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

The NK-92CD16 in the presence of trastuzumab but not NK-92CAR induces regression of the HER2 positive BT474 tumor engrafted in NSG mice. Results presented in (a), (b), and (c) are from the same series of experiments but are presented separately for readability: the group of mice treated with NK-92CD16 + trastuzumab is thus the same for (a), (b), (c), and (d). Six-week-old female NSG mice were injected subcutaneously in the left side with 5 × 106 BT474 cells. When tumors reached a minimal volume of 50 mm3 (within 5–7 days), mice were individually identified and randomly assigned to the control or treated groups (5 to 15 mice per group) and the indicated treatments were initiated.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4664810&req=5

fig4: The NK-92CD16 in the presence of trastuzumab but not NK-92CAR induces regression of the HER2 positive BT474 tumor engrafted in NSG mice. Results presented in (a), (b), and (c) are from the same series of experiments but are presented separately for readability: the group of mice treated with NK-92CD16 + trastuzumab is thus the same for (a), (b), (c), and (d). Six-week-old female NSG mice were injected subcutaneously in the left side with 5 × 106 BT474 cells. When tumors reached a minimal volume of 50 mm3 (within 5–7 days), mice were individually identified and randomly assigned to the control or treated groups (5 to 15 mice per group) and the indicated treatments were initiated.

Mentions: Having shown that NK-92CAR were more effective in vitro than NK-92CD16 against several HER2 positive tumor cells, we developed a mouse xenograft model to test whether such difference could also be evidenced in vivo. Six-week-old female NSG mice were injected subcutaneously in the left side with 5 × 106 BT474 cells, and when the tumor reached a volume of 50 mm3, the mice received the indicated treatment (all mice were sacrificed before the tumor volume reached 2500 mm3). We first tested the capacity of IP injection of NK-92CD16 and trastuzumab to control the growth of a subcutaneous established NK-92 resistant BT474 carcinoma (Figure 4(a)). In the absence of addition of effector cells, weekly injections of trastuzumab (15 mg/kg) alone do not prevent BT474 tumor burden (Figure 4(a)). When 5 × 106 NK-92CD16 cells were used in combination with trastuzumab (15 mg/kg) injected 24 h prior to NK cells, complete tumor regression was observed. Note that tumor regression began after a single dose but that complete regression required 4 injections once per week of 5 × 106 NK-92CD16 cells (Figure 4(a)). To confirm that ADCC was the mechanism responsible for tumor regression, experiments were repeated with the untransduced NK-92NT or with NK-92CD16 in the presence of an irrelevant antibody (rituximab, directed against CD20, an Ag not present at the surface of BT474). As the result of a mechanism of tumor destruction due to ADCC, neither the untransduced NK-92NT in the presence of trastuzumab nor NK-92CD16 in the presence of rituximab were able to control the growth of the BT474 tumor (Figures 4(b) and 4(c), resp.).


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)

The NK-92CD16 in the presence of trastuzumab but not NK-92CAR induces regression of the HER2 positive BT474 tumor engrafted in NSG mice. Results presented in (a), (b), and (c) are from the same series of experiments but are presented separately for readability: the group of mice treated with NK-92CD16 + trastuzumab is thus the same for (a), (b), (c), and (d). Six-week-old female NSG mice were injected subcutaneously in the left side with 5 × 106 BT474 cells. When tumors reached a minimal volume of 50 mm3 (within 5–7 days), mice were individually identified and randomly assigned to the control or treated groups (5 to 15 mice per group) and the indicated treatments were initiated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: The NK-92CD16 in the presence of trastuzumab but not NK-92CAR induces regression of the HER2 positive BT474 tumor engrafted in NSG mice. Results presented in (a), (b), and (c) are from the same series of experiments but are presented separately for readability: the group of mice treated with NK-92CD16 + trastuzumab is thus the same for (a), (b), (c), and (d). Six-week-old female NSG mice were injected subcutaneously in the left side with 5 × 106 BT474 cells. When tumors reached a minimal volume of 50 mm3 (within 5–7 days), mice were individually identified and randomly assigned to the control or treated groups (5 to 15 mice per group) and the indicated treatments were initiated.
Mentions: Having shown that NK-92CAR were more effective in vitro than NK-92CD16 against several HER2 positive tumor cells, we developed a mouse xenograft model to test whether such difference could also be evidenced in vivo. Six-week-old female NSG mice were injected subcutaneously in the left side with 5 × 106 BT474 cells, and when the tumor reached a volume of 50 mm3, the mice received the indicated treatment (all mice were sacrificed before the tumor volume reached 2500 mm3). We first tested the capacity of IP injection of NK-92CD16 and trastuzumab to control the growth of a subcutaneous established NK-92 resistant BT474 carcinoma (Figure 4(a)). In the absence of addition of effector cells, weekly injections of trastuzumab (15 mg/kg) alone do not prevent BT474 tumor burden (Figure 4(a)). When 5 × 106 NK-92CD16 cells were used in combination with trastuzumab (15 mg/kg) injected 24 h prior to NK cells, complete tumor regression was observed. Note that tumor regression began after a single dose but that complete regression required 4 injections once per week of 5 × 106 NK-92CD16 cells (Figure 4(a)). To confirm that ADCC was the mechanism responsible for tumor regression, experiments were repeated with the untransduced NK-92NT or with NK-92CD16 in the presence of an irrelevant antibody (rituximab, directed against CD20, an Ag not present at the surface of BT474). As the result of a mechanism of tumor destruction due to ADCC, neither the untransduced NK-92NT in the presence of trastuzumab nor NK-92CD16 in the presence of rituximab were able to control the growth of the BT474 tumor (Figures 4(b) and 4(c), resp.).

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