Limits...
In Vitro and In Vivo Comparison of Lymphocytes Transduced with a Human CD16 or with a Chimeric Antigen Receptor Reveals Potential Off-Target Interactions due to the IgG2 CH2-CH3 CAR-Spacer.

Clémenceau B, Valsesia-Wittmann S, Jallas AC, Vivien R, Rousseau R, Marabelle A, Caux C, Vié H - J Immunol Res (2015)

Bottom Line: To compare these two mechanisms, we used the same cellular effector (NK-92) and the same signaling domain (FcεRIγ).Analysis of the in vivo xenogeneic system suggested that the human CH2-CH3 IgG2 used as a spacer in our construct was able to interact with the FcR present at the cell surface of the few NSG-FcR+ remaining immune cells.This interaction, leading to blockage of the NK-92(CAR) in the periphery of the engrafted tumor cells, stresses the critical role of the composition of the spacer domain.

View Article: PubMed Central - PubMed

Affiliation: UMR INSERM U892, 8 Quai Moncousu, 44007 Nantes Cedex, France ; Centre Hospitalier Universitaire de Nantes, 1 Place Ricordeau, 44000 Nantes, France.

ABSTRACT
The present work was designed to compare two mechanisms of cellular recognition based on Ab specificity: firstly, when the anti-HER2 mAb trastuzumab bridges target cells and cytotoxic lymphocytes armed with a Fc receptor (ADCC) and, secondly, when HER2 positive target cells are directly recognized by cytotoxic lymphocytes armed with a chimeric antigen receptor (CAR). To compare these two mechanisms, we used the same cellular effector (NK-92) and the same signaling domain (FcεRIγ). The NK-92 cytotoxic cell line was transfected with either a FcγRIIIa-FcεRIγ (NK-92(CD16)) or a trastuzumab-based scFv-FcεRIγ chimeric receptor (NK-92(CAR)). In vitro, the cytotoxic activity against HER2 positive target cells after indirect recognition by NK-92(CD16) was always inferior to that observed after direct recognition by NK-92(CAR). In contrast, and somehow unexpectedly, in vivo, adoptive transfer of NK-92(CD16) + trastuzumab but not of NK-92(CAR) induced tumor regression. Analysis of the in vivo xenogeneic system suggested that the human CH2-CH3 IgG2 used as a spacer in our construct was able to interact with the FcR present at the cell surface of the few NSG-FcR+ remaining immune cells. This interaction, leading to blockage of the NK-92(CAR) in the periphery of the engrafted tumor cells, stresses the critical role of the composition of the spacer domain.

No MeSH data available.


Related in: MedlinePlus

Cytotoxic activity of NK-92CD16 and NK-92CAR. (a) Spontaneous (NK) activity of NK-92 before and after transduction (E/T ratio: 30/1). (b) The effector cells NK-92CD16 and NK-92CAR were tested against the HER2 positive BT474 cell line preincubated in the presence of increasing concentration of trastuzumab (mean of two experiments). (c) Cytotoxic activity of NK-92NT, NK-92CD16, and NK-92CAR and against HER2 negative (MBA-MB-468) or HER2 positive (BT474) cell lines (trastuzumab, 10 mg/mL, E/T ratio: 30/1). Cytotoxicity was evaluated from 51Cr release after 4 hours of incubation. The data represent the means from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4664810&req=5

fig2: Cytotoxic activity of NK-92CD16 and NK-92CAR. (a) Spontaneous (NK) activity of NK-92 before and after transduction (E/T ratio: 30/1). (b) The effector cells NK-92CD16 and NK-92CAR were tested against the HER2 positive BT474 cell line preincubated in the presence of increasing concentration of trastuzumab (mean of two experiments). (c) Cytotoxic activity of NK-92NT, NK-92CD16, and NK-92CAR and against HER2 negative (MBA-MB-468) or HER2 positive (BT474) cell lines (trastuzumab, 10 mg/mL, E/T ratio: 30/1). Cytotoxicity was evaluated from 51Cr release after 4 hours of incubation. The data represent the means from three independent experiments.

