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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

NK-92CAR stimulation after coculture in the presence of NSG splenocytes. (a) Single-cell suspensions from NSG mice spleens were stained with an antibody cocktail and then analyzed by flow cytometry for expression of CD11b (Mac-1) versus Gr-1 (Ly-6G) and CD11b versus CD16/32 (FcRγRIII/FcRγRII). (b) NK-92 (NK-92NT, NK-92CD16, or NK-92CAR) and splenocytes from NSG mice or the HER2 positive cell line BT474 were cocultured at an effector ratio of 5 : 1 and 2 : 1, respectively, in 96-well round bottom culture plates in RPMI 1640 supplemented with 10% FCS and IL2 (100 IU/ML). After 18 h, supernatants were collected and human INF-γ content was estimated using an ELISA kit (Affymetrix, e-biosciences). Data represent the mean with SD from three independent experiments.
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fig7: NK-92CAR stimulation after coculture in the presence of NSG splenocytes. (a) Single-cell suspensions from NSG mice spleens were stained with an antibody cocktail and then analyzed by flow cytometry for expression of CD11b (Mac-1) versus Gr-1 (Ly-6G) and CD11b versus CD16/32 (FcRγRIII/FcRγRII). (b) NK-92 (NK-92NT, NK-92CD16, or NK-92CAR) and splenocytes from NSG mice or the HER2 positive cell line BT474 were cocultured at an effector ratio of 5 : 1 and 2 : 1, respectively, in 96-well round bottom culture plates in RPMI 1640 supplemented with 10% FCS and IL2 (100 IU/ML). After 18 h, supernatants were collected and human INF-γ content was estimated using an ELISA kit (Affymetrix, e-biosciences). Data represent the mean with SD from three independent experiments.

Mentions: As shown in Figure 5(a), 24 h after injection, NK-92NT and NK-92CD16 are present within the tumor, but not NK-92CAR. This was observed whether or not the mice were pretreated with trastuzumab. Because pretreatment of the mice with trastuzumab should have blocked recognition of cross-reactive mice antigens (if any), the interaction between mice macrophages and the CAR was unlikely to be due to the specific VH-VL part of the CAR. In contrast, although NSG mice lack T cells, B cells, and natural killer cells, they nevertheless still have neutrophils, monocyte/macrophages, and dendritic cells. And although these cells are functionally defective because of the NOD/ShiLt genetic background, they nevertheless bear Fc receptors (as shown in Figure 7(a), the CD16-32 staining of the CD11b+/Gr-1+ myeloid NSG splenocytes). And mice Fc receptors can cross-react with the human Fc (see Section 3).


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)

NK-92CAR stimulation after coculture in the presence of NSG splenocytes. (a) Single-cell suspensions from NSG mice spleens were stained with an antibody cocktail and then analyzed by flow cytometry for expression of CD11b (Mac-1) versus Gr-1 (Ly-6G) and CD11b versus CD16/32 (FcRγRIII/FcRγRII). (b) NK-92 (NK-92NT, NK-92CD16, or NK-92CAR) and splenocytes from NSG mice or the HER2 positive cell line BT474 were cocultured at an effector ratio of 5 : 1 and 2 : 1, respectively, in 96-well round bottom culture plates in RPMI 1640 supplemented with 10% FCS and IL2 (100 IU/ML). After 18 h, supernatants were collected and human INF-γ content was estimated using an ELISA kit (Affymetrix, e-biosciences). Data represent the mean with SD from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4664810&req=5

fig7: NK-92CAR stimulation after coculture in the presence of NSG splenocytes. (a) Single-cell suspensions from NSG mice spleens were stained with an antibody cocktail and then analyzed by flow cytometry for expression of CD11b (Mac-1) versus Gr-1 (Ly-6G) and CD11b versus CD16/32 (FcRγRIII/FcRγRII). (b) NK-92 (NK-92NT, NK-92CD16, or NK-92CAR) and splenocytes from NSG mice or the HER2 positive cell line BT474 were cocultured at an effector ratio of 5 : 1 and 2 : 1, respectively, in 96-well round bottom culture plates in RPMI 1640 supplemented with 10% FCS and IL2 (100 IU/ML). After 18 h, supernatants were collected and human INF-γ content was estimated using an ELISA kit (Affymetrix, e-biosciences). Data represent the mean with SD from three independent experiments.
Mentions: As shown in Figure 5(a), 24 h after injection, NK-92NT and NK-92CD16 are present within the tumor, but not NK-92CAR. This was observed whether or not the mice were pretreated with trastuzumab. Because pretreatment of the mice with trastuzumab should have blocked recognition of cross-reactive mice antigens (if any), the interaction between mice macrophages and the CAR was unlikely to be due to the specific VH-VL part of the CAR. In contrast, although NSG mice lack T cells, B cells, and natural killer cells, they nevertheless still have neutrophils, monocyte/macrophages, and dendritic cells. And although these cells are functionally defective because of the NOD/ShiLt genetic background, they nevertheless bear Fc receptors (as shown in Figure 7(a), the CD16-32 staining of the CD11b+/Gr-1+ myeloid NSG splenocytes). And mice Fc receptors can cross-react with the human Fc (see Section 3).

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