Alexander Muik

Purpose: Engagement of the co-stimulatory receptor OX40on activated T cells enhances T-cell activation, survival, and proliferation. First-generation agonistic OX40 antibodies have shown limited clinical efficacy so far. The investigational HexaBody®-OX40 (GEN1055/BNT315) was designed to promote FcγR-independent higher-order OX40 clustering on the cell surface, thereby leveraging receptor biology to achieve optimal stimulation. We assessed the in vitro and in vivo functional activity of HexaBody-OX40, and evaluated the potential of concomitantly blocking PD-1/PD-L1 inhibitory signals to amplify T-cell activity. Experimental Design: Reporter assays and primary human T-cell assays using healthy donor and cancer patient cells were used to study the effects of HexaBody-OX40, alone and in combination with PD-(L)1 blocking antibodies, on T-cell activation and proliferation in vitro. Antitumor activity and pharmacodynamic effects were evaluated in human OX40 knock-in mice and human OX40/PD-1 double knock-in mice bearing MC38 tumors. Results: HexaBody-OX40 exhibited FcγR-independent OX40 agonistic activity in vitro, enhancing T-cell activation, proliferation, and cytokine secretion, and promoted tumor-infiltrating lymphocyte expansion in primary tumor samples ex vivo. In vivo, HexaBody-OX40 demonstrated antitumor activity accompanied by peripheral and intratumoral T-cell immunomodulation. PD-(L)1 blockade further potentiated HexaBody-OX40-induced T-cell proliferation, activation, and cytokine secretion in assays using PBMCs from either healthy donors or cancer patients previously treated with PD-(L)1 blocking antibody. Combined with pembrolizumab, HexaBody-OX40 enhanced antitumor activity and T-cell immunomodulation beyond either monotherapy in vivo. Conclusions: HexaBody-OX40 agonistically enhanced T-cell mediated antitumor immunity through FcγR-independent OX40 agonism. The combination of HexaBody-OX40 with PD-(L)1 blocking antibodies enhanced the effect of both single agent treatments.

The therapeutic potential of interleukin-2 (IL-2) in cancer treatment is limited by toxicity challenges, partly due to an unfavorable pharmacokinetic profile and unintended activation of regulatory T (T _reg ) cells alongside the desired activation of CD8 ⁺ effector T cells. To selectively stimulate CD8 ⁺ T cells over T _reg cells, we engineered an IL-2 variant (IL-2var) with a dual-tuned affinity profile that features reduced binding to IL-2Rα (CD25) and enhanced binding to IL-2Rβ (CD122). To optimize its pharmacokinetics and facilitate tumor enrichment, the variant is fused to albumin and delivered as an mRNA encapsulated in a lipid nanoparticle (Alb–IL-2var RNA-LNP), enabling sustained systemic exposure from hepatic production upon intravenous administration. We show that Alb–IL-2var has a favorable pharmacokinetic profile and is tolerated at biologically active doses in immunocompetent mice and cynomolgus monkeys. Selective enhancement of CD8 ⁺ T cell responses over T _reg cells is demonstrated in vitro in human peripheral blood mononuclear cells and in vivo in mice and cynomolgus monkeys. When combined with an mRNA cancer vaccine in syngeneic subcutaneous mouse tumor models, Alb–IL-2var RNA-LNP stimulates the expansion of tumor-infiltrating and circulating tumor antigen–specific CD8 ⁺ T cells, but not T _reg cells. In advanced and cold syngeneic tumor models, it enhances the efficacy of radiotherapy, checkpoint inhibitors, and cancer vaccines. Combination with checkpoint inhibitors and vaccine induces profound proinflammatory conversion of cold tumors. These preclinical results validate a rational design approach to overcome the limitations of IL-2 therapy and support the clinical evaluation of Alb–IL-2var RNA-LNP for solid cancers.

