CLN-978, a novel half-life extended CD19/CD3/HSA-specific T cell-engaging antibody construct with potent activity against B-cell malignancies with low CD19 expression

Abstract

Background: Despite significant progress in the development of T cell-engaging therapies for various B-cell malignancies, a high medical need remains for the refractory disease setting, often characterized by suboptimal target levels.

Methods: To address this issue, we have developed a 65-kDa multispecific antibody construct, CLN-978, with affinities tuned to optimize the killing of low-CD19 expressing tumor cells. CLN-978 bound to CD19 on B cells with picomolar affinity, and to CD3ε on T cells with nanomolar affinity. A serum albumin binding domain was incorporated to extend serum half-life. In this setting, we biophysically characterize and report the activities of CLN-978 in cell co-culture assays, multiple mouse models and non-human primates.

Results: Human T cells redirected by CLN-978 could eliminate target cells expressing less than 300 copies of CD19 on their surface. The half-life extension and high affinity for CD19 led to significant antitumor activity in murine lymphoma models at very low doses of CLN-978. In primates, we observed a long serum half-life, deep and sustained depletion of normal B cells, and remarkable tolerability, in particular, reduced cytokine release when CLN-978 was administered subcutaneously.

Conclusions: CLN-978 warrants further exploration. An ongoing clinical phase 1 trial is investigating safety, pharmacokinetics, pharmacodynamics, and the initial therapeutic potential of subcutaneously administered CLN-978 in patients with non-Hodgkin's lymphoma.

Keywords: CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cytotoxicity, Immunologic; Drug Evaluation, Preclinical; T-Lymphocytes.

Figure 1

Design and characterization of CLN-978. (A) Design of CLN-978. (B) Summary of Biacore binding data of CLN-978 against targeted proteins. (C) Binding of CLN-978 to human, cynomolgus monkey and mouse peripheral blood mononuclear cells (n=3 donors). BiTE, bispecific T-cell engager; NHP, non-human primate; scFv, single-chain variable fragment; VHH, variable domain of the heavy chain.

Figure 2

CLN-978 potently induces T-cell activation and TDCC. (A–B) RAMOS cells co-cultured with PBMC from six healthy donors at an E:T ratio of 10:1, in the absence of albumin, at the indicated concentrations of CLN-978 for 48 hours. Shown are (A) lysis curves, as flow cytometrically assessed by 7-AAD uptake in RAMOS cells and (B) CD25 and CD69 expression on CD4+ and CD8+ T cells. (C) RAMOS cells co-cultured with PBMC at an E:T ratio of 10:1 and the indicated concentrations of CLN-978, in the presence or absence of human serum albumin, for 48 hours (n=10). (D) Supernatants from co-cultures of RAMOS cells and PBMC in the presence of CLN-978 (2C), also in the presence of albumin were analyzed for the indicated cytokines by Luminex.

Figure 3

CLN-978 is active against low CD19-expressing inducible cell lines. Chemically-induced CD19-expressing CHO cells co-cultured with isolated T cells at an E:T ratio of 10:1, in the presence or absence of the indicated concentrations of CLN-978 for 68 hours (n=4). Shown are: (A) lysis curves, as flow cytometrically assessed by 7-AAD uptake in CD19-expressing CHO cells. (B) CD25 and (C) Ki67 expression profiles as marker for activation of CD8+ T cells. (D) Supernatants from co-cultures of CD19-expressing CHO cells and PBMC in the presence of CLN-978 (4A-C) were analyzed for IFN-γ. (E) Cytotoxicity or IFN-γ production at 225 pM concentration of CLN-978 or blinatumomab at 72 hours (n=10). (F) ROC analysis determined minimal receptor number required for biological effect; Wilcoxon signed-rank test. CHO, Chinese hamster ovary; E, effector cells; IFN, interferon; MFI, mean fluorescence intensity; PBMC, peripheral blood mononuclear cell; ROC, receiver operating characteristic curve; T, target cells; WT, wild type.

Figure 4

CLN-978 is active against low-CD19 expressing engineered cell lines. CD19-expressing A20 cells co-cultured with purified T cells at an E:T ratio of 10:1, in the presence of the indicated concentrations of CLN-978 and human albumin for 48 hours (n=2). Shown are (A) lysis curves, as flow cytometrically assessed by 7-AAD uptake. (B) CD25 and CD69 expression profiles as marker for activation of CD4+and CD8+ T cells. (C) Supernatants from co-cultures of RAMOS cells and PBMC in the presence of CLN-978 (4A) were analyzed for the indicated cytokines by Luminex. E, effector cells; PBMC, peripheral blood mononuclear cell; T, target cells; TNF, tumor necrosis factor; WT, wild type.

Figure 5

In vivo efficacy of CLN-978. (A) hCD3ε-expressing BALB/c mice were inoculated with A20 cells expressing hCD19. Mice were treated once intravenously when tumor volume reached ~100 mm³. (B) Immunodeficient NCG mice were intravenously engrafted with 2×10⁵ Raji cells, then implanted IP with 2×10⁷ PBMCs the following day. Mice were treated intravenously weekly starting on day 1. Statistics were calculated versus vehicle or PBS using ANOVA with multiple comparisons test on d10 in A and d15 in B. (C) Immunodeficient NCG mice were intravenously engrafted with 2×10⁵ Raji cells. On the following day (Day 1), 2×10⁷ PBMCs from a healthy donor were implanted IP. CLN-978 was dosed either intravenously or SC weekly starting on Day 1. All treatment conditions with CLN-978 imparted statistically significant responses relative to controls by ANOVA at d14 (p<0.0001). ANOVA, analysis of variance; IP, intraperitoneal; IV, intravenous; PBMC, peripheral blood mononuclear cell; PBS, phosphate-buffered saline; SC, subcutaneous.

Figure 6

Pharmacokinetics, B-cell depletion, T-cell redistribution and cytokine release in cynomolgus monkeys in response to a single intravenous-administered or SC-administered CLN-978 dose. Female monkeys (n=2) were dosed at 0.1 or 1 mg/kg either intravenous (blue bars) or SC (red bars). Blood samples were collected at predetermined time points. (A) Pharmacokinetics after a single intravenous or SC administration, (B) absolute B cells, (C) absolute T cells, (D) cytokines as measured by Luminex. IFN, interferon; IL, interleukin; IV, intravenous; SC, subcutaneous; TNF, tumor necrosis factor.