DuoBody-CD40x4-1BB induces dendritic-cell maturation and enhances T-cell activation through conditional CD40 and 4-1BB agonist activity

Abstract

Background: Despite the preclinical promise of CD40 and 4-1BB as immuno-oncology targets, clinical efforts evaluating CD40 and 4-1BB agonists as monotherapy have found limited success. DuoBody-CD40×4-1BB (GEN1042/BNT312) is a novel investigational Fc-inert bispecific antibody for dual targeting and conditional stimulation of CD40 and 4-1BB to enhance priming and reactivation of tumor-specific immunity in patients with cancer.

Methods: Characterization of DuoBody-CD40×4-1BB in vitro was performed in a broad range of functional immune cell assays, including cell-based reporter assays, T-cell proliferation assays, mixed-lymphocyte reactions and tumor-infiltrating lymphocyte assays, as well as live-cell imaging. The in vivo activity of DuoBody-CD40×4-1BB was assessed in blood samples from patients with advanced solid tumors that were treated with DuoBody-CD40×4-1BB in the dose-escalation phase of the first-in-human clinical trial (NCT04083599).

Results: DuoBody-CD40×4-1BB exhibited conditional CD40 and 4-1BB agonist activity that was strictly dependent on crosslinking of both targets. Thereby, DuoBody-CD40×4-1BB strengthened the dendritic cell (DC)/T-cell immunological synapse, induced DC maturation, enhanced T-cell proliferation and effector functions in vitro and enhanced expansion of patient-derived tumor-infiltrating lymphocytes ex vivo. The addition of PD-1 blocking antibodies resulted in potentiation of T-cell activation and effector functions in vitro compared with either monotherapy, providing combination rationale. Furthermore, in a first-in-human clinical trial, DuoBody-CD40×4-1BB mediated clear immune modulation of peripheral antigen presenting cells and T cells in patients with advanced solid tumors.

Conclusion: DuoBody-CD40×4-1BB is capable of enhancing antitumor immunity by modulating DC and T-cell functions and shows biological activity in patients with advanced solid tumors. These findings demonstrate that targeting of these two pathways with an Fc-inert bispecific antibody may be an efficacious approach to (re)activate tumor-specific immunity and support the clinical investigation of DuoBody-CD40×4-1BB for the treatment of cancer.

Keywords: T-lymphocytes; dendritic cells; immunotherapy.

Conflict of interest statement

Competing interests: US and ÖT are management board members and employees at BioNTech (Mainz, Germany). AM, FG, KS, AT, BS, JQ, MD and SK are employees at BioNTech. Some of the authors have securities from BioNTech. ES and FV are employees at TRON. HCA III, IA, JMB, SMB, DV, VMS, MF, DPES, SK, TA, ECWB and MJ-K are employees at Genmab and own stock and/or stock options. HCA III, IA, DPES, US, AM and FG are inventors on patents and patent applications related to CD40×4-1BB bispecific antibodies.

© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

DuoBody-CD40×4-1BB reinforces the immunological synapse through target crosslinking. (A) Generation of DuoBody-CD40×4-1BB by cFAE of Fc-silenced mAb-CD40 and mAb-4-1BB. The parental antibodies contain matched mutations in the CH3 domain (F405L/K409R) that drive heterodimerization of the Fab arms and formation of bispecific molecules during cFAE, as well as Fc-silencing mutations (L234F, L235E and D265A) that abrogate binding to FcγR and C1q. (B) Simultaneous binding of DuoBody-CD40×4-1BB to human CD40-transgenic K562 cells labeled with CellTrace Violet and human 4-1BB-transgenic K562 cells labeled with CellTrace FarRed was analyzed by flow cytometry. Double-positive doublets were quantified as percentage within the live cell population (mean±SD; triplicate wells). (C–E) T cells electroporated with a CLDN6-specific TCR were cocultured with CLDN6-electroporated or mock-electroporated autologous iDCs in the presence of 0.125 µg/mL DuoBody-CD40×4-1BB or control antibodies, 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 over time (C), or on average (D), as well as the duration of these DC/T-cell clusters (E) is shown. Error bars represent SD. ****P+ T cells in the presence of Alexa Fluor 647-conjugated DuoBody-CD40×4-1BB (magenta)and LFA-1 (in green) antibodies, on the x and y axes the z-stack of the same picture with the relative zoom in. Nuclei were counterstained with Hoechst (in blue). (G) Representative fluorescent images of cocultures as in (E), in the presence of DuoBody-CD40×4-1BB or control antibodies. The white dashed line represents the interface between the DC and T cell. Scale bars: 10 µm. (H) LUT profile of LFA-1 and DuoBody-CD40×4-1BB or the combination of bsAb-CD40xctrl and bsAb-ctrlx4-1BB at the DC-T-cell interface, indicated by the white dashed line. ANOVA, analysis of variance; cFAE, controlled Fab-arm exchange; iDC, immature dendritic cell; LFA-1, leukocyte functional antigen 1; LUT, look-up table.

