Serial killing of tumor cells by cytotoxic T cells redirected with a CD19‐/CD3‐bispecific single‐chain antibody construct

Abstract

Certain bispecific antibodies exhibit an extraordinary potency and efficacy for target cell lysis by eliciting a polyclonal T-cell response. One example is a CD19-/CD3-bispecific single-chain antibody construct (bscCD19xCD3), which at femtomolar concentrations can redirect cytotoxic T cells to eliminate human B lymphocytes, B lymphoma cell lines and patient-derived malignant B cells. Here we have further explored the basis for this high potency. Using video-assisted microscopy, bscCD19xCD3 was found to alter the motility and activity of T cells from a scanning to a killing mode. Individual T cells could eliminate multiple target cells within a 9 hr time period, resulting in nuclear fragmentation and membrane blebbing of target cells. Complete target cell elimination was observed within 24 hr at effector-to-target cell ratios as low as 1:5. Under optimal conditions, cell killing started within minutes after addition of bscCD19xCD3, suggesting that the rate of serial killing was mostly determined by T-cell movement and target cell scanning and lysis. At all times, T cells remained highly motile, and no clusters of T and target cells were induced by the bispecific antibody. Bystanding target-negative cells were not detectably affected. Repeated target cell lysis by bscCD19xCD3-activated T cells increased the proportion of CD19/CD3 double-positive T cells, which was most likely a consequence of transfer of CD19 from B to T cells during cytolytic synapse formation. To our knowledge, this is the first study showing that a bispecific antibody can sustain multiple rounds of target cell lysis by T cells. ' 2005 Wiley-Liss, Inc.

Development of a Target cell-Biologics-Effector cell (TBE) complex-based cell killing model to characterize target cell depletion by T cell redirecting bispecific agents

“…19 The proposed TBE model was able to simultaneously capture the reported blinatumomab-mediated lysis of CD19+ Nalm-6 cell line by a human T cell-clone at different E:T ratios (10:1 to 1:10) at 4 hr and 24 hr, respectively (Figure 2). The estimated model parameters are summarized in Table 1.…”

“…The low EC 50 value likely reflects the extremely high potency of blinatumomab. 19 Of note, TBEPC R was calculated based on the experimentally determined expression values of T cell CD3 and Nalm-6 cell CD19 and the reported blinatumomab binding affinities to CD3 and CD19 on cell surfaces following 45-minute incubations; 22,23 any potential divergence between the assay measurements and true values of receptor density and binding affinity would affect the derivation of TBEPC R values and the estimation of EC 50 . The baseline killing in the absence of the drug (Base) was fixed at 0, based on the observed data.…”

“…19 Multiple in vitro cytotoxicity assays have showed that, within a reasonable range of E:T ratios (i.e., ≥ 1:80), the effector cells can kill nearly all target cells after a sufficient incubation period. 3,19,24 Accordingly, E max was fixed at 100% in all TBE model analyses. The model-estimated transit time for observable cytotoxicity in the reference system (τ reference ) was 3.49 hours.…”

“…In vitro cytotoxicity data from multiple studies have shown that T cells can conduct serial killing of target cells at lower E:T ratio conditions. 19 Meanwhile, instead of increasing the killing potency of T cells, higher E:T ratios appeared to only accelerate the T cell killing process. 19,23,49–52 Consistently, the data we analyzed also suggested that the higher E:T ratios only accelerate the T cell killing process (τ), but do not affect the intrinsic potency value (EC 50 ).…”

“…19 Meanwhile, instead of increasing the killing potency of T cells, higher E:T ratios appeared to only accelerate the T cell killing process. 19,23,49–52 Consistently, the data we analyzed also suggested that the higher E:T ratios only accelerate the T cell killing process (τ), but do not affect the intrinsic potency value (EC 50 ). Our model used two exponential functions to correlate both effector cell concentrations (k τ1 ) and target-to-effector cell ratios (k τ2 ) to τ.…”