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Chimeric antigen receptor (CAR)-specific monoclonal antibody to detect CD19-specific T cells in clinical trials.

Jena B, Maiti S, Huls H, Singh H, Lee DA, Champlin RE, Cooper LJ - PLoS ONE (2013)

Bottom Line: This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63.This clone can be used to detect CD19-specific CAR(+) T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000.Thus, our CD19-specific CAR mAb (clone no. 136.20.1) will be useful to investigators implementing CD19-specific CAR(+) T cells to treat B-lineage malignancies.

View Article: PubMed Central - PubMed

Affiliation: Division of Pediatrics, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.

ABSTRACT
Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR) with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63). We describe a novel anti-idiotype monoclonal antibody (mAb) to detect CD19-specific CAR(+) T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1) was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19(+) tumor targets. This clone can be used to detect CD19-specific CAR(+) T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1) will be useful to investigators implementing CD19-specific CAR(+) T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.

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Related in: MedlinePlus

Specificity of anti-CD19scFv mAb.(A) Solid phase ELISA shows specificity of mAb (clone no. 136.20.1) as it binds to parental monoclonal antibody (FMC63) with background binding to other antibodies (e.g., CD20-specific mAb or a different CD19-specific mAb). Purified human IgG served as a negative control. (B) Western blot shows clone 136.20.1 detects of CAR protein in T cells genetically modified to express CD19RCD28. Lane A: Unmodified control T cells show endogenous CD3ζ (14 kDa) as detected by commercial CD3ζ-specific antibody and absence of CAR. Lane B: CAR+ T cells show CAR-specific band at 75 kDa as detected by mAb (clone 136.20.1). Lane C: CAR+ T cells show absence of CAR-specific band when the blot is treated with primary antibody after blocking (clone 136.20.1 was blocked with molar excess (1∶5) of parental antibody FMC63). Lane D: CAR+ T cells show the presence of CAR (∼75 kDa) and endogenous CD3ζ (14 KDa) as detected by commercial CD3ζ-specific antibody.
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pone-0057838-g002: Specificity of anti-CD19scFv mAb.(A) Solid phase ELISA shows specificity of mAb (clone no. 136.20.1) as it binds to parental monoclonal antibody (FMC63) with background binding to other antibodies (e.g., CD20-specific mAb or a different CD19-specific mAb). Purified human IgG served as a negative control. (B) Western blot shows clone 136.20.1 detects of CAR protein in T cells genetically modified to express CD19RCD28. Lane A: Unmodified control T cells show endogenous CD3ζ (14 kDa) as detected by commercial CD3ζ-specific antibody and absence of CAR. Lane B: CAR+ T cells show CAR-specific band at 75 kDa as detected by mAb (clone 136.20.1). Lane C: CAR+ T cells show absence of CAR-specific band when the blot is treated with primary antibody after blocking (clone 136.20.1 was blocked with molar excess (1∶5) of parental antibody FMC63). Lane D: CAR+ T cells show the presence of CAR (∼75 kDa) and endogenous CD3ζ (14 KDa) as detected by commercial CD3ζ-specific antibody.