Lung is a major site of metastases for many primary cancers associated with poor outcomes. A central challenge in cancer immunotherapy is overcoming tumor immune evasion, which limits effective antitumor responses. Here, we investigated whether combinatorial mRNA-encoded cytokine therapy can overcome tumor immune evasion by coordinately engaging innate and adaptive immunity, using murine models of pulmonary metastases. We employed intravenously administered cationic nucleoside-modified mRNA-lipoplexes (RNA-LPX) for targeted delivery of mRNA-encoded cytokines to the lung. The cytokine mix containing interferon-α, half-life extended interleukin (IL)-7, and a half-life extended IL-2 variant with reduced CD25-binding modulated the tumor immune microenvironment resulting in a potent and broad anti-tumor response and prolonged survival with good tolerability at the conditions tested. Using cell depletion experiments, we demonstrated that both T and natural killer (NK) cells are crucial mediators of the observed anti-tumor efficacy of the cytokine RNA mix, which induced activation and effector function of NK and T cells, coupled with reduced regulatory T cells (Treg) numbers and Treg activation in the lung. Importantly, antitumor efficacy was maintained in models of impaired antigen presentation, including loss of an immunodominant tumor antigen and MHC class I deficiency, where NK cells served as the primary effectors. The cytokine RNA mix induced immune cell activation in the primary human lung tumor culture, suggesting potential for translational application. Collectively, these findings demonstrate that combinatorial cytokine therapy can drive both antigen-dependent and antigen-independent tumor control for the treatment of lung metastases.

GEN1046 therapeutic efficacy and antitumor immune responses in MC38-hPD-L1 tumor–bearing hPD-L1/h4-1BB dKI mice. A, MC38-hPD-L1 cells (1 × 106 tumor cells) were injected s.c. in the right flank of hPD-L1/h4-1BB dKI mice. After tumor establishment (average tumor volume, ∼80 mm3), mice were randomized and treated with GEN1046 or isotype control antibody (each 5 mg/kg intravenous) at the indicated time points (n = 9 per group). B, Tumor growth of individual mice in each group, with CR defined as number of animals with CR. C, Progression-free survival, defined as the percentage of mice with tumor volume smaller than 500 mm3, is shown as a Kaplan–Meier curve. Mantel–Cox analysis was used to compare survival between treatment groups. ****, P < 0.0001. D, Mice with CR after treatment with GEN1046 (shown in B) were rechallenged by s.c. injection of 1 × 106 MC38-hPD-L1 tumor cells in the left flank on day 164. As a control group, a second cohort of naïve dKI animals was inoculated with 1 × 106 MC38-hPD-L1 tumor cells. Tumor growth of individual mice in each group is shown. E–I, MC38-hPD-L1 or MC38-WT cells (1 × 106 tumor cells) were injected s.c. in the right flank of hPD-L1/h4-1BB dKI mice. After tumor establishment (mean tumor volume, ∼75 mm3), mice were randomized and treated with GEN1046, a durvalumab analogue, or isotype control antibody (all 5 mg/kg intravenous) at the indicated time points. The mice were sacrificed 2 days after the last treatment (n = 5 per treatment group), and the tumors and spleens were excised. F, Sections of resected tumors (4 µm) were stained using anti-CD3, anti-CD8, or anti-FoxP3 antibodies by IHC. The number of positive cells was quantified per mm2. Mann–Whitney U statistical analysis was performed to compare the number of cellular subsets between treatment groups in MC38-hPD-L1 or MC38-WT tumor–bearing mice. *, P < 0.05. G and H, Flow cytometry analysis of dissociated splenocytes. Data from individual mice are shown as well as group mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001, Wilcoxon rank sum test. I, Peripheral blood samples were taken 1 day before treatment (d−1) and 2 days after each treatment. Cytokine analysis was performed by electrochemiluminescence immunoassay. s.c., subcutaneous; Treg, regulatory T cell.

Treatment-related AEs reported in >2 patients treated with GEN1046 (n = 61)

Characteristics of patients treated with GEN1046

GEN1046 antitumor efficacy in patients with advanced solid tumors. A, Waterfall plot of best relative percent change from baseline in tumor size. B–E, CT over time (B and C) and plots of immunologic changes in the periphery (D and E) following the first dose of GEN1046 in a patient with metastatic NSCLC treated with 200 mg GEN1046 (B and D) and a patient with metastatic ovarian cancer treated with 80 mg GEN1046 (C and E). Numbers (percentages) below CT scans are sum of diameter of target lesion (percent change from baseline). NA, not applicable; NE, not evaluable. aArchival formalin-fixed paraffin-embedded block of tumor tissue obtained >5 years prior was evaluated; loss of PD-L1 immunoreactivity may have occurred.

Pharmacokinetics and pharmacodynamics of GEN1046 in patients with advanced solid tumors. A, Mean plasma concentration of GEN1046 during the first two dosing cycles with administration Q3W. B and C, Maximal fold change from baseline in pharmacodynamic markers measured in peripheral blood during cycle 1 in patients receiving low (≤200 mg) and high (≥400 mg) doses of GEN1046. P values from the Wilcoxon–Mann–Whitney test. LLOQ, lower limit of quantification.

