Specific Lysis of Melanoma Cells by Receptor Grafted T Cells is Enhanced by Anti-Idiotypic Monoclonal Antibodies Directed to the scFv Domain of the Receptor
1999; Elsevier BV; Volume: 112; Issue: 5 Linguagem: Inglês
10.1046/j.1523-1747.1999.00586.x
ISSN1523-1747
AutoresUwe Reinhold, Wolfgang Tilgen, Lide Liu, Hans-Christian Lüdtke-Handjery, Claudia Heuser, Andreas Hombach, Xinhui Wang, Soldano Ferrone, Hinrich Abken,
Tópico(s)Immunotherapy and Immune Responses
ResumoMalignant transformation of melanocytes is frequently associated with abnormalities in antigen processing and in human leukocyte antigen class I antigen expression. Here, we evaluated a human leukocyte antigen class I antigen-independent approach to target cytotoxic T lymphocytes to melanoma cells by grafting cytotoxic T lymphocytes with a chimeric receptor that consists of both a domain binding to high molecular weight-melanoma associated antigen and a cellular activation domain. The binding domain is a single-chain antibody fragment (scFv) derived from the monoclonal anti-high molecular weight-melanoma associated antigen antibody 763.74 by phage display techniques. The cellular activation domain is the signaling unit of the FcεRI receptor γ chain. Both domains constitute the chimeric receptor scFv763.74-γR. Cytotoxic MD45 T cells grafted with the scFv763.74-γR receptor bind specifically to high molecular weight-melanoma associated antigen-positive melanoma cells and lyse melanoma cells in a human leukocyte antigen class I independent fashion. Pre-incubation of receptor grafted T cells with immobilized anti-idiotypic (id) monoclonal antibody MK2–23 binding to the scFv domain of the receptor enhanced the lysis of melanoma cells indicating that the specific cytolytic activity of receptor grafted T cells can be increased by costimulation with cross-linked anti-idiotypic monoclonal antibodies that recognize the antigen binding domain of the chimeric receptor. Malignant transformation of melanocytes is frequently associated with abnormalities in antigen processing and in human leukocyte antigen class I antigen expression. Here, we evaluated a human leukocyte antigen class I antigen-independent approach to target cytotoxic T lymphocytes to melanoma cells by grafting cytotoxic T lymphocytes with a chimeric receptor that consists of both a domain binding to high molecular weight-melanoma associated antigen and a cellular activation domain. The binding domain is a single-chain antibody fragment (scFv) derived from the monoclonal anti-high molecular weight-melanoma associated antigen antibody 763.74 by phage display techniques. The cellular activation domain is the signaling unit of the FcεRI receptor γ chain. Both domains constitute the chimeric receptor scFv763.74-γR. Cytotoxic MD45 T cells grafted with the scFv763.74-γR receptor bind specifically to high molecular weight-melanoma associated antigen-positive melanoma cells and lyse melanoma cells in a human leukocyte antigen class I independent fashion. Pre-incubation of receptor grafted T cells with immobilized anti-idiotypic (id) monoclonal antibody MK2–23 binding to the scFv domain of the receptor enhanced the lysis of melanoma cells indicating that the specific cytolytic activity of receptor grafted T cells can be increased by costimulation with cross-linked anti-idiotypic monoclonal antibodies that recognize the antigen binding domain of the chimeric receptor. cytotoxic T cells high molecular weight melanoma-associated antigen idiotypic single-chain antibody fragment tumor-associated antigen In recent years there has been a growing interest in the application of immunotherapy for the treatment of melanoma. This trend reflects, at least in part, the disappointing results of conventional chemotherapy (Koh, 1991Koh H.K. Cutaneous melanoma.N Engl J Med. 1991; 325: 171-182Crossref PubMed Scopus (438) Google Scholar), the identification of melanoma-associated antigens (MAA) suitable to be used as targets for immunotherapy (Boon, 1994Boon T. Tumor antigens recognized by T lymphocytes.Annu Rev Immunol. 