Attenuating CD3 affinity in a PSMAxCD3 bispecific antibody enables killing of prostate tumor cells with reduced cytokine release

Abstract

Background: Therapeutic options currently available for metastatic castration-resistant prostate cancer (mCRPC) do not extend median overall survival >6 months. Therefore, the development of novel and effective therapies for mCRPC represents an urgent medical need. T cell engagers (TCEs) have emerged as a promising approach for the treatment of mCRPC due to their targeted mechanism of action. However, challenges remain in the clinic due to the limited efficacy of TCEs observed thus far in solid tumors as well as the toxicities associated with cytokine release syndrome (CRS) due to the usage of high-affinity anti-CD3 moieties such as OKT3.

Methods: Using genetically engineered transgenic rats (UniRat and OmniFlic) that express fully human IgG antibodies together with an NGS-based antibody discovery pipeline, we developed TNB-585, an anti-CD3xPSMA TCE for the treatment of mCRPC. TNB-585 pairs a tumor-targeting anti-PSMA arm together with a unique, low-affinity anti-CD3 arm in bispecific format. We tested TNB-585 in T cell-redirected cytotoxicity assays against PSMA⁺ tumor cells in both two-dimensional (2D) cultures and three-dimensional (3D) spheroids as well as against patient-derived prostate tumor cells. Cytokines were measured in culture supernatants to assess the ability of TNB-585 to induce tumor killing with low cytokine release. TNB-585-mediated T cell activation, proliferation, and cytotoxic granule formation were measured to investigate the mechanism of action. Additionally, TNB-585 efficacy was evaluated in vivo against C4-2 tumor-bearing NCG mice.

Results: In vitro, TNB-585 induced activation and proliferation of human T cells resulting in the killing of PSMA⁺ prostate tumor cells in both 2D cultures and 3D spheroids with minimal cytokine release and reduced regulatory T cell activation compared with a positive control antibody that contains the same anti-PSMA arm but a higher affinity anti-CD3 arm (comparable with OKT3). In addition, TNB-585 demonstrated potent efficacy against patient-derived prostate tumors ex vivo and induced immune cell infiltration and dose-dependent tumor regression in vivo.

Conclusions: Our data suggest that TNB-585, with its low-affinity anti-CD3, may be efficacious while inducing a lower incidence and severity of CRS in patients with prostate cancer compared with TCEs that incorporate high-affinity anti-CD3 domains.

Keywords: T-lymphocytes; cytokines; immunotherapy; prostatic neoplasms.

Figure 1

TNB-585 is a stable molecule with favorable developability characteristics. (A) TNB-585 stability was assessed following incubation at 37°C for 1 month. High and low molecular weight species were analyzed by CE-SDS and SDS-PAGE. (B) SE-UPLC chromatograms of TNB-585 stressed at 37°C for 1, 3, and 6 months compared with 4°C are shown. (C) TNB-585 activity was measured using a T cell activation bioassay after heat stress at 37°C at 1 week, 3 months, and 6 months. (D) PK in cynomolgus monkeys was evaluated by ELISA following a single dose intravenous administration of TNB-585 at 0.07, 0.97, or 10.39 mg/kg.

Figure 2

TNB-585 binds to PSMA and CD3 and induces antigen-dependent cytotoxicity and cytokine release. (A) Dose–response curves of TNB-585 binding to multiple on-target (22RV1, LNCaP, MDA-PCa-2b and PC3-PSMA) and off-target (HT1376, DU145, PC3 and IGROV-1) cell lines as assessed by flow cytometry are shown. (B) TNB-585-mediated cytotoxicity and cytokine release was evaluated against either PSMA-transfected (PC3-PSMA) or non-transfected (PC3-WT) PC3 tumor cells. Cytotoxicity was evaluated by annexin V staining using flow cytometry. IFNγ and IL-2 concentrations were measured by ELISA. Data are reported as mean±SD. *p<0.05 compared with PC3-WT.

