Differentiated agonistic antibody targeting CD137 eradicates large tumors without hepatotoxicity

Abstract

CD137 (4-1BB) is a member of the TNFR superfamily that represents a promising target for cancer immunotherapy. Recent insights into the function of TNFR agonist antibodies implicate epitope, affinity, and IgG subclass as critical features, and these observations help explain the limited activity and toxicity seen with clinically tested CD137 agonists. Here, we describe the preclinical characterization of CTX-471, a fully human IgG4 agonist of CD137 that engages a unique epitope that is shared by human, cynomolgus monkey, and mouse and is associated with a differentiated pharmacology and toxicology profile. In vitro, CTX-471 increased IFN-γ production by human T cells in an Fcγ receptor-dependent (FcγR-dependent) manner, displaying an intermediate level of activity between 2 clinical-stage anti-CD137 antibodies. In mice, CTX-471 exhibited curative monotherapy activity in various syngeneic tumor models and showed a unique ability to cure mice of very large (~500 mm3) tumors compared with validated antibodies against checkpoints and TNFR superfamily members. Extremely high doses of CTX-471 were well tolerated, with no signs of hepatic toxicity. Collectively, these data demonstrate that CTX-471 is a unique CD137 agonist that displays an excellent safety profile and an unprecedented level of monotherapy efficacy against very large tumors.

Keywords: Immunology; Immunotherapy; Oncology.

Figure 1. CTX-471 binds to a unique epitope on CD137.

(A) Binding traces from Bio-Layer Interferometry (BLI) experiments testing ability of recombinant CD137L to bind to preformed complexes of CD137 and stated antibody. (B) Max BLI response measured for binding of full-length or truncated forms of human CD137 to tested antibodies. (C) Mapping of identified contact residues for binding of CTX-471 (red) or urelumab (blue) onto crystal structure of human CD137/CD137L complex (PDB 6CPR) based on mutational analyses. (D and E) BLI measurements for binding of mouse CD137 truncations and point mutations to tested antibodies with mapping of identified contact residues onto crystal structure of the mouse CD137/CD137L complex (PDB 6MKZ).

Figure 2. CTX-471 costimulates primary T cells with FcγR-dependent activity.

(A–C) IFN-γ secretion after a 3-day coculture of primary T cells and CHO cells engineered to express FcγRIIb (CD32b). Freshly isolated human T cells were stimulated with soluble anti-CD3 (clone OKT3) and anti-CD137 antibodies in the presence of CHO-CD32b (A and B) or CHO (C) cells. Representative data from n = 3–4 experiments (A and C) . All data presented as mean ± SEM.

Figure 3. CTX-471–induced activity is further enhanced by CD137 ligand.

(A) HEK-SplitCD137 cells were incubated with cross-linked anti-CD137 antibodies for 4 hours. The cells were then lysed and the level of CD137 cross-linking was determined by measuring luminescence of firefly luciferase. (B–D) HEK-293T cells expressing CD137 and a minimal NF-κB reporter were cocultured in the presence of plate-bound isotype or CD137L-Fc, along with cross-linked CTX-471 (B), urelumab (C), or utomilumab (D) for 4 hours. The cells were then lysed, and the level of NF-κB signaling was determined by measuring the luminescence of firefly luciferase. All data are the average of n = 3 independent experiments, presented as mean ± SEM. (E) IFN-γ secretion after a 3-day coculture of anti-CD3 activated primary T cells with CHO-CD32b cells and CTX-471 with or without CD137L-Fc.

Figure 4. CTX-471-AF achieves high rates of complete tumor regression in mouse models.

(A) Tumor growth rates of individual mice with established (50–75 mm³) CT-26 tumors following i.p. administration of CTX-471-AF on days 0, 3, 6, and 9. (B) Kaplan-Meier survival curves of mice in A and growth curves from tumor rechallenge of cured mice 88 days after last treatment. (C) Tumor growth rates of individual mice with established (75–100 mm³) A20 tumors following i.p. administration of CTX-471-AF on days 0, 7, 14, 21 and 28. (D) Kaplan-Meier survival curves of mice in C and growth curves from tumor rechallenge of cured mice 86 days after last treatment. Log-rank test followed by Bonferroni’s multiple comparison analysis was performed to determine the statistical significance of overall survival with CTX-471-AF versus control (*P<0.05, ***P<0.001).

Figure 5. CTX-471 profoundly reprograms the tumor immune environment.

