Blockade of the PD-1/PD-L1 axis and stimulation of 4-1BB have been shown to enhance immune responses to cancer, and to synergize when used in combination. To maximize the potential for synergy, Muik et al. used the DuoBody technology platform to develop GEN1046, a full-length IgG1 bispecific antibody targeting PD-L1 and 4-1BB (with abrogated FcγR binding) and tested it in both preclinical and early clinical settings. The results of these studies were recently published in Cancer Discovery.
In a coculture of dendritic cells (DCs) and T cells, the addition of GEN1046 increased and prolonged DC–T cell contacts, consistent with bridging of the bispecific between PD-L1 on DCs and 4-1BB (CD137) on T cells. Interestingly, both PD-L1 and 4-1BB needed to be present for effective 4-1BB agonism (i.e., the effect was dependent on cross-linking to PD-L1+ cells), but not for effective PD-L1 blockade (i.e., the effect was independent of cross-linking). The PD-L1 blockade acted as a classical checkpoint blockade, reducing PD-1-mediated inhibition of TCR signaling.
To evaluate whether GEN1046 could induce T cell proliferation, Muik et al. utilized CD3-activated PBMCs as a polyclonal model (CD3-activated PBMCs) and PD-L1+ immature DCs expressing CLDN6 and CLDN6-TCR+ CD8+ T cells as an antigen-specific model. In both settings, GEN1046 enhanced proliferation of activated (but not unactivated) CD8+ T cells, and to a lesser extent, CD4+ T cells. GEN1046 also induced strong, dose-dependent production of proinflammatory cytokines, most prominently IFNγ. The effects induced by GEN1046 were stronger than those induced by anti-PD-L1, or a combination of anti-PD-L1 and agonist 4-1BB. An additional culture of CLDN6-TCR+ CD8+ T cells with CLDN6+PD-L1+ MDA-MB-231 target cells showed that GEN1046 increased the frequency of granzyme B+CD107a+ CD8+ T cells, increased cellular expression of granzyme B and CD107a, and enhanced T cell-mediated cytotoxicity against the PD-L1+ target cells compared to controls.
Moving into mouse models, Muik et al. tested GEN1046 in double knock-in transgenic mice expressing the extracellular domains of human PD-L1 and human 4-1BB. Mice were engrafted with MC38 tumors transfected with human PD-L1. Administration of GEN1046 twice weekly for 3 cycles induced complete tumor regression in all animals and significantly increased PFS. Six of the nine animals that achieved complete regression also rejected rechallenge.
Immunophenotyping of tumors and spleens resected from hPD-L1/h4-1BB dKI mice bearing either wild-type or hPD-L1 MC38 tumors showed that treatment with GEN1046 increased infiltration of T cells, mostly CD8+ T cells, into the hPD-L1 MC38 tumors. In the spleen, GEN1046 increased T cells, and the percentage of CD8+ (but not CD4+) T cells, central memory CD8+ T cells, and PD-1+ CD8+ and CD4+ T cells. Tregs were also increased in the spleen, but not in tumors. In peripheral blood, increases in IFNγ, TNFα, IL-2, and CXCL10 were observed, in line with increased T cell activity.
Using tumor samples from three patients with NSCLC, Muik et al. found that administration of GEN1046 and low-dose IL-2 to ex vivo cultures enhanced the expansion of tumor-reactive TILs, including specific T cell clonotypes, in all samples. Two of the three samples had baseline expression of 4-1BB, and in these two samples, expansion of CD8+ T cells was also enhanced by GEN1046. When GEN1046-treated TILs were re-treated with autologous tumor cells, more TILs upregulated 4-1BB, IFNγ, and CD107a compared to TILs treated with anti-PD-L1 or IL-2.
Following a toxicity study in cynomolgus monkeys, which showed that GEN1046 was well tolerated, Muik et al. initiated a first-in-human phase I/IIa dose-escalation trial in 61 patients with advanced, heavily pre-treated solid tumors. The most common tumor types were colorectal, ovarian, pancreatic, and lung. Patients in this trial were given GEN1046 at 9 escalating dose levels. Biopsies taken from 52 patients prior to treatment showed that 84.6% of tumors were PD-L1- per TPS (tumor proportion score) and 50% of those were also PD-L1- per CPS (combined positive score).
Analysis of the pharmacokinetics of GEN1046 in patients showed that systemic levels peaked shortly after each infusion. Consistent with preclinical markers, IFNγ, IFN-responsiveness chemokines CXCL9–11, proliferating and total effector memory CD8+ T cells, and activated NK cells increased in peripheral blood. Low doses (≤200mg) of the drug had greater increases in immune modulation compared to higher doses (≥400mg), consistent with known bell curves for trimer-forming bsAbs, which cannot effectively cross-link in conditions of agent excess.
Overall, GEN1046 exhibited a manageable safety profile. Cases of dose-limiting toxicity were observed in 6 patients (9.8%), but each case was manageable and toxicity was not seen upon re-exposure. The maximum-tolerated dose was not reached. To identify the expansion dose, Muik et al. used an integrated model to evaluate receptor occupancy of PD-L1 in tumors, and trimer formation between GEN1046, PD-L1+ tumor cells, and 4-1BB+ immune cells. This showed an expected bell curve for average trimer formation that was consistent with the greater immune effects observed at lower doses.
Adverse events (AEs) were observed in 43 (70.5%) patients. Most AEs were low-grade, though 17 patients (27.9%) experienced at least one grade 3-4 adverse event. 6 patients (9.8%) discontinued treatment due to AEs; 3 of which were due to grade 3 transaminase elevations.
At the time of data cutoff, 4 patients remained on treatment. At a median follow-up of 9.4 months, disease control (stable disease or better) had been achieved in 40 (65.6%) patients. 2 patients achieved partial responses (PR), including one patient with TNBC and one with ovarian cancer. Neither patient had received prior treatment targeting PD-L1. Two additional unconfirmed PRs (uPRs) were observed in patients with NSCLC. Both patients had progressed previously on PD-L1-targeted treatments.
Looking at specific patient cases, the researchers highlighted one of the patients with NSCLC who achieved a uPR in NSCLC after 11 weeks. Treatment was continued past the point of progression, but was eventually discontinued. They also highlighted the patient with ovarian cancer who achieved a PR at week 19, which was maintained until week 60, when SD was observed. As of August 2021, the patient had received 30 cycles and remained on treatment.
Overall, Muik et al. demonstrated the safety and potential efficacy of GEN1046 in simultaneously targeting PD-L1 and 4-1BB using a single bispecific antibody without FcγR binding. This strategy offers simplicity and simultaneous targeting, which could enhance immune responses to cancer. Trials for GEN1046 are continuing.
By Lauren Hitchings