Figure 3

Activation of T cells by TNB-585 induces CD69 upregulation, proliferation, and perforin and granzyme production. (A) CD69 upregulation was assessed on CD4⁺ and CD8⁺ T cells by flow cytometry following incubation of LNCaP or DU145 tumor cells and T cells in the presence of increasing concentrations of TNB-585, PC, or NC for 48 hours at 37°C. (B) proliferation of CFSE-labeled CD4⁺ and CD8⁺ T cells was assessed following incubation with 22RV1 or DU145 tumor cells in the presence of increasing concentrations of TNB-585, PC, or NC for 48 hours at 37°C. Percent proliferation was measured by CFSE dilution using flow cytometry. (C) Perforin and granzyme concentrations in culture supernatants were measured by ELISA after 48 hours incubation of TNB-585, PC, or NC with T cells and LNCaP tumor cells. (D) Upregulation of CD69 on T cells from three healthy donors was measured by flow cytometry after 48 hours incubation with 22RV1 tumor cells and either TNB-585, PC, or NC. Activated T cells (CD69⁺) were further subgated on CD25⁺Foxp3⁺ expression to evaluate the percentage of Tregs that comprise the total activated T cell population. Data are reported as mean±SD. *P<0.05, **p<0.01 compared with PC. CSFE, carboxyfluorescein succinimidyl ester; NC, negative control; PC, positive control.

Figure 4

TNB-585 induces tumor cell killing with reduced cytokine release. TNB-585 mediated tumor killing and cytokine release was evaluated against four PSMA⁺ cell lines (22RV1, LNCaP, MDA-PCa-2b, and PC3-PSMA) and one PSMA⁻ cell line (DU145) using T cells isolated from three healthy donors. T cells were incubated with prostate tumor cells at a 10:1 E:T ratio in the presence of increasing concentrations of TNB-585, PC, or NC. Cytotoxicity was measured using either WST-1 (LNCaP) or flow cytometry (22Rv1, MDA-PCa-2b, PC3-PSMA, and DU145). Cytokine (IL-2, IFNγ, IL-6, and TNFα) concentrations in culture supernatants were measured using MSD technology. Representative dose response curves from a single donor are shown. Data are reported as mean±SD. NC, negative control; PC, positive control.

Figure 5

TNB-585 mediates cytotoxicity of PSMA⁺ LNCaP spheroids with minimal cytokine production. LNCaP spheroids were incubated with huPBMCs at an E:T ratio of 1+1 in the presence of increasing concentrations of TNB-585, PC, or NC for 4 days at 37°C. (A) Images were taken daily to capture morphological changes in the spheroids (100 nM antibody concentration is shown). Cytotoxicity (Bi) was assessed by LDH release, and cytokine concentrations (Bii–iv) were measured using MSD technology. (C) Spheroids were analyzed by IHC using anti-PSMA and anti-CD3 antibodies to evaluate T cell infiltration. Scale: top panel: 100 µm; bottom panel: 200 µm. IHC, Immunohistochemistry; LDH, lactate dehydrogenase; NC, negative control; PC, positive control.

Figure 6

TNB-585 mediates lysis of patient-derived prostate tumors with minimal cytokine production. TNB-585, PC, or NC were added to dissociated tumor cells from freshly procured prostatic adenocarcinoma tissue (i–iv) or thawed previously dissociated prostatic adenocarcinoma (v–vii) and incubated without additional human PBMCs (i, ii) or with either unmatched huPBMC (iii–iv) or donor matched huPBMC (v–vii) at an effector to target cell ratio (E:T=1:2) for 24 hours at 37°C and 8% CO₂. Percent cytotoxicity of PSMA-positive cells (A) and IFNγ concentration in the corresponding wells (B) are shown. NC, negative control; PBMCs, Peripheral blood mononuclear cellsl; PC, positive control.

Figure 7

TNB-585 induces immune cell infiltration and dose-dependent tumor regression in an NCG mouse xenograft model. (A) A schematic representation of the study designs is shown. NCG mice were engrafted with 2×10⁶ C4-2 tumor cells followed by injection of either resting T cells or preactivated PBMCs. Biweekly treatment began when the average tumor volume was ~50 mm³. (B) Tumor growth inhibition was evaluated in C4-2 tumor bearing mice comparing treatment with TNB-585, PC, and NC (150 µg/dose) using either resting T cells or preactivated PBMCs as effectors cells. (C) Tumor growth inhibition was evaluated in C4-2 tumor bearing mice using preactivated PBMCs at TNB-585 doses ranging from 5.5 to 450 µg per dose. tumor volumes were measured twice per week (i). At the end of study, tumors were harvested and stained by IHC using an anti-CD45 antibody to evaluate immune cell infiltration into the tumor. Representative images for each dose group are shown (ii). Tumor volumes are reported as mean±SEM. *P<0.05 compared with negative or vehicle treated control. NC, negative control; PC, positive control.