Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy

Abstract

Endogenous costimulatory molecules on T cells such as 4-1BB (CD137) can be leveraged for cancer immunotherapy. Systemic administration of agonistic anti-4-1BB antibodies, although effective preclinically, has not advanced to phase 3 trials because they have been hampered by both dependency on Fcγ receptor-mediated hyperclustering and hepatotoxicity. To overcome these issues, we engineered proteins simultaneously targeting 4-1BB and a tumor stroma or tumor antigen: FAP-4-1BBL (RG7826) and CD19-4-1BBL. In the presence of a T cell receptor signal, they provide potent T cell costimulation strictly dependent on tumor antigen-mediated hyperclustering without systemic activation by FcγR binding. We could show targeting of FAP-4-1BBL to FAP-expressing tumor stroma and lymph nodes in a colorectal cancer-bearing rhesus monkey. Combination of FAP-4-1BBL with tumor antigen-targeted T cell bispecific (TCB) molecules in human tumor samples led to increased IFN-γ and granzyme B secretion. Further, combination of FAP- or CD19-4-1BBL with CEA-TCB (RG7802) or CD20-TCB (RG6026), respectively, resulted in tumor remission in mouse models, accompanied by intratumoral accumulation of activated effector CD8⁺ T cells. FAP- and CD19-4-1BBL thus represent an off-the-shelf combination immunotherapy without requiring genetic modification of effector cells for the treatment of solid and hematological malignancies.

Fig. 1.. Design of a bispecific tumor-targeted 4-1BB ligand (TA–4-1BBL), which features IgG-like pharmacokinetics.

(A) TA–4-1BBL consists of an Fc, devoid of FcγR binding (huIgG1PGLALA) (22), a trimeric hu4-1BBL, and a Fab, binding to FAP or CD19. Heterodimerization and correct assembly are achieved by knob into hole (kih) mutation in the Fc, CH1-CL domain crossover, and mutations in CH1(EE) and CL(RK) (49, 50). Class averages based on negative stain transmission electron microscopy of the whole molecule and x-ray crystallography (for data collection and refinement statistics, see table S1) of the trimeric 4-1BBL (missing CH1/CL parts in the structure are shown in dashed lines) were used to further characterize TA–4-1BBL. (B) Pharmacokinetic profile of TA–4-1BBL after a single dose injected intravenously, 12 mice per group. After therapy, three mice per group were bled at indicated time points. Concentration of drug in serum was measured by enzyme-linked immunosorbent assay (ELISA). Shown is mean (SD) of n = 3 mice per time point and group. FAP–4-1BBL and CD19–4-1BBL were tested once in two different experiments and combined in this graph. (C) Simultaneous binding by surface plasmon resonance to both targets, 4-1BB and FAP (left) or 4-1BB and CD19 (right). Results were confirmed in two independent experiments.

Fig. 2.. FAP–4-1BBL targets to FAP⁺tumor tissue and LN in a CRC-bearing rhesus monkey.

(A) Immunohistochemistry staining of FAP in the peritumoral LN and in tumor tissue after necropsy (n = 1). (B) Maximum intensity projection of a CRC-bearing rhesus monkey 120 hours after intravenous injection of FAP–4-1BBL (0.5 mg/kg) mixed with tracer amounts of ⁸⁹Zr-labeled FAP–4-1BBL (165 MBq). Tissue accumulation of radioactive tracer is described on a minimum-maximum standard uptake value (SUV) scale. (C) SUV of indicated organs determined by positron emission tomography after 48 and 120 hours. Organs of FAP expression, perfusion, and excretion (including organs of antibody metabolism) are indicated. Targeting and retention of FAP–4-1BBL are defined as an increase of SUV from 48 to 120 hours.

Fig. 3.. FAP–4-1BBL induces in vitro T cell activation in the presence of signal 1.