Mentions: First, we showed that the spontaneous cytotoxic activity (tested against K562, E/T ratio: 30/1) of the untransduced and the transduced NK-92 was not significantly modified by the retroviral transduction (Figure 2(a)). Next, ADCC activity of the NK-92CD16 against the BT474 cell line was tested in the presence of increasing concentration of trastuzumab (as in the example shown in Figure 2(b)) and demonstrated a plateau (close to 50% specific lysis) at 10 μg/mL (E/T ratio: 30/1). No ADCC was observed in the presence of rituximab and NK-92CD16 or NK-92NT. Note that a background lysis was observed for NK-92NT + trastuzumab (compared to NK-92NT + rituximab), likely due to background expression of CD16 by the NK-92 cell line. For all other in vitro experiments, trastuzumab was used at a concentration of 10 μg/mL. Next, we compared the efficiency of target cell lyses induced either after direct recognition of the HER2 Ag by the NK-92CAR alone or after indirect recognition by the NK-92CD16 in the presence of trastuzumab. To this end, target cells (the HER2 negative MDA-MB-468 and the HER2 positive BT474) were coated or not with trastuzumab, and the untransduced NK-92NT was used as a negative control. Cytotoxic activities of NK-92CAR and NK-92CD16 against trastuzumab coated or not coated MDA-MB-468 and BT474 are summarized in Figure 2(c) (at an effector-to-target ratio of 30 : 1). Neither NK-92CAR nor NK-92CD16 presented a significant level of cytotoxicity against the HER2 negative MDA-MB-468 cell line (Figure 2(c), left panel). When tested against the BT474, NK-92CAR showed a high level of cytotoxic activity (101 ± 3% at 30 : 1). Killing of BT474 was also observed by NK-92CD16 in the presence of trastuzumab, although this occurred at a level below that observed by NK-92CAR (46 ± 8% at the same effector-to-target ratio of 30 : 1). In addition, the preincubation of the BT474 in the presence of trastuzumab drastically reduced the killing by NK-92CAR (Figure 2(c), right panel). To confirm the differences in cytotoxic performance between NK-92CD16 and NK-92CAR, further comparison was performed against 4 different HER2 positive cell lines: BT474, BT474 scid (a subclone of BT474 used for the in vivo experiments), MCF7, and MDA-MB-231 (Figure 3). These data showed that, in vitro, in these experimental conditions, based upon 4 hr cytotoxicity assays, the direct pathway of killing (by NK-92CAR) was always more efficient than the indirect pathway (by NK-92CD16) (Figures 3(a), 3(b), 3(c), and 3(d)).


In Vitro and In Vivo Comparison of Lymphocytes Transduced with a Human CD16 or with a Chimeric Antigen Receptor Reveals Potential Off-Target Interactions due to the IgG2 CH2-CH3 CAR-Spacer.

Clémenceau B, Valsesia-Wittmann S, Jallas AC, Vivien R, Rousseau R, Marabelle A, Caux C, Vié H - J Immunol Res (2015)

Cytotoxic activity of NK-92CD16 and NK-92CAR. (a) Spontaneous (NK) activity of NK-92 before and after transduction (E/T ratio: 30/1). (b) The effector cells NK-92CD16 and NK-92CAR were tested against the HER2 positive BT474 cell line preincubated in the presence of increasing concentration of trastuzumab (mean of two experiments). (c) Cytotoxic activity of NK-92NT, NK-92CD16, and NK-92CAR and against HER2 negative (MBA-MB-468) or HER2 positive (BT474) cell lines (trastuzumab, 10 mg/mL, E/T ratio: 30/1). Cytotoxicity was evaluated from 51Cr release after 4 hours of incubation. The data represent the means from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4664810&req=5

