cells stained by SytoxGreen was measured after 4 h by flow cytometry. (E) PBMCs from healthy donors were co-incubated with OVCAR-3 cells for up to 13 days in the presence of 50 IU/ml rIL-2. Evobody ETA-067 was added at 10 or 1 nM concentration while the culture with rIL-2 alone (Medium) served as negative control. The Vδ2 γδ T cells were counted via FACS staining and the acquisition of a specific volume (n=3 donors). (F) Ex vivo isolated Vδ2 TIL within tumor tissue were cultured in medium or stimulated with ETA-067 together with repetitive addition of IL-2 (every 3 days). After 10 to 14 days, the absolute number of expanded Vδ2 TIL in medium or ETA-067 stimulated cultures were determined and x-fold Vδ2 TIL expansion of the four donors in four different experiments was calculated.

Figure 3

Two different patient-derived ex vivo models. (A, B) Freshly isolated tumor tissue from ovarian cancer patients was minced and dissociated for two different models. (A) Dissociated tumor tissue was cultured in complete medium overnight. After 24 h, adherent cells were collected and stained for EpCAM and FOLR1 expression. Autologous PBL were isolated from the blood and stained for CD3-positive Vδ2 T cells. Adherent tumor cells and autologous PBL were cultured in the presence of 1 nM ETA-067 and 50 IU/mL rIL-2 with an E/T ratio of 1:13 over 4 days to determine Vδ2 T cells cytotoxicity, expansion and the percentage of activation (CD69, CD25) and inhibitory check point molecules (PD-1 and TIGIT) on Vδ2 T cells and other T cells (CD4, CD8, Treg). (B) For the novel patient-derived ex vivo tumor tissue model (exTuTiMo) complete freshly isolated, dissociated tumor tissue including an immunosuppressive tumor-microenvironment was plated and stimulated with or 1 nM control construct ETA-090 (spontaneous lysis) or 1 nM ETA-067 together with 50 IU/mL rIL-2 in complete medium with an unchanged physiological effector/target ratio of 1:240. Cytotoxicity was measured over the whole time and flow cytometric analysis of the absolute cell number (cell count > proliferation) and expression of activation (CD25) and inhibitory markers (PD-1 and TIGIT) markers as well as of central and effector memory cells (CM, EM) was determined initially and at an endpoint on Vδ2, CD4, CD8 or Treg TILs (10 days after culture).