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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

Tissue distribution of NK-92NT, NK-92CD16, and NK-92CAR in NSG mice bearing an established BT474 tumor. NK-92NT, NK-92CAR, or NK-92CD16 CSFE labelled cells (20 × 106) were injected into NSG mice bearing BT474 tumors (tumor size range of 50 to 100 mm3). Twenty-four hours later, mouse blood, liver, tumor, lung, and spleen were collected, and the percentages of CD45 positive cells were analyzed by FACS analysis (percentage of CD45 was calculated from 50,000 events, and the means of two independent experiments are presented).
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fig5: Tissue distribution of NK-92NT, NK-92CD16, and NK-92CAR in NSG mice bearing an established BT474 tumor. NK-92NT, NK-92CAR, or NK-92CD16 CSFE labelled cells (20 × 106) were injected into NSG mice bearing BT474 tumors (tumor size range of 50 to 100 mm3). Twenty-four hours later, mouse blood, liver, tumor, lung, and spleen were collected, and the percentages of CD45 positive cells were analyzed by FACS analysis (percentage of CD45 was calculated from 50,000 events, and the means of two independent experiments are presented).

Mentions: To investigate the unexpected total lack of in vivo efficacy of NK-92CAR, we compared the dissemination abilities of NK-92CAR and NK-92CD16 in the NSG mouse model. For this purpose, 2 × 107 NK-92NT, NK-92CAR, or NK-92CD16 cells were injected into NSG mice bearing BT474 tumors (tumor size ranging between 50 and 100 mm3). Twenty-four hours later, mouse blood, liver, tumor, lung, and spleen were collected, and the percentages of CD45 positive cells were analyzed by FACS analysis using specific Ab. As shown in Figures 5(b), 5(c), and 5(d), no major differences between NK-92NT, NK-92CAR, and NK-92CD16 spreading were found within blood, spleen, and liver, suggesting no major differences in their ability to recirculate within the NSG mice after IP injection. However, whereas NK-92NT and NK-92CD16 can be found in tumors even at a low but significant ratio, no NK-92CAR could be found inside the tumor (Figure 5(a)). We then analyzed whether preinjection of either trastuzumab, competing for HER2 binding with CAR-HER2, or Cetuximab, recognizing HER1 also expressed at the surface of the BT474 cells without interfering with CAR-HER2 binding, could induce a chemoattractive signal for NK cells inside the tumor. As shown in Figure 5(a), opsonization of tumors with specific Ab does not attract NK-92CAR effectors. To confirm these data, we injected 3 × 107 CSFE labelled NK-92NT, NK-92CAR, or NK-92CD16 cells into NSG mice bearing BT474 tumors (tumor size ranging between 50 and 100 mm3) and collected the tumors 72 hours later. We then performed IHC on frozen sections by direct reading of CFSE labelled NK effectors and visualization of the tumor cell nuclei by counterstaining with DAPI. As shown in Figure 6(a), NK-92NT and NK-92CD16 could be found in different parts of the tumors, either as a cluster or as single cells. Preinjection of trastuzumab did not affect the NK-92CD16 frequency within the tumor (Figure 6(a), panels 2 and 3). In support of the previous FACS analysis, NK-92CAR effectors could never be found within the tumors. We observed that where present at all, most NK-92CAR were blocked at the edge of the tumor in the form of clusters (Figure 6(b), right panel) whereas NK-92CD16 could be found everywhere inside the tumor (Figure 6(b), left panel). Suspecting a possible interaction between mice macrophages and NK-92CAR, we counterstained our frozen IHC sections with anti-Iba1 antibodies specific to mouse macrophages (Wako). As shown in Figure 6(c) right panel, a yellow merge staining always located at the edge of the tumor with NK-92CAR was observed, suggesting that mice macrophages (in red) could aggregate with NK-92CAR effectors (in green). This was never observed inside the tumor, or with NK-92CD16 for which isolated macrophages are distributed everywhere inside the tumor (Figure 6(c), left panel, red arrow). Taken together, these data suggest that mice macrophages aggregating NK-92CAR might have prevented them from infiltrating inside the tumor, explaining the lack of efficacy for these type of effectors.