Mentions: The specificity of mAb (clone no. 136.20.1) towards CD19scFv was initially evaluated by an indirect ELISA with wells coated with purified mAbs. Except for parental anti-human CD19 mAb (FMC63) all other antibodies exhibited negligible cross reactivity to CD19scFv specific mAb (Fig. 2A). Anti-CD19scFv binds to its parental mAb at concentration as low as 2 µg/mL with binding saturated at 4 µg/mL. The specificity of this mAb towards clone FMC63 was confirmed as it did not cross react to another anti-human CD19 antibody (Invitrogen, clone MHCD 1900). Further, it did not bind to purified human IgG and CD20-specific mAb. The ability of mAb (clone no. 136.20.1) to detect CD19+ CAR protein was verified by western blot using whole protein lysates obtained from ex vivo-propagated CD19-specific CAR+ T cells. MAb (clone no. 136.20.1) detected a ∼75 kDa protein in CD19RCD28 modified cell lysates run in reducing SDS-PAGE and electroblotted onto PVDF membrane (Fig. 2B, lane B). The specificity was verified on parallel by a commercial available CD3ζ antibody which detects CD3ζ signaling domain in CD19RCD28 expressing CAR (Fig. 2B, lane D) and endogenous CD3ζ in unmodified control T cells (Fig. 2B, lane A). The ability of this mAb to detect the surface expression of CD19+ CAR was verified by flow cytometry analysis using CD19RCD28-expressing primary T cells and Jurkat cells (Fig. 3A) and (Fig. 3B) respectively. MAb (clone 136.20.1) detected expression of CD19scFv on surface of genetically modified T cells (85%) as well as in Jurkat cells (99%) which matches with detection level of anti-Fc antibodies that binds to CH2–CH3 stalk of the CAR. Indeed, the staining pattern was compared with commercial IgG-γ chain specific antibodies (Fc-PE Invitrogen) that binds to CH2–CH3 region of the CAR and by mAb (clone no. 2D3 developed in-house) which binds to the CH2–CH3 hinge region of the CAR (Fc-FITC) [13]. A panel of CAR+ T cells that target different TAAs (CD33, CD123, ROR1, HERV-K) were used to verify if the clone no. 136.20.1 cross reacts with any of these CAR transgenes. Each CAR construct was expressed from Sleeping Beauty vector backbone and contains different signaling endodomains in addition to the common IgG4-derived hinge/CH2–CH3 linker. It did not cross react with any of other CARs tested other than those with specificity for CD19 (Fig. S2). No background binding to mock-electroporated control T cells was observed. Additional evidence supporting specificity of mAb (clone no. 136.20.1) are provided as supplementary data (Fig. S3 and Fig. S4, kindly provided by Dr. Gianpietro Dotti at Baylor College of Medicine, Houston and Dr. Stephen Forman at City of Hope National Medical Center). In both cases CAR scaffold differs either by presence of human CD4 transmembrane region or IgG1 Fc stalk that connects the scFv with the signaling endodomains. Furthermore, our mAb has been used to detect CD19-specific CAR+ T cells that employ an extracellular domain derived from CD8α [5], [20]. These results demonstrate that irrespective of extra-cellular scaffolding and transmembrane domains, our mAb (clone no. 136.20.1) detects CD19scFv within CAR expressing scFv derived from FMC63.


Chimeric antigen receptor (CAR)-specific monoclonal antibody to detect CD19-specific T cells in clinical trials.

Jena B, Maiti S, Huls H, Singh H, Lee DA, Champlin RE, Cooper LJ - PLoS ONE (2013)

Specificity of anti-CD19scFv mAb.(A) Solid phase ELISA shows specificity of mAb (clone no. 136.20.1) as it binds to parental monoclonal antibody (FMC63) with background binding to other antibodies (e.g., CD20-specific mAb or a different CD19-specific mAb). Purified human IgG served as a negative control. (B) Western blot shows clone 136.20.1 detects of CAR protein in T cells genetically modified to express CD19RCD28. Lane A: Unmodified control T cells show endogenous CD3ζ (14 kDa) as detected by commercial CD3ζ-specific antibody and absence of CAR. Lane B: CAR+ T cells show CAR-specific band at 75 kDa as detected by mAb (clone 136.20.1). Lane C: CAR+ T cells show absence of CAR-specific band when the blot is treated with primary antibody after blocking (clone 136.20.1 was blocked with molar excess (1∶5) of parental antibody FMC63). Lane D: CAR+ T cells show the presence of CAR (∼75 kDa) and endogenous CD3ζ (14 KDa) as detected by commercial CD3ζ-specific antibody.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585808&req=5

