Engineering of a trispecific tumor-targeted immunotherapy incorporating 4-1BB co-stimulation and PD-L1 blockade

Abstract

Co-stimulatory 4-1BB receptors on tumor-infiltrating T cells are a compelling target for overcoming resistance to immune checkpoint inhibitors, but initial clinical studies of 4-1BB agonist mAbs were accompanied by liver toxicity. We sought to engineer a tri-specific antibody-based molecule that stimulates intratumoral 4-1BB and blocks PD-L1/PD-1 signaling without systemic toxicity and with clinically favorable pharmacokinetics. Recombinant fusion proteins were constructed using scMATCH3 technology and humanized antibody single-chain variable fragments against PD-L1, 4-1BB, and human serum albumin. Paratope affinities were optimized using single amino acid substitutions, leading to design of the drug candidate NM21-1480. Multiple in vitro experiments evaluated pharmacodynamic properties of NM21-1480, and syngeneic mouse tumor models assessed antitumor efficacy and safety of murine analogues. A GLP multiple-dose toxicology study evaluated its safety in non-human primates. NM21-1480 inhibited PD-L1/PD-1 signaling with a potency similar to avelumab, and it potently stimulated 4-1BB signaling only in the presence of PD-L1, while exhibiting an EC₅₀ that was largely independent of PD-L1 density. NM21-1480 exhibited high efficacy for co-activation of pre-stimulated T cells and dendritic cells. In xenograft models in syngeneic mice, NM21-1480 induced tumor regression and tumor infiltration of T cells without causing systemic T-cell activation. A GLP toxicology study revealed no evidence of liver toxicity at doses up to 140 mg/kg, and pharmacokinetic studies in non-human primates suggested a plasma half-life in humans of up to 2 weeks. NM21-1480 has the potential to overcome checkpoint resistance by co-activating tumor-infiltrating lymphocytes without liver toxicity.

Keywords: Immune checkpoint inhibitor; T-cell stimulation; antibody fragment; cancer immunotherapy; fusion protein; non-human primate; trispecific antibodies; xenograft model.

Figure 1.

Engineering of the molecular scaffold and calibration of the 4–1BB and PD-L1 binding affinities. (a) Schematic representation (left) of the trispecific scMATCH3 molecular scaffold and a structural model (right) derived from X-ray crystallography structures. αPD-L1 (VL1/VH1) in blue, α4-1BB (VH2/VL2) in green, αHSA (VL3/VH3) in orange, and Gly₄-Ser peptide linkers in red. (b) Surface representation of a structural model based on X-ray crystallography complex structures showing the trimer of 4–1BB extracellular domain (dark green), its natural trimeric ligand, 4–1BB-L (gray), and the bound 4–1BB-binding domains of urelumab as an Fab fragment (purple), as scFv targeting the proximal part of 4–1BB (38–02-A04 in red) and an scFv targeting the distal part of 4–1BB (38–27-A11 in light green). (c) Drug concentration–response curves for 12 scMATCH3 analogues with the indicated α4-1BB and αPD-L1 affinities. Responses were measured from Jurkat cells containing NFAT (PD-L1, blue) or NF-κB (4–1BB, green) reporters. Markers indicate mean (± SD) of two technical replicates of one experiment. (d) Contour plots showing the percentage activity of scMATCH3 constructs to stimulate 4–1BB activity measured by NF-ĸB (in shades of green) at 99% relative PD-L1 antagonistic activity. Data are shown for distal 4–1BB epitope-targeting scMATCH3 molecules (left) and proximal 4–1BB epitope-targeting molecules (right). The contour plot was generated using the distance method for interpolation in Minitab. (e) Maximum secretion of IL-2 from SEA pre-activated human PBMCs measured by ELISA after addition of trispecific scMATCH3 molecules binding to the membrane distal or membrane-proximal epitope of 4–1BB (mean ± SD of four independent experiments). (f) Concentration–response curves for stimulation of 4–1BB signaling in Jurkat NF- κB reporter cells (green) and PD-L1 antagonism in Jurkat NFAT reporter cells (blue) of a bivalent, bispecific mAb (PRO1928) employing the same α4-1BB and αPD-L1 paratopes as NM21-1480 (left graph) and NM21-1480 (right graph). Mean (± SD) of one representative experiment of two independent experiments

Figure 2.

NM21-1480 stimulation of 4–1BB requires PD-L1, but its EC₅₀ is largely independent of PD-L1 density. (a) Concentration–response curve for NM21-1480-induced stimulation of 4–1BB relative to maximal urelumab-induced activation in a NF-κB Jurkat reporter cell line co-incubated with IFN-γ pre-stimulated cancer cell-lines expressing different PD-L1 densities. PD-L1 density on the cancer cell lines is represented as the average αPD-L1 antibody-binding capacity quantified by flow cytometry. Markers represent mean (± SD) of one representative experiment of ≥ 4 independent experiments. (b) EC₅₀ values for NM21-1480 stimulation of 4–1BB measured by NF-κB Jurkat reporter cells plotted against PD-L1 density of cell lines co-incubated within the assay. Every cell line was tested in ≥ 4 independent experiments and all obtained individual EC₅₀ values were plotted against PD-L1 density. A linear regression model is shown as solid line to indicate constant EC₅₀. (c) Normalized concentration–response curve for NM21-1480 or urelumab stimulation of NF-κB in Jurkat reporter cells co-incubated with PD-L1-negative CHO cells (left) or PD-L1-positive HCC827 cells (right). Markers represent mean (± SD) of one representative experiment of ≥ 4 independent experiments. (d) Human PBMCs were pre-activated with an αCD3 antibody then exposed to NM21-1480. Concentration–response curves for IL-2 secretion in response to NM21-1480 stimulation in the presence of PD-L1-positive HCC827 cells (left) or PD-L1-negative CHO cells (right). No measurable Il-2 secretion was observed in non-pre-activated PBMCs. Mean (± SD) of one representative experiment of 3 independent experiments

Figure 3.