1994; 12: 337-365Crossref PubMed Google Scholar;Rosenberg, 1996Rosenberg S. Development of cancer immunotherapies based on identification of the genes encoding cancer regression antigens.J Natl Cancer Inst. 1996; 88: 1635-1644Crossref PubMed Scopus (124) Google Scholar), and the significant progress in our understanding of the molecular steps leading to the development of an immune response (Brodsky et al., 1996Brodsky F.M. Lem L. Bresnahan P.A. Antigen processing and presentation.Tissue Antigens. 1996; 47: 464-471Crossref PubMed Scopus (33) Google Scholar). Because of the general belief that T cell immunity plays a major part in the control of tumor growth (McMichael, 1992McMichael A. Cytotoxic T lymphocytes and immune surveillance.Cancer Surv. 1992; 13: 5-21PubMed Google Scholar), the emphasis has been on the application of T cell based immunotherapy. The latter is negatively affected by abnormalities in human leukocyte antigen (HLA) class I antigen presentation and in antigen processing which occur frequently in malignant cells (Ferrone and Marincola, 1995Ferrone S. Marincola F.M. Loss of HLA class I antigens by melanoma cells: molecular mechanisms, functional significance and clinical relevance.Immunol Today. 1995; 16: 487-494Abstract Full Text PDF PubMed Scopus (455) Google Scholar;Seliger et al., 1997Seliger B. Maeurer M.J. Ferrone S. TAP off–tumors on.Immunol Today. 1997; 18: 292-299Abstract Full Text PDF PubMed Google Scholar). Therefore, methods are being developed which target T cells to tumor cells bypassing the requirements of presentation of tumor-associated antigen (TAA) derived peptides by HLA class I antigens to T cell receptors. Among these approaches some were developed by Eshhar and his collaborators (Gross and Eshhar, 1992Gross G. Eshhar Z. Endowing T cells with antibody specificity using chimeric T cell receptors.Faseb J. 1992; 6: 3370-3378PubMed Google Scholar), which has attracted much interest. Using this methodology cytotoxic T lymphocytes (CTL) are targeted to tumor cells by grafting a chimeric receptor. Its extracellular moiety is composed of the variable regions of the heavy and light chain of an antibody recognizing a TAA. Its intracellular moiety consists of a signal transduction chain for specific cellular activation. Early studies proved the validity of the approach by showing that grafting with a chimeric receptor specific for TNP endowed CTL with a non-major histocompatibility complex restricted recognition of TNP bearing target cells (Gross et al., 1989Gross G. Waks T. Eshhar Z. Expression of immunoglobulin–T cell receptor chimeric molecules as functional receptors with antibody-type specificity.Proc Natl Acad Sci USA. 1989; 86: 10024-10028Crossref PubMed Scopus (987) Google Scholar). More recently, chimeric receptors have been constructed with single chain fragments of variable regions (scFv) derived from monoclonal antibody (MoAb) recognizing human TAA (Abken et al., 1998Abken H. Hombach A. Reinhold U. Ferrone S. Can combined T-cell- and antibody-based immunotherapy outsmart tumor cells?.Immunol Today. 1998; 19: 2-5Abstract Full Text PDF PubMed Scopus (35) Google Scholar). They include ErbB-2 (Stancovski et al., 1993Stancovski I. Schindler D.G. Waks T. Yarden Y. Sela M. Eshhar Z. Targeting of T lymphocytes to Neu/HER2-expressing cells using chimeric single chain Fv receptors.J Immunol. 