TNB-486 induces potent tumor cell cytotoxicity coupled with low cytokine release in preclinical models of B-NHL

Abstract

The therapeutic potential of targeting CD19 in B cell malignancies has garnered attention in the past decade, resulting in the introduction of novel immunotherapy agents. Encouraging clinical data have been reported for T cell-based targeting agents, such as anti-CD19/CD3 bispecific T-cell engager blinatumomab and chimeric antigen receptor (CAR)-T therapies, for acute lymphoblastic leukemia and B cell non-Hodgkin lymphoma (B-NHL). However, clinical use of both blinatumomab and CAR-T therapies has been limited due to unfavorable pharmacokinetics (PK), significant toxicity associated with cytokine release syndrome and neurotoxicity, and manufacturing challenges. We present here a fully human CD19xCD3 bispecific antibody (TNB-486) for the treatment of B-NHL that could address the limitations of the current approved treatments. In the presence of CD19+ target cells and T cells, TNB-486 induces tumor cell lysis with minimal cytokine release, when compared to a positive control. In vivo, TNB-486 clears CD19+ tumor cells in immunocompromised mice in the presence of human peripheral blood mononuclear cells in multiple models. Additionally, the PK of TNB-486 in mice or cynomolgus monkeys is similar to conventional antibodies. This new T cell engaging bispecific antibody targeting CD19 represents a novel therapeutic that induces potent T cell-mediated tumor-cell cytotoxicity uncoupled from high levels of cytokine release, making it an attractive candidate for B-NHL therapy.

Keywords: Bispecific antibody; CD19XCD3; T-cell engager; TNB-486; cytokine release syndrome; low cytokine release.

Figure 1.

CD19 Expression on normal and malignant B cells (a) Antigen density of CD19 was measured on the indicated human B cell lines by quantitative flow cytometry. Error bars indicate standard error mean (SEM) of 2–4 independent experiments. (b) Antigen density of CD19 was enumerated on normal human PBMCs and malignant B cells from 4 CLL and 2 DLBCL DTCs or PBMCs. Error bars represent SEM of independent samples. Unpaired Student’s t-test was used to determine significance

Figure 2.

TNB-486 is a fully human bispecific antibody engaging CD19 and CD3 (a) TNB-486 was constructed using knobs-into-holes technology. The format of the antibody is depicted. (b) Cell binding dose curves of TNB-486 on three CD19+ B cell lines Daudi, Raji and Nalm-6, and one CD19- cell line, K562 is shown. (c) Cell surface affinity of TNB-486 to CD19 expressed on Nalm-6 cells was determined by Scatchard analysis

Figure 3.

TNB-486 mediates T cell activation, proliferation, and cytotoxic granule release. CD19+ RI-1 tumor cells were incubated with T cells from a single healthy donor at an E:T ratio of 10:1 and serially diluted indicated test antibody (a) Activation of CD4+ or CD8+ T cells were determined by flow cytometric measurement of the activation marker CD69 after 24 hours of co-culture (b) T cell proliferation was measured by labeling T cells with CFSE and monitoring CD4 or CD8 proliferation after 5 days of co-culture (c) Perforin and granzyme B levels were measured in the cell culture supernatants of co-culture after 48 hours by ELISA

Figure 4.

TNB-486 induces lysis of CD19+ B cells (a) T cells and B cells sorted from normal PBMCs were co-cultured at an E:T ratio of 10:1 and incubated with varying doses of TNB-486. Cytotoxicity of B cells was evaluated by flow cytometry at 24 h, 42 h, 72 h and 96 h. (b) PBMCs from three independent donors (the shades of blue represent three independent donors for TNB-486, shades of red and gray/black represent the three donors for PC and NC, respectively) were incubated with increasing doses of TNB-486 without adjusting the E:T ratio. Cytotoxicity of B cells was measured by flow cytometry at 48 h. (c) CD19+ tumor cells Daudi, Raji, Ramos, SU-DHL-4, WSU-DLCL2 and RI-1 or a CD19- K562 cell line were co-cultured with T cells from a healthy donor (at an E:T ratio of 5:1) and increasing doses of TNB-486. TDCC was measured at 48 hours by enumerating the live CD20+ B cells by flow cytometry (d) TNB-486 mediated cytotoxicity of CD19+ RI-1 tumor cells and CD4+ or CD8+ T cells as effectors was determined as in (C)

Figure 5.

TNB-486 induced lysis is accompanied by low cytokine release. TDCC of TNB-486 was evaluated as in (Figure 4) but using T cells from three independent donors. Raji or RI-1 tumor cells were used as target cells. IL-2 levels were measured from culture supernatants of the co-culture assay by MSD

Figure 6.

TNB-486 induces tumor regression in multiple xenograft models of established tumors. (a) 1 × 10⁶ Raji-luc cells were injected i.v via tail vein into CIEA-NOG mice (N = 5/group). On day 6 post-implantation (pi), 10 × 10⁶ human PBMCs were injected i.v. TNB-486, NC or PC, at the respective doses shown, was injected on days 7, 14 and 21. GvHD/GvT onset was observed by day 15 pi and hence data points after day 14 are not shown. (b and c) CIEA-NOG mice (N = 10/group) received 10 × 10⁶ SUDHL-10 cells (b) or Nalm-6 cells (c) with 50% Matrigel subcutaneously in the lower right flank. When tumor volume reached 200 mm3, mice received 10 × 10⁶ human PBMCs i. v followed by antibody treatment Q4D as shown in the figure. Statistical analyses were performed by Two-way ANOVA on GraphPad Prism. TNB-486 treated animals were compared with PBMC + vehicle treated animals. A value of p < .05 was considered significant

Figure 7.

TNB-486 pharmacokinetics in mice and cynomolgus monkeys (a) PK parameters of TNB-486 was evaluated in normal Balb/c mice at 1 or 10 mg/kg (b) PK parameters of TNB-486 was evaluated in cynomolgus monkeys at 0.1, 1 or 10 mg/kg. Serum concentration of TNB-486 in mice were determined by an IgG4 specific AlphaLISA kit (mice) or antigen specific ELISA (cynomolgus monkeys)