GEN1046 induces dose-dependent, conditional T-cell proliferation and cytokine production and enhances antigen-specific T-cell–mediated cytotoxicity in vitro. A and B, Activated T cells were cocultured with autologous iDCs in the presence of GEN1046 or control antibodies (0.125 µg/mL), and T-cell/iDC clusters were visualized over time by live-cell imaging. Quantification of the number of T cells in contact with a given DC on average (A), or over time (B; left), as well as the duration of these DC/T-cell clusters (B; right) is shown. ****, P < 0.0001; ns, not significant; Mann–Whitney U test. C, Induction of 4-1BB signaling by GEN1046 or control antibodies was assessed using a 4-1BB reporter assay. D, Blockade of the PD-1/PD-L1 interaction by GEN1046 or control antibodies was assessed using a PD-1/PD-L1 blockade bioassay. E, CFSE-labeled human PBMCs were stimulated with anti-CD3 (0.1 µg/mL) and incubated with GEN1046 or control antibodies (0.2 µg/mL) for 4 days. CFSE dilution in CD8+ T cells was analyzed by flow cytometry, and the expansion index was calculated. Data shown are the fold change in expansion index of treatment groups, relative to untreated cells of individual donors, as well as mean ± standard deviation (n = 11). ****, P < 0.0001; **, P < 0.01; *, P < 0.05, Friedman test with Dunn multiple comparisons test. F, CD8+ T cells were electroporated with RNA encoding a CLDN6-specific TCR and PD-1, labeled with CFSE, and cocultured with autologous DCs electroporated with CLDN6-encoding RNA in the presence of GEN1046 or control antibodies (0.2 µg/mL) for 4 days. Proliferation was measured by CFSE dilution as described in E. ****, P < 0.0001, Friedman test with Dunn multiple comparisons test. G, IFNγ concentrations in supernatant taken after 48 hours from cultures as described in F. Data shown are mean concentration ± standard deviation of triplicate wells from one representative donor (n = 3 donors). H–K, CD8+ T cells were electroporated with RNA encoding a CLDN6-specific TCR and were preactivated for 24 hours in coculture with CLDN6-expressing MDA-MB-231 cells (MDA-MB-231 hCLDN6) to induce 4-1BB expression. Subsequently, the preactivated CD8+ T cells were transferred to cocultures with previously seeded MDA-MB-231 hCLDN6 tumor cells (hCLDN6+ PD-L1+) in the presence of GEN1046 or control antibodies (0.2 µg/mL). H–I, After 48 hours, expression of cytotoxic mediator GZMB and degranulation marker CD107a by the CD8+ T cells was assessed; pooled data from four experiments (n = 3–12) are depicted. MFIs are expressed as relative values compared with the isotype control condition. J, Cytotoxicity of the CD8+ T cells toward MDA-MB-231 CLDN6 cells was monitored by electrical impedance measurement over 5 days using the xCELLigence real-time cell analysis. Representative data (mean ± standard deviation of triplicate wells) from cocultures derived from one individual donor are shown (n = 12). Data were normalized to the time point of coculture start and expressed relative to tumor-cell cultures without T cells (without T cells set to 100%). K, AUC (total area) analysis of cytotoxicity data (n = 11–12). ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05, mixed-effect analysis with Dunnett multiple comparisons test. MFI, median fluorescence intensity; ns, not significant.

GEN1046 promotes TIL expansion from patient-derived tumor tissue. Tumor tissues resected from patients with NSCLC were cut into pieces of 1 to 2 mm3 and cultured in the presence of IL2 (10–50 U/mL) and GEN1046 (or a GEN1046 surrogate comprising the PD-L1–specific Fab arm of GEN1046 and a nonhumanized variant of the 4-1BB–specific Fab arm or atezolizumab (0.2 µg/mL), or with IL2 only for 14–17 days. A, Cell numbers after expansion were determined by flow cytometry, and total TILs, CD8+ T cells, CD4+ T cells, and NK cells are shown for three patients. Tumor PD-L1 expression and 4-1BB expression by CD8+ T cells in the specimen at baseline are indicated. B, TCR repertoire analysis was performed by TRB RNA sequencing of the expanded TILs and the tumor fragments. Cumulative frequency of shared clonotypes, the 20 most abundant clonotypes in the GEN1046 surrogate–treated cultures, is shown. C, TILs expanded as in A were restimulated with enzymatically digested autologous tumor (TC) in the presence or absence of an MHC I–blocking antibody. Expression of 4-1BB and intracellular expression of IFNγ and CD107a in CD8+ T cells were analyzed by flow cytometry.