(A) HeLa-hu4-1BB-NFκB-luc reporter cells were cocultured with or without huFAP-expressing cells, 4-1BB agonists, or control proteins. Luciferase activity was measured as units of released light (URLs) (n = technical duplicates). Significance was calculated, comparing the area under the curve (AUC) by unpaired one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. EC₅₀ values for FAP–4-1BBL are indicated as mean (SD). (B) Carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled human peripheral blood mononuclear cells (huPBMCs) were cocultured with irradiated NIH/3T3-huFAP fibroblasts in the presence or absence of agonistic 2 nM CD3 huIgG1 and titrated 4-1BB agonists. Proliferation (CFSE dilution), 4-1BB, and CD25 expression of CD4⁺ or CD8⁺ T cells after 5 days [mean (SD), n = technical triplicates] are shown. Significance was calculated as in (A). (C) CFSE-labeled huPBMCs were cocultured with irradiated FAP-expressing cells (NIH/3T3-huFAP or U87MG), 2 nM CD3 huIgG1 alone, or in combination with DP47–4-1BBL or FAP–4-1BBL. CD4⁺ and CD8⁺ T cells were characterized for their expression of CD45RO, CD45RA, and CD62L by flow cytometry. Total counts of 4-1BB–expressing T cells divided in CM, EM, naive, and T_eff/TEMRA subpopulations are shown [mean (SD), n = technical triplicates]. Unpaired one-way ANOVA with Tukey’s multiple comparison test was used. (D) CFSE-labeled HLA-A*02:01/NLV peptide–specific EM CD8⁺ T cells were cocultured with NLV peptide pulsed (1 or 10 nM) or unpulsed HLA-A*02:01⁺ FAP⁺ MV-3 melanoma cells and 4-1BB agonists or controls. After 24 hours, brefeldin A and monesin were added for 4 hours and cells were analyzed for 4-1BB and IFN-γ expression by flow cytometry [mean (SD), n = technical triplicates]. Significance was calculated as in (A). (E and F) CFSE-labeled huPBMCs of five different donors were cocultured with irradiated NIH/3T3-huFAP and carcinoembryonic antigen (CEA)⁺ PD-L1⁺ gastric tumor cells (MKN45-huPD-L1), FAP–4-1BBL, carcinoembryonic antigen-targeted T cell bispecific antibody (CEA-TCB) or atezolizumab alone or in combination [atezolizumab shown only in (F)]. After 4 days, proliferation (CFSE dilution), CD25, and 4-1BB expression of CD8⁺ T cells [mean (SD), n = technical triplicates] were analyzed by flow cytometry. IFN-γ in supernatant was measured by multiplex (n = technical duplicates). Significance was analyzed as in (C). Each shown experiment represents results of at least three independent and similar experiments. For huPBMC-based assays, at least three different donors were tested, but one representative donor is shown. n.s., not significant.

Fig. 4.. FAP–4-1BBL in combination with CD3 activation induces cytokine release from FAP-expressing human cancer samples.

(A) Single-cell suspensions of EOC tumor lesions were tested for FAP expression. (B) Single-cell suspensions of EOC patients’ tumor tissue were cultured unstimulated, with agonistic CD3 huIgG1 alone, or in combination with FAP–4-1BBL. Shown are the frequencies of 4-1BB⁺ and Ki67⁺ CD8⁺ T cells determined by flow cytometry. Significance was calculated using paired one-way ANOVA with Tukey’s multiple comparison test. (C) Tumor suspension of patient 4 was incubated in medium, with CD3 huIgG1 or FAP–4-1BBL alone or in combination. Supernatant was analyzed for cytokines by multiplex. Technical duplicates are shown as individual curves. Significance was calculated, comparing the AUC using unpaired one-way ANOVA with Tukey’s multiple comparison test. (D) Melanoma digests from three patients were tested for frequency of FAP- and CD3-expressing cells by flow cytometry. (E) FAP⁺ tumor digests were cultured unstimulated or with CD3 huIgG1, FAP–4-1BBL, or DP47–4-1BBL alone or in combination. Secreted cytokines were analyzed using cytometric bead array (CBA). Depending on tumor size sample, n = 2 (patient 5 and 6) or n = 1 (patient 7) technical replicate per condition was measured. Significance was calculated using unpaired twoway ANOVA with Tukey’s multiple comparison test. (F) Colon adenocarcinoma, lung adenocarcinoma, lung adenosquamous carcinoma, or melanoma tumor lesions were analyzed for FAP, CEA, or tyrosinase-related protein (TYRP1) expression. (G) Fresh tumor tissue from lesions shown in (F) were placed in bioreactors (each symbol represents one bioreactor), supplied with 8 ml of medium/reactor, and for patient 8, with autologous huPBMCs. Bioreactors were incubated with medium alone (untreated) or stimulatory molecules as indicated. Supernatant was analyzed for IFN-γ using CBA. Significance was analyzed using unpaired one-way ANOVA multiple comparison, uncorrected Fisher’s LSD test. Because of the limited available patient material, all performed assays are shown.

Fig. 5.. Combination of FAP–4-1BBL and CEA-TCB in vivo decreases tumor growth and increases intratumoral CD8⁺ T cell accumulation.

