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. 2012 Feb 13:10:29.
doi: 10.1186/1479-5876-10-29.

Protein L: a novel reagent for the detection of chimeric antigen receptor (CAR) expression by flow cytometry

Zhili Zheng  1 , Nachimuthu Chinnasamy, Richard A Morgan
Affiliations

Protein L: a novel reagent for the detection of chimeric antigen receptor (CAR) expression by flow cytometry

Zhili Zheng et al. J Transl Med. .

Abstract

Background: There has been significant progress in the last two decades on the design of chimeric antigen receptors (CAR) for adoptive immunotherapy targeting tumor-associated antigens. Structurally CARs consist of a single chain antibody fragment directed against a tumor-associated antigen fused to an extracellular spacer and transmembrane domain followed by T cell cytoplasmic signaling moieties. Currently several clinical trials are underway using gene modified peripheral blood lymphocytes (PBL) with CARs directed against a variety of tumor associated antigens. Despite the improvements in the design of CARs and expansion of the number of target antigens, there is no universal flow cytometric method available to detect the expression of CARs on the surface of transduced lymphocytes.

Methods: Currently anti-fragment antigen binding (Fab) conjugates are most widely used to determine the expression of CARs on gene-modified lymphocytes by flow cytometry. The limitations of these reagents are that many of them are not commercially available, generally they are polyclonal antibodies and often the results are inconsistent. In an effort to develop a simple universal flow cytometric method to detect the expression of CARs, we employed protein L to determine the expression of CARs on transduced lymphocytes. Protein L is an immunoglobulin (Ig)-binding protein that binds to the variable light chains (kappa chain) of Ig without interfering with antigen binding site. Protein L binds to most classes of Ig and also binds to single-chain antibody fragments (scFv) and Fab fragments.

Results: We used CARs derived from both human and murine antibodies to validate this novel protein L based flow cytometric method and the results correlated well with other established methods. Activated human PBLs were transduced with retroviral vectors expressing two human antibody based CARs (anti-EGFRvIII, and anti-VEGFR2), two murine antibody derived CARs (anti-CSPG4, and anti-CD19), and two humanized mouse antibody based CARs (anti-ERBB2, and anti-PSCA). Transduced cells were stained first with biotin labeled protein L followed by phycoerythrin (PE)-conjugated streptavidin (SA) and analyzed by flow cytometry. For comparison, cells were stained in parallel with biotin conjugated goat-anti-mouse Fab or CAR specific fusion proteins. Using protein L, all CAR transduced lymphocytes exhibited specific staining pattern ranging from 40 to 80% of positive cells (compared to untransduced cells) and staining was comparable to the pattern observed with anti-Fab antibodies.

Conclusion: Our data demonstrate the feasibility of employing Protein L as a general reagent for the detection of CAR expression on transduced lymphocytes by flow cytometry.

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Figures

Figure 1
Figure 1
A. Illustration showing the binding sites (arrows) of Protein A, Protein G, and Protein L to the heavy and light chain regions of the antibody. Protein L binding is restricted to those antibodies that contain kappa light chains. B. Schema showing Protein L binding to the kappa light chain of a single chain variable fragment (scFv) portion of a chimeric antigen receptor (CAR). TM, transmembrane region of CAR; vH, variable heavy chain; vL, variable light chain.
Figure 2
Figure 2
Titration of Protein L concentration required for optimal FACS analysis. Activated PBLs were transduced with retroviral vector expressing anti-PSCA-CAR or anti-EGFRvIII-CAR and analyzed for CAR expression on day 8. One million cells were stained with 0.01, 0.05, 0.1, 1.0, 1.5, 2.0 or 3.0 μg/sample of biotinylated Protein L. The cells were then washed and stained with phycoerythrin (PE)-conjugated streptavidin (SA). Cells were analyzed using a FACScan flow cytometer and the data analyzed with FlowJo software. Result of a representative experiment from four independent experiments is presented. No-Prot-L, samples stained with SA-PE alone.
Figure 3
Figure 3
A. FACS analysis of PBL transduced with various human or murine monoclonal antibody (mAb) based CARs. Five days following the transduction of PBLs with retroviral vectors expressing CARs targeting ERBB2, EGFRvIII, VEGFR2, CSPG4 antigen or CD19, cells were first stained with biotinylated protein L followed by PE-conjugated streptavidin (SA). Untransduced (UT) cells and T cell receptor (TCR) transduced cells were used as negative controls. Cells were analyzed using a FACScan flow cytometer and the data analyzed with FlowJo software. B. Comparison of Protein L staining to other established methods of detection of CAR expression. PBL expressing murine mAb-based anti-CSPG4 CAR were analyzed for CAR expression using goat-anti-mouse fragment antigen binding (Fab) versus protein L. These results are representative of five independent determinations. C. Humanized mAb-based anti-ERBB2 CAR transduced PBL were analyzed for CAR expression using a CAR-specific ERBB2-fragment crystallizable (Fc) fusion protein versus protein L. The result presented here is a representative experiment of three independent determinations.
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