Protein L: a novel reagent for the detection of Chimeric Antigen Receptor (CAR) expression by flow cytometry

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Multicistronic lentiviral vectors containing the FMDV 2A cleavage factor demonstrate robust expression of encoded genes at limiting MOI

BACKGROUND: A number of gene therapy applications would benefit from vectors capable of expressing multiple genes. In this study we explored the feasibility and efficiency of expressing two or three transgenes in HIV-1 based lentiviral vector. Bicistronic and tricistronic self-inactivating lentiviral vectors were constructed employing the internal ribosomal entry site (IRES) sequence of encephalomyocarditis virus (EMCV) and/or foot-and-mouth disease virus (FMDV) cleavage factor 2A. We employed enhanced green fluorescent protein (eGFP), O(6)-methylguanine-DNA-methyltransferase (MGMT), and homeobox transcription factor HOXB4 as model genes and their expression was detected by appropriate methods including fluorescence microscopy, flow cytometry, immunocytochemistry, biochemical assay, and western blotting. RESULTS: All the multigene vectors produced high titer virus and were able to simultaneously express two or three transgenes in transduced cells. However, the level of expression of individual transgenes varied depending on: the transgene itself; its position within the construct; the total number of transgenes expressed; the strategy used for multigene expression and the average copy number of pro-viral insertions. Notably, at limiting MOI, the expression of eGFP in a bicistronic vector based on 2A was ~4 times greater than that of an IRES based vector. CONCLUSION: The small and efficient 2A sequence can be used alone or in combination with an IRES for the construction of multicistronic lentiviral vectors which can express encoded transgenes at functionally relevant levels in cells containing an average of one pro-viral insert

A mechanistic study of immune system activation by fusion of antigens with the ligand-binding domain of CTLA4

Fusion proteins consisting of the ligand-binding domain of CTLA4 covalently attached to an antigen (Ag) are potent immunogens. This fusion strategy effectively induces Ag-specific immunity both when introduced as a DNA-based vaccine and as a recombinant protein. CTLA4 is a ligand for B7 molecules expressed on the surface of antigen-presenting cells (APCs), and this interaction is critical for the fusion protein to stimulate Ag-specific immunity. We show that interaction of the fusion protein with either B7-1 or B7-2 is sufficient to stimulate immune activity, and that T cells are essential for the development of IgG responses. In addition, we demonstrate that human dendritic cells (DCs) pulsed with CTLA4-Ag fusion proteins can efficiently present Ag to T cells and induce an Ag-specific immune response in vitro. These studies provide further mechanistic understanding of the process by which CTLA4-Ag fusion proteins stimulate the immune system, and represent an efficient means of generating Ag-specific T cells for immunotherapy

Ex vivo generation of genetically modified dendritic cells for immunotherapy: implications of lymphocyte contamination

Genetically modified dendritic cell (DC) vaccines expressing tumor-associated antigens are currently used for cancer immunotherapy. Peripheral blood (PB) monocyte precursors are a relatively convenient source of DCs for use in clinical studies, but are often contaminated by lymphocytes. The current study was conducted to examine the impact of T-lymphocyte contamination on genetically modified DC product. PB monocyte-derived DCs were efficiently transduced (75-95%) with an HIV-1-based self-inactivating lentiviral vector encoding a model antigen, the enhanced green fluorescent protein (eGFP). The lymphocyte-free DC culture transduced with Lenti-eGFP showed stable expression of eGFP without measurable decline in viability. In contrast, the eGFP-positive DCs disappeared rapidly in transduced DC cultures containing lymphocyte contaminants, concurrent with detectable activation and expansion of T-lymphocytes. Upon antigen recall, these T cells elicited major histocompatability complex-restricted antigen-specific cytotoxicity against eGFP-positive autologous DCs and mitogen-stimulated T lymphoblasts, mainly through the perforin-mediated pathway. In summary, this study demonstrate that the relative purity of DC cultures could determine the persistence of gene-modified DC, which may affect the induction of effective immune responses by DC vaccination strategies

Analysis of recognition of other genes by TCR-5.

<p>Peripheral blood T cells expressing TCR-5 were cocultured overnight with T2 cells previously pulsed with the serial dilutions of the indicated peptides. Results of IFNg concentration in culture supernatants are expressed as average of duplicates in a representative experiment. Sequence alignment of the tested peptides is shown in the figure legend for A) SSX-family genes and B) non-SSX genes with overlapping sequences. IGSF22: immunoglobulin superfamily member 22, ARHGAP1: Rho GTPase-activating protein 1, GPR82: Probable G-protein coupled receptor 82, PHF8: histone lysine demethylase PHF8, LIPM: lipase member M, SYT14: synaptotagmin-14, TCOF1: treacle protein, RBL2: retinoblastoma-like protein 2, FRAS1: extracellular matrix protein FRAS1. Prediction of binding affinity to HLA-A2*0201 is shown for each peptide, expressed as dissociation constant (K_D, nM).</p

Generation of retroviral vectors for the expression of SSX2-specific TCRs.

<p>The coding region of each TCR alpha-chain was amplified by PCR using primers flanked by an NcoI restriction site in the 5′ end and an overhanging sequencing containing the element in the 3′ end. In parallel, each TCR beta-chain was amplified by PCR using a forward primer containing a 5′ overhang that overlaps with the 3′ overhang present in the primer used for the amplification of the alpha-chain. The reverse primer used for amplification of the beta-chain contained a stop codon and an <i>Eco</i>RI restriction site. In a second round of PCR, the products of the alpha- and beta-chain amplifications were pooled, and ligation of both cDNA fragments through the overlapping overhangs was achieved by PCR using external primers. The resulting PCR products were cloned in pMSGV1 vector for retrovirus production. LTR: long terminal repeat, sd: splice donor, sa: splice acceptor, ψ: retrovirus encapsidation signal.</p

IFNg secretion (pg/mL) by TCR-5 transduced lymphocytes upon coculture with indicated targets.

<p>IFNg secretion (pg/mL) by TCR-5 transduced lymphocytes upon coculture with indicated targets.</p

Codon optimization and replacement of TCR constant regions with murine sequences.

<p>A) Schematic representation of the three constructs generated for the expression of TCR-5 and derivatives. LTR: long terminal repeat, sd: splice donor, sa: splice acceptor, ψ: retrovirus encapsidation signal, MC: mouse TCR constant region, 2A: linker peptide. B) Analysis of expression of TCR-5 variants by tetramer staining. OKT3-stimulated lymphocytes were transduced twice with the corresponding TCR-expressing vector and stained with anti-CD3, anti-CD8 and SSX2_41-49 tetramers one week after transduction. Representative results from three independent experiments. Values in parentheses represent the mean fluorescence intensity of tetramer staining within the CD8 T cell population. C) ⁵¹Cr-release assay for the evaluation of antigen-specific cytolysis induced by TCR-5-transduced lymphocytes after four-hour coculture with the indicated target cells. Percentage of lysis depicted for each target cell line at different effector:target ratios is the average of duplicates in a representative experiment of three independent experiments. UT: untransduced T cells used as negative control of unspecific lysis, WT: Wild-type TCR, Co Op: copon-optimized TCR, MCR: codon-optimized TCR with mouse constant region.</p