1993; 151: 6577-6582PubMed Google Scholar;Moritz et al., 1994Moritz D. Wels W. Mattern J. Groner B. Cytotoxic T lymphocytes with a grafted recognition specificity for ErbB2-expressing tumor cells.Proc Natl Acad Sci USA. 1994; 91: 4318-4322Crossref PubMed Scopus (186) Google Scholar), 38 kDa folate binding protein (Hwu et al., 1993Hwu P. Shafer G.E. Treisman J. et al.Lysis of ovarian cancer cells by human lymphocytes redirected with a chimeric gene composed of an antibody variable region and the Fc receptor γ chain.J Exp Med. 1993; 178: 361-366Crossref PubMed Scopus (222) Google Scholar), G250 (Weijtens et al., 1996Weijtens M.E.M. Willemsen R.A. Valerio D. Stam K. Bolhuis R.L.H. Single chain Ig/γ gene-redirected human T lymphocytes produce cytokines, specifically lyse tumor cells, and recycle lytic capacity.J Immunol. 1996; 157: 836-843PubMed Google Scholar), CD30 (Hombach et al., 1998aHombach A. Heuser C. Sircar R. Tillmann T. Diehl V. Pohl C. Abken H. An anti-CD30 chimeric receptor that mediates CD3-zeta independent T-cell activation against Hodgkin's lymphoma cells in presence of soluble CD30.Cancer Res. 1998; 58: 1116-1119PubMed Google Scholar), TAG72 (Hombach et al., 1997Hombach A. Heuser C. Sircar R. et al.Specific T cell targeting of TAG72+ gastrointestinal tumor cells by a chimeric receptor with antibody-like specificity.Gastroenterology. 1997; 113: 1163-1170Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), membrane-bound CEA (Hombach et al., 1998bHombach A. Koch D. Sircar R. et al.Chimeric receptor that selectively targets membrane-bound carcinoembryonic antigen (mCEA) in presence of soluble CEA.Gene Ther. 1998Google Scholar), and CD44v6 splice variants (Hekele et al., 1996Hekele A. Dall P. Moritz D. Growth retardation of tumors by adoptive transfer of cytotoxic T lymphocytes reprogrammed by CD44v6-specific scFv: zeta-chimera.Int J Cancer. 1996; 68: 232-238Crossref PubMed Scopus (50) Google Scholar). Effector cells transfected with the respective chimeric receptor have been shown to acquire the ability to lyse tumor cells expressing the appropriate TAA in a major histocompatibility complex unrestricted fashion. In this study we have constructed a melanoma cell specific, chimeric receptor utilizing scFv fragments derived from the mouse anti-human high molecular weight-melanoma associated antigen (HMW-MAA) MoAb 763.74. This antigen seems to be suitable for an immunotherapeutic approach as it is expressed in a large percentage of melanoma lesions (Ferrone et al., 1988Ferrone S. Temponi M. Gargiulo D. Scassellati G.A. Cavaliere R. Natali P.G. Selection and utilization of monoclonal antibody defined melanoma associated antigens of immunoscintigraphy in patients with melanoma.in: Srivastava S.C. Radiolabeled Monoclonal Antibodies for Imaging and Therapy. Plenum Press, New York and London1988Crossref Google Scholar), has a restricted distribution in normal tissues, and has already been used successfully as target of active and specific immunotherapy in melanoma (Spitler et al., 1987Spitler L.E. Del Rio M. Khentigan A. et al.Therapy of patients with malignant melanoma using a monoclonal antimelanoma antibody-ricin A chain immunotoxin.Cancer Res. 1987; 47: 1717-1723PubMed Google Scholar;Mittelman et al., 1990Mittelman A. Chen Z.J. Kageshita T. et al.Active specific immunotherapy in patients with melanoma: a clinical trial with mouse antiidiotypic monoclonal antibodies elicited with syngeneic anti-high-molecular-weight-melanoma-associated antigen monoclonal antibodies.J Clin Invest. 1990; 86: 2136-2144Crossref PubMed Scopus (116) Google Scholar). We therefore demonstrate that cytotoxic T cells grafted with the anti-HMW-MAA receptor are activated to specific, major histocompatibility complex-unrestricted cellular cytotoxicity against HMW-MAA+ melanoma cells and that the cytolytic activity can be increased by anti-idiotypic (id) antibodies that recognize the antigen-binding domain of the grafted chimeric receptor. The mouse hybridoma cells which secrete the anti-HMW-MAA MoAb 763.74, the human melanoma cell lines Colo 38 and SK-MEL-5, a subclone of the human melanoma cell line A375, the MD45 alloreactive cytotoxic T hybridoma cells (Kaufmann et al., 1981Kaufmann