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)

Tissue distribution of NK-92NT, NK-92CD16, and NK-92CAR in NSG mice bearing an established BT474 tumor. NK-92NT, NK-92CAR, or NK-92CD16 CSFE labelled cells (20 × 106) were injected into NSG mice bearing BT474 tumors (tumor size range of 50 to 100 mm3). Twenty-four hours later, mouse blood, liver, tumor, lung, and spleen were collected, and the percentages of CD45 positive cells were analyzed by FACS analysis (percentage of CD45 was calculated from 50,000 events, and the means of two independent experiments are presented).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Tissue distribution of NK-92NT, NK-92CD16, and NK-92CAR in NSG mice bearing an established BT474 tumor. NK-92NT, NK-92CAR, or NK-92CD16 CSFE labelled cells (20 × 106) were injected into NSG mice bearing BT474 tumors (tumor size range of 50 to 100 mm3). Twenty-four hours later, mouse blood, liver, tumor, lung, and spleen were collected, and the percentages of CD45 positive cells were analyzed by FACS analysis (percentage of CD45 was calculated from 50,000 events, and the means of two independent experiments are presented).
Mentions: To investigate the unexpected total lack of in vivo efficacy of NK-92CAR, we compared the dissemination abilities of NK-92CAR and NK-92CD16 in the NSG mouse model. For this purpose, 2 × 107 NK-92NT, NK-92CAR, or NK-92CD16 cells were injected into NSG mice bearing BT474 tumors (tumor size ranging between 50 and 100 mm3). Twenty-four hours later, mouse blood, liver, tumor, lung, and spleen were collected, and the percentages of CD45 positive cells were analyzed by FACS analysis using specific Ab. As shown in Figures 5(b), 5(c), and 5(d), no major differences between NK-92NT, NK-92CAR, and NK-92CD16 spreading were found within blood, spleen, and liver, suggesting no major differences in their ability to recirculate within the NSG mice after IP injection. However, whereas NK-92NT and NK-92CD16 can be found in tumors even at a low but significant ratio, no NK-92CAR could be found inside the tumor (Figure 5(a)). We then analyzed whether preinjection of either trastuzumab, competing for HER2 binding with CAR-HER2, or Cetuximab, recognizing HER1 also expressed at the surface of the BT474 cells without interfering with CAR-HER2 binding, could induce a chemoattractive signal for NK cells inside the tumor. As shown in Figure 5(a), opsonization of tumors with specific Ab does not attract NK-92CAR effectors. To confirm these data, we injected 3 × 107 CSFE labelled NK-92NT, NK-92CAR, or NK-92CD16 cells into NSG mice bearing BT474 tumors (tumor size ranging between 50 and 100 mm3) and collected the tumors 72 hours later. We then performed IHC on frozen sections by direct reading of CFSE labelled NK effectors and visualization of the tumor cell nuclei by counterstaining with DAPI. As shown in Figure 6(a), NK-92NT and NK-92CD16 could be found in different parts of the tumors, either as a cluster or as single cells. Preinjection of trastuzumab did not affect the NK-92CD16 frequency within the tumor (Figure 6(a), panels 2 and 3). In support of the previous FACS analysis, NK-92CAR effectors could never be found within the tumors. We observed that where present at all, most NK-92CAR were blocked at the edge of the tumor in the form of clusters (Figure 6(b), right panel) whereas NK-92CD16 could be found everywhere inside the tumor (Figure 6(b), left panel). Suspecting a possible interaction between mice macrophages and NK-92CAR, we counterstained our frozen IHC sections with anti-Iba1 antibodies specific to mouse macrophages (Wako). As shown in Figure 6(c) right panel, a yellow merge staining always located at the edge of the tumor with NK-92CAR was observed, suggesting that mice macrophages (in red) could aggregate with NK-92CAR effectors (in green). This was never observed inside the tumor, or with NK-92CD16 for which isolated macrophages are distributed everywhere inside the tumor (Figure 6(c), left panel, red arrow). Taken together, these data suggest that mice macrophages aggregating NK-92CAR might have prevented them from infiltrating inside the tumor, explaining the lack of efficacy for these type of effectors.

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