pone-0057838-g002: Specificity of anti-CD19scFv mAb.(A) Solid phase ELISA shows specificity of mAb (clone no. 136.20.1) as it binds to parental monoclonal antibody (FMC63) with background binding to other antibodies (e.g., CD20-specific mAb or a different CD19-specific mAb). Purified human IgG served as a negative control. (B) Western blot shows clone 136.20.1 detects of CAR protein in T cells genetically modified to express CD19RCD28. Lane A: Unmodified control T cells show endogenous CD3ζ (14 kDa) as detected by commercial CD3ζ-specific antibody and absence of CAR. Lane B: CAR+ T cells show CAR-specific band at 75 kDa as detected by mAb (clone 136.20.1). Lane C: CAR+ T cells show absence of CAR-specific band when the blot is treated with primary antibody after blocking (clone 136.20.1 was blocked with molar excess (1∶5) of parental antibody FMC63). Lane D: CAR+ T cells show the presence of CAR (∼75 kDa) and endogenous CD3ζ (14 KDa) as detected by commercial CD3ζ-specific antibody.
Mentions: The specificity of mAb (clone no. 136.20.1) towards CD19scFv was initially evaluated by an indirect ELISA with wells coated with purified mAbs. Except for parental anti-human CD19 mAb (FMC63) all other antibodies exhibited negligible cross reactivity to CD19scFv specific mAb (Fig. 2A). Anti-CD19scFv binds to its parental mAb at concentration as low as 2 µg/mL with binding saturated at 4 µg/mL. The specificity of this mAb towards clone FMC63 was confirmed as it did not cross react to another anti-human CD19 antibody (Invitrogen, clone MHCD 1900). Further, it did not bind to purified human IgG and CD20-specific mAb. The ability of mAb (clone no. 136.20.1) to detect CD19+ CAR protein was verified by western blot using whole protein lysates obtained from ex vivo-propagated CD19-specific CAR+ T cells. MAb (clone no. 136.20.1) detected a ∼75 kDa protein in CD19RCD28 modified cell lysates run in reducing SDS-PAGE and electroblotted onto PVDF membrane (Fig. 2B, lane B). The specificity was verified on parallel by a commercial available CD3ζ antibody which detects CD3ζ signaling domain in CD19RCD28 expressing CAR (Fig. 2B, lane D) and endogenous CD3ζ in unmodified control T cells (Fig. 2B, lane A). The ability of this mAb to detect the surface expression of CD19+ CAR was verified by flow cytometry analysis using CD19RCD28-expressing primary T cells and Jurkat cells (Fig. 3A) and (Fig. 3B) respectively. MAb (clone 136.20.1) detected expression of CD19scFv on surface of genetically modified T cells (85%) as well as in Jurkat cells (99%) which matches with detection level of anti-Fc antibodies that binds to CH2–CH3 stalk of the CAR. Indeed, the staining pattern was compared with commercial IgG-γ chain specific antibodies (Fc-PE Invitrogen) that binds to CH2–CH3 region of the CAR and by mAb (clone no. 2D3 developed in-house) which binds to the CH2–CH3 hinge region of the CAR (Fc-FITC) [13]. A panel of CAR+ T cells that target different TAAs (CD33, CD123, ROR1, HERV-K) were used to verify if the clone no. 136.20.1 cross reacts with any of these CAR transgenes. Each CAR construct was expressed from Sleeping Beauty vector backbone and contains different signaling endodomains in addition to the common IgG4-derived hinge/CH2–CH3 linker. It did not cross react with any of other CARs tested other than those with specificity for CD19 (Fig. S2). No background binding to mock-electroporated control T cells was observed. Additional evidence supporting specificity of mAb (clone no. 136.20.1) are provided as supplementary data (Fig. S3 and Fig. S4, kindly provided by Dr. Gianpietro Dotti at Baylor College of Medicine, Houston and Dr. Stephen Forman at City of Hope National Medical Center). In both cases CAR scaffold differs either by presence of human CD4 transmembrane region or IgG1 Fc stalk that connects the scFv with the signaling endodomains. Furthermore, our mAb has been used to detect CD19-specific CAR+ T cells that employ an extracellular domain derived from CD8α [5], [20]. These results demonstrate that irrespective of extra-cellular scaffolding and transmembrane domains, our mAb (clone no. 136.20.1) detects CD19scFv within CAR expressing scFv derived from FMC63.

Bottom Line: This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63.This clone can be used to detect CD19-specific CAR(+) T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000.Thus, our CD19-specific CAR mAb (clone no. 136.20.1) will be useful to investigators implementing CD19-specific CAR(+) T cells to treat B-lineage malignancies.

View Article: PubMed Central - PubMed

Affiliation: Division of Pediatrics, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.

ABSTRACT
Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR) with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63). We describe a novel anti-idiotype monoclonal antibody (mAb) to detect CD19-specific CAR(+) T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1) was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19(+) tumor targets. This clone can be used to detect CD19-specific CAR(+) T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1) will be useful to investigators implementing CD19-specific CAR(+) T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.

Show MeSH
Related in: MedlinePlus