Y. Berke G. Eshhar Z. Cytotoxic T lymphocyte hybridomas that mediate specific tumor-cell lysis in vitro.Proc Natl Acad Sci USA. 1981; 78: 2502-2506Crossref PubMed Scopus (51) Google Scholar) of BALB/C origin and the MD45 cells transfected with the unrelated scFv CD30R (chimeric receptor clone HRS-3γC1) (Hombach et al., 1998aHombach A. Heuser C. Sircar R. Tillmann T. Diehl V. Pohl C. Abken H. An anti-CD30 chimeric receptor that mediates CD3-zeta independent T-cell activation against Hodgkin's lymphoma cells in presence of soluble CD30.Cancer Res. 1998; 58: 1116-1119PubMed Google Scholar) were cultured in medium RPMI 1640 (Gibco, Paisley, U.K.) supplemented with 10% fetal bovine serum (PAA Laboratories, Linz, Austria), penicillin (100 U per ml; Biochrom, Berlin, Germany), streptomycin (100 μg per ml; Biochrom), and 2 mM L-glutamine (Biochrom). All cell lines were maintained in a 5% CO2 humidified incubator at 37°C. A lysate of melanoma cells Colo 38 was prepared by incubating cells (1 × 107) for 30 min on ice in 1.2 ml of lysis buffer (ethylenediamine tetraacetic acid 1 mM, NaCl 150 mM, Tris base 10 mM, 1% NP40) containing 10 mM phenylmethylsulfonyl fluoride. Supernatant was collected after centrifugation at 10,000 × g for 30 min and stored at –20°C. Primers used for polymerase chain reaction amplification of cDNA of VH and VL domains of MoAb 763.74 were purchased from Pharmacia (Freiburg, Germany). Oligonucleotide primers VH54 (5′GCG GCC CAG TCT AGA ATG GCC CAG 3′) and E-tag/VL34 (5′GGT TCC AGC GGA TCC GGA TAC GCG 3′) used for the introduction of restriction sites at the ends of the scFv were synthesized by the phosphoamidite procedure (Caruthers et al., 1987Caruthers M.H. Barone A.D. Beaucage S.L. et al.Chemical synthesis of deoxyoligonucleotides by the phosphoramidite method.Methods Enzymol. 1987; 154: 287-313Crossref PubMed Scopus (313) Google Scholar) using an Expedite Synthesizer (Millipore, Eschborn, Germany). DNA sequences were determined by the chain termination method (Sanger et al., 1977Sanger F. Nicklen S. Coulson A. DNA sequencing with chain-terminating inhibitors.Proc Natl Acad Sci USA. 1977; 74: 5463-5467Crossref PubMed Scopus (52610) Google Scholar) using the ABI Model 377 automatic sequencing system. The pRSV-γ vector (Eshhar et al., 1993Eshhar Z. Waks T. Gross G. Schindler D.G. Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the γ or ζ subunits of the immunoglobulin and T-cell receptors.Proc Natl Acad Sci USA. 1993; 90: 720-724Crossref PubMed Scopus (1062) Google Scholar) containing the FcεRI γ chain under the control of the RSV LTR was kindly provided by Dr. Z. Eshhar (Weizman Institute of Sciences, Rehovot, Israel). The 120 kDa MAA binding scFv W34 was isolated from a semisynthetic scFv library by panning with cultured human melanoma cells WM1158 (X. Wang and S. Ferrone, unpublished results). scFv HRS-3 fragments were generated from anti-CD30 MoAb HRS-3 as described (Hombach et al., 1998aHombach A. Heuser C. Sircar R. Tillmann T. Diehl V. Pohl C. Abken H. An anti-CD30 chimeric receptor that mediates CD3-zeta independent T-cell activation against Hodgkin's lymphoma cells in presence of soluble CD30.Cancer Res. 1998; 58: 1116-1119PubMed Google Scholar). The mouse MoAb 225.28, TP41.2, and 763.74 recognizing spatially distant antigenic determinants of HMW-MAA, the mouse anti-id MoAb MK2–23 generated from a BALB/C mouse immunized with MoAb 763.74 and the mouse anti-id MoAb TK7–371 recognizing an idiotope in the antigen combining site of the immunizing syngeneic anti-HMW-MAA MoAb TP61.5 were developed as described (Wilson et al., 1981Wilson B.S. Imai K. Natali P.G. Ferrone S. Distribution and molecular characterization of a cell-surface and a cytoplasmic antigen detectable in human melanoma cells with monoclonal antibodies.Int J Cancer. 