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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

CD16 and CAR vector design and expression in NK-92 cells. (a) Schematic representation of the chimeric human FcγRIII-human FcεRIγ molecule and the trastuzumab- (4D5-) based CAR against HER2: the CD16/γ chimeric cDNA comprised the leader (S) and the two extracellular domains (EC1 and EC2) of human CD16H48V158, two amino acids (aa) of the extracellular domain of human FcεRIγ, and the intact transmembrane (TM) and intracellular (IC) domains. The trastuzumab- (4D5-) based CAR contains the VL: hum Ab 4D5-8 light chain; a linker; VH: hum Ab 4D5-8 heavy chain and two amino acids (aa) of the extracellular domain of the human FcεRIγ; and the intact transmembrane (TM) and intracellular (IC) domains. (b) Transgene expression of NK-92 cell line transduced with CD16-γ chimeric receptor or trastuzumab-γ CAR. Anti-CD16 (clone 3G8) was used to determine CD16 expression on CD16-γ transduced NK-92 cell line (solid line), and an isotype Ab was used as negative control (dotted line). Anti-human IgG2a-Fc (clone HP6002) was used to determine trastuzumab-γ CAR expression on trastuzumab-γ CAR transduced NK-92 cell line (solid line), and an isotype Ab was used as negative control (dotted line).
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fig1: CD16 and CAR vector design and expression in NK-92 cells. (a) Schematic representation of the chimeric human FcγRIII-human FcεRIγ molecule and the trastuzumab- (4D5-) based CAR against HER2: the CD16/γ chimeric cDNA comprised the leader (S) and the two extracellular domains (EC1 and EC2) of human CD16H48V158, two amino acids (aa) of the extracellular domain of human FcεRIγ, and the intact transmembrane (TM) and intracellular (IC) domains. The trastuzumab- (4D5-) based CAR contains the VL: hum Ab 4D5-8 light chain; a linker; VH: hum Ab 4D5-8 heavy chain and two amino acids (aa) of the extracellular domain of the human FcεRIγ; and the intact transmembrane (TM) and intracellular (IC) domains. (b) Transgene expression of NK-92 cell line transduced with CD16-γ chimeric receptor or trastuzumab-γ CAR. Anti-CD16 (clone 3G8) was used to determine CD16 expression on CD16-γ transduced NK-92 cell line (solid line), and an isotype Ab was used as negative control (dotted line). Anti-human IgG2a-Fc (clone HP6002) was used to determine trastuzumab-γ CAR expression on trastuzumab-γ CAR transduced NK-92 cell line (solid line), and an isotype Ab was used as negative control (dotted line).

Mentions: The chimeric cDNAs were synthesized by GeneCust (Dudelange, Luxembourg). The CD16/γ chimeric cDNA comprises the leader (S) and the two extracellular domains (EC1 and EC2) of human CD16H48V158 and two amino acids (aa) of the extracellular domain of the human FcεRIγ (Pro4-Gln5), as well as the intact transmembrane (TM) and intracellular (IC) domains (Figure 1(a)). The trastuzumab-based CAR contains the VL and VH from the mAb (Ab4D5-8), separated by a linker, the human CH2-CH3 IgG2 as a spacer, and the same signaling domain as that of CD16 (Figure 1(a)). After transduction (see Section 4), 41% of the NK-92 expressed CD16 and 36% expressed the CAR. After immunomagnetic purification using anti-CD16 and anti-human IgG2a-Fc-specific mAb (see Section 4), essentially pure populations of NK-92CD16 and NK-92CAR were obtained (Figure 1(b)).


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)

CD16 and CAR vector design and expression in NK-92 cells. (a) Schematic representation of the chimeric human FcγRIII-human FcεRIγ molecule and the trastuzumab- (4D5-) based CAR against HER2: the CD16/γ chimeric cDNA comprised the leader (S) and the two extracellular domains (EC1 and EC2) of human CD16H48V158, two amino acids (aa) of the extracellular domain of human FcεRIγ, and the intact transmembrane (TM) and intracellular (IC) domains. The trastuzumab- (4D5-) based CAR contains the VL: hum Ab 4D5-8 light chain; a linker; VH: hum Ab 4D5-8 heavy chain and two amino acids (aa) of the extracellular domain of the human FcεRIγ; and the intact transmembrane (TM) and intracellular (IC) domains. (b) Transgene expression of NK-92 cell line transduced with CD16-γ chimeric receptor or trastuzumab-γ CAR. Anti-CD16 (clone 3G8) was used to determine CD16 expression on CD16-γ transduced NK-92 cell line (solid line), and an isotype Ab was used as negative control (dotted line). Anti-human IgG2a-Fc (clone HP6002) was used to determine trastuzumab-γ CAR expression on trastuzumab-γ CAR transduced NK-92 cell line (solid line), and an isotype Ab was used as negative control (dotted line).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4664810&req=5

fig1: CD16 and CAR vector design and expression in NK-92 cells. (a) Schematic representation of the chimeric human FcγRIII-human FcεRIγ molecule and the trastuzumab- (4D5-) based CAR against HER2: the CD16/γ chimeric cDNA comprised the leader (S) and the two extracellular domains (EC1 and EC2) of human CD16H48V158, two amino acids (aa) of the extracellular domain of human FcεRIγ, and the intact transmembrane (TM) and intracellular (IC) domains. The trastuzumab- (4D5-) based CAR contains the VL: hum Ab 4D5-8 light chain; a linker; VH: hum Ab 4D5-8 heavy chain and two amino acids (aa) of the extracellular domain of the human FcεRIγ; and the intact transmembrane (TM) and intracellular (IC) domains. (b) Transgene expression of NK-92 cell line transduced with CD16-γ chimeric receptor or trastuzumab-γ CAR. Anti-CD16 (clone 3G8) was used to determine CD16 expression on CD16-γ transduced NK-92 cell line (solid line), and an isotype Ab was used as negative control (dotted line). Anti-human IgG2a-Fc (clone HP6002) was used to determine trastuzumab-γ CAR expression on trastuzumab-γ CAR transduced NK-92 cell line (solid line), and an isotype Ab was used as negative control (dotted line).
Mentions: The chimeric cDNAs were synthesized by GeneCust (Dudelange, Luxembourg). The CD16/γ chimeric cDNA comprises the leader (S) and the two extracellular domains (EC1 and EC2) of human CD16H48V158 and two amino acids (aa) of the extracellular domain of the human FcεRIγ (Pro4-Gln5), as well as the intact transmembrane (TM) and intracellular (IC) domains (Figure 1(a)). The trastuzumab-based CAR contains the VL and VH from the mAb (Ab4D5-8), separated by a linker, the human CH2-CH3 IgG2 as a spacer, and the same signaling domain as that of CD16 (Figure 1(a)). After transduction (see Section 4), 41% of the NK-92 expressed CD16 and 36% expressed the CAR. After immunomagnetic purification using anti-CD16 and anti-human IgG2a-Fc-specific mAb (see Section 4), essentially pure populations of NK-92CD16 and NK-92CAR were obtained (Figure 1(b)).

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