Figure 2

DuoBody-CD40×4-1BB exhibits conditional agonist activity. (A) CD40 bioluminescent cell-based reporter assay in the absence or presence of 4-1BB-expressing K562 cells. Cells were incubated with DuoBody-CD40×4-1BB, recombinant CD40L or control antibodies. (B) Human peripheral blood B cells were cocultured with 4-1BB-transgenic or wild type K562 cells in the presence of DuoBody-CD40×4-1BB for 48 hours. Expression of CD69 and CD86 on CD20+ B cells was analyzed by flow cytometry. Data shown are mean percentages of CD69+ or CD86+ B cells from one representative donor (n=3). Error bars represent SD. (C) 4-1BB bioluminescent cell-based reporter assay in the absence or presence of CD40-expressing K562 cells. Fold luminescence-induction of cells incubated with DuoBody-CD40×4-1BB or control antibodies, compared with treatment control (isotype control antibody or media only) is shown. Error bars represent SD. RLU, relative luminescence units.

Figure 3

DuoBody-CD40×4-1BB induces DC maturation. Immature DCs from healthy donors were cocultured with purified, allogeneic CD8+ T cells in the presence of DuoBody-CD40×4-1BB, Fc-inert analogs of clinical mAbs mitazalimab and urelumab (0.001–1 µg/mL) or control antibodies (1 µg/mL; for combination of control antibodies 1 µg/mL of both antibodies was added) for 5 days. The percentage of HLA-DR+CD86+ DCs was measured by flow cytometry. Data from one donor are shown. Dotted line shows percentage of HLA-DR+CD86+ DCs in DC-T-cell cultures in the absence of treatment. DC, dendritic cell.

Figure 4

DuoBody-CD40×4-1BB enhances T-cell activation in vitro. (A) CFSE-labeled human PBMCs were stimulated with 0.03 µg/mL anti-CD3 and incubated with DuoBody-CD40×4-1BB or control antibodies (0.2 µg/mL) for 4 days. CFSE dilution in CD8+ T cells was analyzed by flow cytometry. Expansion index of individual donors and mean±SD (n=7) is shown. (B) CD8+ T cells were electroporated with RNA encoding an HLA-A2/CLDN6-specific TCR, labeled with CFSE and cocultured with autologous DCs electroporated with CLDN6-encoding RNA in presence of the DuoBody-CD40×4-1BB or control antibodies (0.2 µg/mL; for combination of control antibodies 0.2 µg/mL of both antibodies was added) for 4 days. Proliferation was measured as described in (A). Expansion index of individual donors and mean±SD (n=9) is shown. ***P<0.001; *p<0.05; Friedman test. (C–D) Polyclonal (C) and antigen-specific T-cell proliferation assays (D) as described in A–B with increasing concentrations of DuoBody-CD40×4-1BB or isotype control antibody. Data shown are mean expansion index (±SD) of triplicate wells of one representative donor (n=12 for polyclonal assay, n=5 for antigen-specific assay). (E) Cytokine concentrations in supernatant taken after 48 hours from cultures as described in A–B. Data shown are mean concentration ±SD of triplicate wells from one representative donor (n=3 for polyclonal assay, n=5 for antigen-specific assay). (F–G) CD8+ T cells electroporated with a CLDN6-specific TCR were cocultured with CLDN6-expressing K562 cells for 48 hours. The percentage of IFN-γ+ cells or GzmB+CD107a+ cells (F), and expression levels of GzmB and CD107a normalized to the isotype control (G) were analyzed by flow cytometry. Data from individual donors and mean±SD (n=8) are shown. ***P<0.001; **p<0.01; One-way ANOVA with Dunnett’s multiple comparisons test (F) or paired t-test (G). ANOVA, analysis of variance; CFSE, carboxyfluorescein succinimidyl ester; DC, dendritic cell; IFN, interferon; PBMC, peripheral blood mononuclear cell.