1981; 28: 293-300Crossref PubMed Scopus (227) Google Scholar;Giacomini et al., 1983Giacomini P. Ng A.K. Kantor R.R.S. Natali P.G. Ferrone S. Double determinant immunoassay to measure a human high-molecular-weight melanoma-associated antigen.Cancer Res. 1983; 43: 3586-3590PubMed Google Scholar;Kusama et al., 1989Kusama M. Kageshita T. Chen Z.J. Ferrone S. Characterization of syngeneic antiidiotypic monoclonal antibodies to murine anti-human high molecular weight melanoma-associated antigen monoclonal antibodies.J Immunol. 1989; 143: 3844-3852PubMed Google Scholar;Chen et al., 1991Chen Z.J. Yang H. Kageshita T. Ferrone S. Human high-molecular-weight melanoma-associated antigen mimicry by mouse antiidiotypic monoclonal antibody TK7–371.Cancer Res. 1991; 51: 4790-4797PubMed Google Scholar;Temponi et al., 1992Temponi M. Gold A.M. Ferrone S. Binding parameters and idiotypic profile of the whole immunoglobulin and Fab′ fragments of murine monoclonal antibody to distinct determinants of the human high molecular weight-melanoma associated antigen.Cancer Res. 1992; 52: 2497-2503PubMed Google Scholar). MoAb were purified from ascitic fluid by sequential precipitation with caprylic acid and ammonium sulfate (Temponi et al., 1989Temponi M. Kageshita T. Perosa F. Ono R. Okada H. Ferrone S. Purification of murine IgG monoclonal antobodies by precipitation with caprylic acid: Comparison with other methods of purification.Hybridoma. 1989; 8: 85-95Crossref PubMed Scopus (127) Google Scholar). The purity and activity of MoAb preparations were monitored by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Laemmli, 1970Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (207012) Google Scholar) and by binding assays with the corresponding antigen, respectively. Anti-E Tag MoAb and horseradish peroxidase (HRP)-conjugated anti-E Tag MoAb were purchased from Pharmacia. Rat anti-mouse interleukin (IL)-2 MoAb and biotinylated rat anti-mouse IL-2 MoAb were purchased from Pharmingen (San Diego, CA). Goat anti-mouse IgG antibodies, fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG xenoantibodies and HRP-conjugated anti-mouse IgG xenoantibodies were purchased from Immunotech (Hamburg, Germany). RNA was extracted from the 763.74 hybridoma cells by the acid–guanidium–thiocyanate–chloroform method. First-strand cDNA synthesis and amplification of the Fv-encoding regions were carried out utilizing the recombinant phage antibody system (Pharmacia) following the supplier's instructions. The VH and VL cDNA fragments were fused via the (Gly4Ser)3 peptide linker in VH-VL order, introducing an Sfi and a NotI restriction site at the 5′ and 3′ end of the scFv, respectively. The scFv cDNA was amplified by polymerase chain reaction utilizing primer VH54 and E-tag/VL, digested with Sfi and NotI, and inserted into the phagemid pCANTAB5E DNA (Pharmacia). Phages expressing the 763.74 scFv were selected by panning on immobilized anti-id MoAb MK2–23. To this end, cell culture flasks were coated with anti-id MoAb MK2–23 (10 μg per ml) at 4°C overnight. Following three washings the culture flasks were blocked with phosphate-buffered saline (PBS)/bovine serum albumin (BSA) (5%) and then incubated for 2 h at room temperature with the supernatant containing the phages. Finally, the flask was washed 40 times with PBS/Tween 20 (0.1%). TG1 cells were infected with phages and plated out on 2 × YT-medium containing 100 mM glucose and ampicillin (100 μg per ml). Individual colonies were transferred to microtiter plates and phages were rescued by overnight incubation in 2 × YT-medium (without glucose) supplemented with ampicillin (100 μg per ml) and kanamycin (50 μg per ml). The scFv protein was expressed by inducing with 1.0 mM isopropyl-β-D-thiogalactopyranoside at 25°C a colony of Escherichia coli HB2151 cells transformed with the recombinant phagemid and grown at 37°C in 2xyT medium containing 0.1% glucose and 100 μg ampicillin per ml to an OD600 of 