fig2: Cytotoxic activity of NK-92CD16 and NK-92CAR. (a) Spontaneous (NK) activity of NK-92 before and after transduction (E/T ratio: 30/1). (b) The effector cells NK-92CD16 and NK-92CAR were tested against the HER2 positive BT474 cell line preincubated in the presence of increasing concentration of trastuzumab (mean of two experiments). (c) Cytotoxic activity of NK-92NT, NK-92CD16, and NK-92CAR and against HER2 negative (MBA-MB-468) or HER2 positive (BT474) cell lines (trastuzumab, 10 mg/mL, E/T ratio: 30/1). Cytotoxicity was evaluated from 51Cr release after 4 hours of incubation. The data represent the means from three independent experiments.
Mentions: First, we showed that the spontaneous cytotoxic activity (tested against K562, E/T ratio: 30/1) of the untransduced and the transduced NK-92 was not significantly modified by the retroviral transduction (Figure 2(a)). Next, ADCC activity of the NK-92CD16 against the BT474 cell line was tested in the presence of increasing concentration of trastuzumab (as in the example shown in Figure 2(b)) and demonstrated a plateau (close to 50% specific lysis) at 10 μg/mL (E/T ratio: 30/1). No ADCC was observed in the presence of rituximab and NK-92CD16 or NK-92NT. Note that a background lysis was observed for NK-92NT + trastuzumab (compared to NK-92NT + rituximab), likely due to background expression of CD16 by the NK-92 cell line. For all other in vitro experiments, trastuzumab was used at a concentration of 10 μg/mL. Next, we compared the efficiency of target cell lyses induced either after direct recognition of the HER2 Ag by the NK-92CAR alone or after indirect recognition by the NK-92CD16 in the presence of trastuzumab. To this end, target cells (the HER2 negative MDA-MB-468 and the HER2 positive BT474) were coated or not with trastuzumab, and the untransduced NK-92NT was used as a negative control. Cytotoxic activities of NK-92CAR and NK-92CD16 against trastuzumab coated or not coated MDA-MB-468 and BT474 are summarized in Figure 2(c) (at an effector-to-target ratio of 30 : 1). Neither NK-92CAR nor NK-92CD16 presented a significant level of cytotoxicity against the HER2 negative MDA-MB-468 cell line (Figure 2(c), left panel). When tested against the BT474, NK-92CAR showed a high level of cytotoxic activity (101 ± 3% at 30 : 1). Killing of BT474 was also observed by NK-92CD16 in the presence of trastuzumab, although this occurred at a level below that observed by NK-92CAR (46 ± 8% at the same effector-to-target ratio of 30 : 1). In addition, the preincubation of the BT474 in the presence of trastuzumab drastically reduced the killing by NK-92CAR (Figure 2(c), right panel). To confirm the differences in cytotoxic performance between NK-92CD16 and NK-92CAR, further comparison was performed against 4 different HER2 positive cell lines: BT474, BT474 scid (a subclone of BT474 used for the in vivo experiments), MCF7, and MDA-MB-231 (Figure 3). These data showed that, in vitro, in these experimental conditions, based upon 4 hr cytotoxicity assays, the direct pathway of killing (by NK-92CAR) was always more efficient than the indirect pathway (by NK-92CD16) (Figures 3(a), 3(b), 3(c), and 3(d)).

Bottom Line: To compare these two mechanisms, we used the same cellular effector (NK-92) and the same signaling domain (FcεRIγ).Analysis of the in vivo xenogeneic system suggested that the human CH2-CH3 IgG2 used as a spacer in our construct was able to interact with the FcR present at the cell surface of the few NSG-FcR+ remaining immune cells.This interaction, leading to blockage of the NK-92(CAR) in the periphery of the engrafted tumor cells, stresses the critical role of the composition of the spacer domain.

View Article: PubMed Central - PubMed

Affiliation: UMR INSERM U892, 8 Quai Moncousu, 44007 Nantes Cedex, France ; Centre Hospitalier Universitaire de Nantes, 1 Place Ricordeau, 44000 Nantes, France.

ABSTRACT
The present work was designed to compare two mechanisms of cellular recognition based on Ab specificity: firstly, when the anti-HER2 mAb trastuzumab bridges target cells and cytotoxic lymphocytes armed with a Fc receptor (ADCC) and, secondly, when HER2 positive target cells are directly recognized by cytotoxic lymphocytes armed with a chimeric antigen receptor (CAR). To compare these two mechanisms, we used the same cellular effector (NK-92) and the same signaling domain (FcεRIγ). The NK-92 cytotoxic cell line was transfected with either a FcγRIIIa-FcεRIγ (NK-92(CD16)) or a trastuzumab-based scFv-FcεRIγ chimeric receptor (NK-92(CAR)). In vitro, the cytotoxic activity against HER2 positive target cells after indirect recognition by NK-92(CD16) was always inferior to that observed after direct recognition by NK-92(CAR). In contrast, and somehow unexpectedly, in vivo, adoptive transfer of NK-92(CD16) + trastuzumab but not of NK-92(CAR) induced tumor regression. Analysis of the in vivo xenogeneic system suggested that the human CH2-CH3 IgG2 used as a spacer in our construct was able to interact with the FcR present at the cell surface of the few NSG-FcR+ remaining immune cells. This interaction, leading to blockage of the NK-92(CAR) in the periphery of the engrafted tumor cells, stresses the critical role of the composition of the spacer domain.

No MeSH data available.


Related in: MedlinePlus