NM21-1480 potently inhibits PD-L1 interactions and signaling. (a) Competition ELISA quantifying PD-L1/PD-1 interaction in the presence of different concentrations of NM21-1480 or avelumab. Mean (± SD) of two technical replicates of one experiment. (b) Competition ELISA quantifying PD-L1/B7.1 interaction in the presence of different concentrations of NM21-1480 or avelumab. Markers represent mean (± SD) of two technical replicates of one experiment. (c) Jurkat NFAT reporter cells co-incubated with PD-L1-positive CHO cells and a T-cell receptor activator were exposed to different concentrations of NM21-1480 or avelumab. Both agents inhibited NFAT expression with similar EC₅₀. Representative results from 9 independent experiments

Figure 4.

Potent stimulation of 4–1BB and immune cell co-stimulation by NM21-1480. (a) Human PBMCs were pre-stimulated with SEA to induce T- cell activation and expression of PD-L1 and 4-1BB expression. IL-2 secretion was then measured by ELISA in response to NM21-1480 or combinations of αPD-L1/PD-1 mAbs plus α4-1BB mAbs (p < .001 by one-way ANOVA and Tukey’s multiple comparisons test). In non-pre-stimulated PBMCs, IL-2 secretion was near or below the lower limit of detection. Bars indicate mean of maximum IL-2 levels ± SD from ≥3 independent experiments. (b) Cytokine release from quiescent human PBMCs in response to 3 concentrations of NM21-1480 and positive (OKT3, PHA, Rituxan) and negative (infliximab) controls. Colors indicate cytokine, and individual data points for each condition and cytokine represent each of 6 healthy donors. (c) Viability of 4 indicated classes of SEA pre-stimulated PBMCs after exposure to NM21-1480, avelumab, or urelumab. Viability was assessed by annexin V-APC staining. Mean (± SD) of three technical replicates of one experiment), representative of 2 experiments. (d) Markers of T-cell activation (TNFα, IL-2, IFNγ) or DC activation (IL12p40, CD86, HLA-DR) in response to stimulation in co-cultures of T cells and monocyte-derived dendritic cells (MoDCs). MoDCs were prepared from CD14+ cells cultured for 7 days. MoDCs were then cultured together with T cells from a separate donor for 5 days in the presence of NM21-1480, avelumab plus urelumab, or IgG1 plus IgG4. Supernatants and cells were collected at the end of culture (or 48 hours for IL2) and cytokine production was measured by ELISA or cell surface markers measured by flow cytometry. Data are presented as mean ± SEM (n = 6 biological replicates). **p˂0.01, ***p˂0.001, ****p˂0.0001 comparing to NM21-1480, as determined using a repeated measures two-way ANOVA with Dunnett’s multiple comparisons test

Figure 5.

Activity of NM21-1480 in xenograft models. (a), (b) and (c), CD34+ stem cell-substituted NOG mice were engrafted with HCC-827 NSCLC cells. Mice were randomized at day 0 when tumor volumes reached between 80 and 100 mm³, and were treated on days 0, 5, 10, 15, and 20 as indicated by the dotted vertical lines. (a) Relative tumor volume at the indicated times after randomization. Each marker represents mean of 10 animals. (b) Relative tumor volume at the end of the study. Horizontal bars indicate mean (±95% CI). Statistical analysis was performed by one-way ANOVA with Bonferroni correction. The results are representative of two experiments. (c) At the end of the study, tumors were harvested and assessed for infiltration of human T cells by flow cytometry. Each colored marker represents the normalized CD8 + T-cell count in the tumor microenvironment of one mouse (n = 10). Horizontal bars indicate mean (±95% CI). Statistical analysis was performed by one-way ANOVA with Bonferroni correction. (d) NCG mice were injected subcutaneously (s.c.) with H292 cells and human PBMCs on day 0. Mice were treated on days 5, 10, 15, 20 and 25 with a control IgG, a CD3.TAA scMATCH3 molecule, NM21-1601 or the combination of CD3.TAA and NM21-1601. The graph displays tumor volume at the indicated times after randomization. Each point represents mean and standard deviation of 8 animals. Statistical analysis was performed using a two-way ANOVA with Tukey’s multiple comparison test, * p < .05; ** p < .005 compared to control IgG treatment group at day 28

Figure 6.

Preclinical pharmacology of NM21-1480 and GLP toxicology study in cynomolgus monkey. (a) Organ distribution of ¹²⁵I-labeled NM21-1601 at the indicated times after administration to BalbC nude mice bearing a PD-L1-positive tumor (MDA-MB-231). Each marker represents the scintillation count (mean ± SD) per gram of tissue from the indicated organ of 3 animals. (b) Serum concentration (mean ± SD) of NM21-1480 from a single-dose pharmacokinetic study of 3 dose levels in 3 cynomolgus monkeys. C-G, A GLP toxicology study in cynomolgus monkeys was conducted with 5 doses of NM21-1480 administered weekly at doses levels of 0, 20, 60 and 140 mg/kg. (c) Serum transaminase (ALT and ALT) levels before first dose and at days 15 and 29. (d) Representative liver photomicrographs at the end of the study. (e) Memory cell populations from blood samples measured by flow cytometry on the indicated days. Each marker represents an individual animal. Horizontal bars indicate mean ± SD. (f) Plasma levels of NM21-1480 during repeated dosing. Each marker represents an individual animal. (g) Unoccupied PD-L1 binding sites on circulating NK and B cells before the first dose of NM21-1480 and at the indicated times during repeated dosing. Each marker represents an individual animal