1.0. Cells were harvested from 1 liter of bacterial culture by centrifugation at 4000 × g for 15 min at 4°C and lysed. The lysate was centrifuged at 8000 × g for 30 min at 4°C. The supernatant was dialyzed against PBS and tested for content of scFv fragments. Proteins of bacteria extracts containing the scFv were electrophoretically separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and blotted on to nitrocellulose membrane. Following an overnight incubation at room temperature in 4% BSA/PBS, the blot was incubated for 1 h at room temperature with HRP-conjugated anti-E tag MoAb solution (1 μg 4% BSA/PBS per ml; Pharmacia). Following six washings with PBS, binding of HRP-labeled anti-E-tag MoAb was visualized using 4-chloro-1-naphtol substrate (Sigma, Deisenhofen, Germany) and 0.03% H2O2. Ninety-six well microtiter plates (Nunc, Roskilde, Denmark) were coated with HMW-MAA as previously described (Chen et al., 1991Chen Z.J. Yang H. Kageshita T. Ferrone S. Human high-molecular-weight melanoma-associated antigen mimicry by mouse antiidiotypic monoclonal antibody TK7–371.Cancer Res. 1991; 51: 4790-4797PubMed Google Scholar). Briefly, microtiter plates were coated with anti-HMW-MAA MoAb 225.28 by adding 100 μl of a MoAb solution (either 10 μg NaHCO3 per ml, pH 9.6 or 2 μg PBS per ml, pH 7.4) to each well. Following an overnight incubation at room temperature, plates were washed three times with PBS and blocked with 2% BSA/PBS for 1 h at room temperature. A melanoma cell lysate (100 μl) was added to each well following an overnight incubation at room temperature, plates were washed three times with PBS. Plates were coated with the whole IgG or F(ab′)2 fragments of MoAb or with scFv fragments by adding to each well 100 μl of a MoAb whole IgG or F(ab′)2 fragment solution (10 μg NaHCO3 per ml, pH 9.6) or of a scFv fragment solution (20 μg PBS per ml, pH 7.4), respectively. At the end of an overnight incubation at 4°C, plates were washed twice with PBS and blocked with 4% BSA/PBS. The enzyme-linked immunosorbent assay (ELISA) to measure the reactivity of scFv763.74 fragments with HMW-MAA and with anti-id MoAb was performed in 96 well microtiter immunoplates (Nunc) coated with HMW-MAA or with scFv fragments. One hundred microliters of 2-fold dilutions of a scFv763.74 solution (20 μg PBS per ml) or of spent medium of anti-id MoAb secreting hybridomas were added to each well and the mixture was incubated for 2 h at room temperature. Following three washings, HRP-conjugated anti-E-tag MoAb or HRP-conjugated anti-mouse IgG Fc xenoantibodies were added and incubation was continued for an additional 2 h at room temperature. Following three washings, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (Sigma) and 0.03% H2O2 in citrate buffer were added. Following a 30 min incubation at room temperature the reaction was stopped by adding 100 liters of sodium dodecyl sulfate/N,N-dimethyl formamide solution to each well. Absorbance was read at 405 nm utilizing a microtiter plate ELISA reader (SLT-Labinstruments, Crailsheim, Germany). Results are expressed as mean optical density (OD) units. All assays were done in triplicate. The inhibition assay to measure the ability of anti-id MoAb to inhibit the binding of MoAb 763.74 and scFv763.74 to melanoma cells Colo 38 and to anti-id MoAb MK2–23 was performed as described (Perosa et al., 1989Perosa F. Kageshita T. Ono R. Ferrone S. Serological methods to detect anti-idiotypic antibodies.Methods Enzymol. 