Figure 5

DuoBody-CD40×4-1BB enhances TIL expansion. Tumor tissues resected from patients were cut into pieces of 1–2 mm3 and cultured in the presence of IL-2 (10 U/mL) and DuoBody-CD40×4-1BB (0.0008, 0.003, 0.0125, 0.05 or 0.2 µg/mL), a non-humanized variant of DuoBody-CD40×4-1BB (0.01–1 µg/mL), control antibodies (0.2 µg/mL) or IL-2 only for 10–15 days. (A–D) Cell numbers after expansion were determined by flow cytometry, and normalized to reference beads. Total TIL, CD8+, CD4+ T-cell and NK-cell numbers are shown for a patient with colon cancer (A) and three patients with NSCLC (B–D) *p<0.05. One-way ANOVA with Dunnett’s multiple comparisons test. (E) TCR repertoire analysis was performed by TRB RNA sequencing of TIL expanded in (D), in the presence of a non-humanized variant of DuoBody-CD40×4-1BB (0.1 µg/mL) or IL-2 only. Cumulative frequency of shared clonotypes, the 20 most abundant clonotypes in the DuoBody-CD40×4-1BB-treated cultures are shown. ANOVA, analysis of variance; NK, natural killer; NSCLC, non-small cell lung cancer; TCR, T-cell receptor; TIL, tumor-infiltrating lymphocyte.

Figure 6

Combination of DuoBody-CD40×4-1BB with pembrolizumab amplifies the magnitude of the immune response. (A, B) Purified CD8+ T cells from healthy donors were cocultured with LPS-matured allogeneic DCs in the presence of DuoBody-CD40×4-1BB, research-grade pembrolizumab (either alone or in combination (concurrent treatment)) or control antibodies for 5 days. (A) Cytokine concentrations in supernatant taken after 5 days of culture in the presence of indicated antibodies. Data shown are mean concentration±SD of duplicate wells from one representative donor pair (donor pair two is shown in online supplemental figure 5). Dashed line indicates cocultures that were not treated with antibody (No Tx). (B) The percentage of 4-1BB+ CD8+ T cells and CD27+ CD8+ T cells was measured by flow cytometry. Data shown are the percentage of positive cells within the total CD8+ T-cell population from one representative donor pair (donor pair two is shown in online supplemental figure 5). Dashed line indicates cocultures on Day 0 (before antibodies were added to the treated cocultures) and cocultures that were not treated with antibody at Day 5 of the MLR assay (No Tx). (C) CD8+ T cells were electroporated with RNA encoding an HLA-A2/CLDN6-specific TCR and PD-1, labeled with CFSE and cocultured with autologous DCs electroporated with CLDN6-encoding RNA in presence of the DuoBody-CD40×4-1BB (0.0022, 0.0067 or 0.2 µg/mL), IgG1 isotype control (0.8 µg/mL), clinical-grade pembrolizumab (0.8 µg/mL) or combinations thereof for 5 days. CFSE dilution in CD8+ T cells was analyzed by flow cytometry. Mean expansion index±SD (n=4) is shown. Dashed line indicates CD8+ T cells that were cocultured with DCs that were not electroporated with CLDN6 in the presence of the IgG1 isotype control antibody as negative control. CFSE, carboxyfluorescein succinimidyl ester; DC, dendritic cell; LPS, lipopolysaccharide; TCR, T-cell receptor.

Figure 7

DuoBody-CD40×4-1BB mediates broad immune modulation of peripheral APCs and T cells in patients. Patients with advanced solid tumors were treated with DuoBody-CD40×4-1BB in a first-in-human clinical trial (NCT04083599). Data shown are preliminary data from the dose-escalation phase of the ongoing study from patients treated with 30 mg or higher doses of DuoBody-CD40×4-1BB (cut-off date: 20 August 2021). Blood samples were collected on the indicated time points and analyzed for cytokines and immune cell subsets. For each parameter, the mean fold change compared with Cycle 1 Day 1 predose is shown. Numbers in the top right corners denote the number of patients that were included for each parameter. (A) Mean fold change±SEM of IFN-γ and TARC compared with Cycle 1 Day 1 predose. (B) Mean fold change±SEM of absolute CD19+ B-cell counts (cells/μL; calculated using the following formula: (B cells (event count)/leukocytes (event count)*LEUKOCYTES (103 cells/µL))*1000) and the percentage of CD86+ B cells within the CD19+ B-cell population compared with Cycle 1 Day 1 predose. C. Mean fold change±SEM of the percentage of CD8+Ki67+ T cells within the CD8+ T-cell population and CD8+ Ki67+ effector memory T cells within the CD8+CD45RA-CCR7- effector memory T-cell population compared with Cycle 1 Day 1 predose. APC, antigen presenting cell; IFN, interferon.