(A–H) Mice with established CT-26 tumors (n = 4 per group) were treated with CTX-471-AF on days 0, 3, 6, and 9. Tissues were harvested on day 11, and dissociated tumors were analyzed by flow cytometry. CD45⁺ tumor infiltrating leukocytes (TILs; A); fraction of CD8⁺ T cells (B), CD4⁺ T cells (C), and Tregs (D) within TILs; TIGIT⁺PD-1⁺ double-positive cells within CD8⁺ T cells (E), and CD4⁺ T cells (F); tumor associated macrophages (TAM; G) and M1 polarized TAMs (H) are shown. Statistical significance was determined using 1-way ANOVA followed by Bonferroni’s multiple comparison compared with control treatment groups (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). All data are presented as mean ± SEM. (I) t-SNE plot showing unsupervised clustering of the scRNA profiles of CD8⁺ TILs from CT-26 tumor–bearing mice treated with isotype, CTX-471-AF, or 3H3. t-SNE plot highlighting cells from isotype- (red), CTX-471-AF– (orange), and 3H3-treated (gray) mice. (J) Heatmap of differentially expressed genes between clusters (0, 1, and 2) that show differential enrichment in cells from isotype-, CTX-471-AF–, and 3H3-treated mice. (K) Violin plots showing expression of a memory-precursor CD8⁺ T cell signature (left), effector CD8⁺ T cell signature (middle), and a dysfunction/exhaustion signature (right) in the scRNA profiles of CD8⁺ TILs from isotype- (blue), CTX-471-AF– (orange), and 3H3-treated (green) mice. ***P < 0.001 by Benjamini-Hochberg corrected Tukey’s multiple comparisons test.

Figure 6. CTX-471 efficacy requires T and NK cells, as well as FcγR engagement.

(A) Effect of CTX-471 on tumor growth in the absence of T cells. Mice (n = 10 per group) were depleted of T cells by administering 500 μg/mouse of CD4 (GK1.5) or CD8 (YTS 169.4) antibodies on days –1, 0, 5, 10, 15, and 20 after CT-26 tumor cell implantation. Mice received 150 μg/mouse CTX-471 on days 6, 9, 12, 19, and 26. (B) Effect of CTX-471 on tumor growth in the absence of NK cells. Mice (n = 8 per group) were depleted of NK cells by administering 50 μL/mouse of asialo-GM1 antibody (anti-ASGM1) on days –1, 0, 5, 10, 15, and 20 after CT-26 tumor cell implantation. Mice received 150 μg/mouse CTX-471 on days 7, 10, 13, 20, and 27. (C) Effect of CTX-471 on overall survival in the absence of FcγR engagement. Mice with established CT-26 tumors were treated with 150 μg/mouse of CTX-471-AF or CTX-471 with human IgG4, aglycosylated human IgG4, or rat IgG2a isotypes on days 0, 3, 6, and 9. (D) Flow cytometric analysis of CT-26 tumor infiltrating Tregs on day 9 following administration of CTX-471 with hIgG4 or rIgG2a isotype on days 0, 3, and 6. Statistical significance was determined using Log-rank test (C) or 1-way ANOVA (D) followed by Bonferroni’s multiple comparison test compared with control treatment groups (*P<0.05, **P<0.01, ****P<0.0001). All data presented as mean ± SEM.

Figure 7. High doses of CTX-471 and CTX-471-AF do not induce hepatic inflammation.

(A–I) Nontumor-bearing mice (n = 4 in each treatment group) were administered with 10–80 mg/kg CTX-471, CTX-471-AF, or 3H3 i.v. on days 0, 7, 14, and 21. Plasma, spleens, and livers were collected on day 28. Alanine aminotransferase (ALT) activity (A) and TNF-α (B) concentrations were determined from plasma. CD8⁺ T cell (C) levels in the livers were determined by flow cytometry. Formalin-fixed paraffin-embedded tissues were stained for H&E (D) or with antibodies against CD8 (E and F) and F4/80 (G–I). Mean cell densities of CD8⁺ cells (F) and F4/80⁺ cells (H), and number of F4/80 clusters (I; defined as > 10 cells), are shown. Scale bars: 200 μm. Arrowheads indicate lymphocyte clusters. (J) Mouse BM-derived macrophages were stimulated with CpG ODN (10 μg/mL), along with the indicated soluble antibodies (50 nM), for 48 hours, and levels of TNF-α and IL-27 were measured in supernatants. (K) Primary human monocyte–derived macrophages were stimulated with CpG ODN (10 μg/mL), along with the indicated soluble antibodies (50 nM) for 48 hours, and levels of TNF-α and IL-27 are measured in supernatants. One-way ANOVA with Bonferroni’s post hoc test was performed to determine the statistical significance of treatment versus control (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

Figure 8. CTX-471-AF eradicates large tumors.

(A) Overall survival and tumor growth rates of individual mice with large (~500 mm³) CT-26 tumors following i.p. administration of 150 μg/mouse CTX-471, CTX-471-AF, and CTX-471-AF2 on days 0, 3, 6, and 9. (B) Tumor growth rates of individual mice with large (~450 mm³) CT-26 tumors following i.p. administration of CD137 agonists (CTX-471-AF or 3H3; 25 μg/mouse on days 0, 7, and 14), checkpoint inhibitors (Avelumab, RMP1-14, or 9H10; 200 μg/mouse on days 0, 3, and 6), or an OX40 agonist (OX-86; 200 μg/mouse on days 0, 3, and 6). (C) Kaplan-Meier survival curves of mice in B. Statistical significance was determined using Log-rank test (A and C) followed by Bonferroni’s multiple comparison compared with control treatment groups (*P<0.05, **P<0.01). (D and E) Histological analysis of CT-26 tumors on days 7, 10, and 14 following i.p. administration of 25 μg/mouse CTX-471-AF or isotype control on day 0. Formalin-fixed paraffin-embedded tissues were stained for H&E (D) or with antibodies against CD8 (E). Scale bars: 200 μm.