(A) A representative example of FAP expression in the tumor at the start of therapy (200 mm³). (B and C) Shown are two of three independent experiments. CEA⁺ human gastric cancer cells MKN45 and NIH/3T3-huFAP fibroblasts were coinjected subcutaneously into human stem cell–engrafted NSG (HSC-NSG) mice. At an average tumor size of 200 mm³, mice were treated with vehicle, CEA-TCB, FAP–4-1BBL, or DP47–4-1BBL alone or in combination as indicated (n = 10 per group). Shown is mean (SEM). Statistical significance of tumor volumes at the end point was calculated using unpaired one-way ANOVA with Tukey’s multiple comparison test (B) or unpaired, two-tailed Student’s t test (C). (D) Digested tumor tissues (day 52) were analyzed by flow cytometry gating on living human CD4⁺ or CD8⁺ T cells. Regulatory T (T_reg) cells were defined as FoxP3⁺ CD25⁺ CD4⁺ T cells. Each symbol represents one individual mouse; bars indicate mean (SD). Significance was calculated using unpaired one-way ANOVA with Tukey’s multiple comparison test. (E) Representative immunohistochemistry staining for CD3⁺ and CD8⁺ cells is shown. Tissue sections were scanned, and whole scans were analyzed by Definiens. Tumors were taken from the experiment shown in (C) at termination. (F) CD3⁺ and CD8⁺ T cells count per square millimeter of immunohistochemistry analyzed by Definiens for the whole section. Each symbol represents one individual mouse; shown is mean (SD). Significance was calculated as in (D). (G) Ten HSC-NSG mice per group were treated weekly with vehicle, FAP–4-1BBL, or DP47–4-1BBL intravenously. Three days after first (day 3) and second injection (day 10), bone marrow from femur (n = 5 per group) was analyzed for cell counts by flow cytometry. Tumor experiments were repeated in three independent experiments; bone marrow evaluation experiment was assessed once.

Fig. 6.. CD19–4-1BBL induces in vitro T cell activation and localizes with CD20-TCB in the immunological synapse.

(A) HeLa-hu4-1BB-NFκB-luc reporter cells were cocultured in the absence or presence of huCD19-expressing cell lines and CD19–4-1BBL or DP47–4-1BBL. Shown is the luciferase activity after 6 hours as URLs (n = technical duplicates). Significance was calculated comparing AUC by unpaired two-tailed Student’s t test. EC₅₀ values are indicated as mean (SD). (B) huPBMCs (n = technical duplicates) were incubated with 1.2 × 10⁶ CD3/CD28 beads/ml (top) or PHA-L (1 μg/ml) (bottom) and CD19–4-1BBL or DP47–4-1BBL, whereby CD19–4-1BBL is cross-linked via B cells. IFN-γ secretion was analyzed by ELISA after 2 days. Significance was calculated as in (A), and EC₅₀ values are indicated as mean (SD). (C) Human CD3⁺ T cells isolated from buffy coat (n = technical duplicates) were cocultured with WSU-DLCL2 cells and CD20-TCB. Expression of 4-1BB and CD69 on CD8⁺ T cells was determined by flow cytometry. (D) WSU-DLCL2–bearing HSC-NSG mice (n = 4) were treated weekly intravenously with vehicle or CD20-TCB. Tumors were isolated 2 days after second injection, and tumor-infiltrating CD8⁺ T cells were analyzed by flow cytometry for 4-1BB expression. Each symbol indicates one individual mouse. Shown is mean (SD); significance was calculated by unpaired two-tailed Student’s t test. (E) CD3⁺ T cells were cocultured with WSU-DLCL2 cells, CD19–4-1BBL, and CD20-TCB as indicated. After 18 hours, supernatants were analyzed by ELISA for IFN-γ secretion. Shown is the mean (SD) of n = technical duplicates, indicated with symbols. Significance was calculated using an unpaired one-way ANOVA with Tukey’s multiple comparison test. (F) Localization of CD19–4-1BBL (magenta), WSU-DLCL2 cells (blue), and activated 4-1BB–expressing CD8⁺ T cells (green) by confocal fluorescence microscopy. (G) The localization of CD19–4-1BBL, WSU-DLCL2 cells, and CD20-TCB–activated 4-1BB–expressing CD8⁺ T cells was monitored for up to 90 frames. In the graph, the intensity of AF647-labeled CD19–4-1BBL in the surface contact area between 4-1BB⁺ CD8⁺ T cells and CD19⁺ WSU-DLCL2 cells is shown. Experiments shown in (A) to (D) were repeated at least three times in independent experiments. If primary cells were used, then at least three different donors were tested. Experiments shown in (E) to (G) were performed only once.

Fig. 7.. Combination of CD19–4-1BBL and CD20-TCB in vivo inhibits tumor growth and increases intratumoral CD8⁺ T cell accumulation and activation.

(A) Human WSU-DLCL2 cells were injected subcutaneously in HSC-NSG mice. At 250 mm³ tumor size, mice were treated with vehicle or CD20-TCB or CD19–4-1BBL alone or in combination. Shown is the mean (SEM) of the number of mice per group as indicated. Statistical significance of tumor volumes at the end point was calculated using unpaired, two-tailed Student’s t test. (B) Digested tumor single-cell suspensions from animals euthanized at day 20 (n = 4 per group indicated by symbols) were analyzed by flow cytometry. Shown is mean (SD), and significance was calculated using unpaired one-way ANOVA with Tukey’s multiple comparison test. (C) Representative immunohistochemistry staining of tumor tissue from animals euthanized at day 20 (n = 4 per group) for huCD3⁺, granzyme B (GnzB)⁺, huCD20⁺, and huCD19⁺ cells is shown for each group. Tissue sections were scanned, and whole scans were analyzed by Definiens. The shown experiment represents results of three independent experiments.