1989; 178: 74-90Crossref PubMed Scopus (22) Google Scholar). The competition ELISA to map the antigenic determinant recognized by scFv763.74 was performed by adding 100 μl of a MoAb 763.74 solution (1 μg PBS per ml) to MoAb MK2–23 coated wells. Following a 1 h incubation at room temperature and two washings 100 μl of 2-fold dilutions of a scFv763.74 solution (1 μg PBS per ml) were added to a microtiter plate. Results are expressed as mean OD units. The specificity of the inhibition was monitored utilizing HRP-conjugated anti-E-tag MoAb. Binding of scFv fragments was detected by adding sequentially HRP-conjugated anti-E-Tag MoAb and citrate buffer supplemented with 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid and 0.03% H2O2. The reaction was then stopped and absorbance was read at 405 nm with an ELISA reader. Indirect immunofluorescence (IIF) staining was performed by incubating cells (1 × 105) sequentially either with scFv fragments (1 μg per ml) and FITC-conjugated anti-E tag MoAb (1 μg per ml) or with anti-id MoAb and FITC-conjugated anti-mouse IgG Fc xenoantibodies. Each incubation was performed for 30 min at 4°C. Anti-id MoAb to unrelated MoAb were used as controls. Fluorescence was excited at 488 nm with a 15-mW Argon laser (FACScan; Becton Dickinson, Heidelberg, Germany). Fluorescence intensity of 5000 cells per probe was monitored. The double determinant immunoassay to measure IL-2 content in medium was performed in microtiter immuno plates (Nunc) coated with anti-IL-2 MoAb (2 μg PBS per ml). Supernatants from lymphocyte cultures (100 μl per well) were added to plates in triplicate. At the end of a 4 h incubation at room temperature, plates were washed and incubated for 1 h at room temperature with 100 μl per well of a biotinylated rat anti-mouse IL-2 MoAb solution (1 μg PBS per ml). Plates were then washed five times with PBS and incubated for 1 h at room temperature with avidin-conjugated peroxidase. Reaction was developed in presence of 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid in citrate buffer containing 0.03% H2O2. The absorbance at 405 nm was determined with a microtiter plate ELISA reader. Results are expressed as OD units. The cDNA coding for the chimeric scFv763.74-γR was constructed by introducing the Xbal and the BamHI restriction sites in the flanking regions of the 763.74 scFv cDNA and by ligation into the pRSV-γ vector. The scFv763.74 DNA was amplified from RNA of 763.74 hybridoma cells by reverse transcription–polymerase chain reaction using the VH54 and E-tag/VL34 oligonucleotides as primers. The latter primers introduce a Xbal site and a BamHI site, respectively. After digestion with BamHI/XbaI, the scFv763.74 DNA was ligated in frame to the γ-chain cDNA resulting in a DNA coding for scFv763.74-γ chimeric receptor. The DNA sequences were determined by the chain termination method (Sanger et al., 1977Sanger F. Nicklen S. Coulson A. DNA sequencing with chain-terminating inhibitors.Proc Natl Acad Sci USA. 1977; 74: 5463-5467Crossref PubMed Scopus (52610) Google Scholar) using the ABI 100 Model 377 automatic sequencing system. The MD45 hybridoma cells were transfected by single pulse electroporation (250 V, 2400 μF; BioRad, Munich, Germany) with 100 μg of the recombinant DNA. Cells were grown for 2 wk in the presence of G418 (geneticin, 2 mg per ml; Sigma). Subsequently, transfected cells were cloned by limiting dilution at 0.3 cells per well. Growing cells were cultured in medium RPMI 1640 supplemented with 10% fetal bovine serum and geneticin (2 mg per ml). Cultures were subcultured when the cell density was higher than 1 × 106 per ml. Target cells (3 × 106 cells) were labeled with 150 μCi of 51Cr (Amersham Buchler, Braunschweig, Germany) for 1 h at 37°C. Following three washings with PBS, 51Cr-labeled target cells (5 × 103 cells) were incubated for 16 h at 37°C with effector cells at the ratio of 20:1, 10:1, 1:1, and 0.1:1 in a total volume of 200 μl. Supernatant (100 μl) was then harvested by centrifugation and assayed for radioactivity by γ-spectrophotometry. Specific cytotoxicity (%) was calculated by the following formula: % cytotoxicity = (experimental cpm – spontaneous cpm) × 100/(total cpm – spontaneous cpm). Maximum and spontaneous release were measured by counting the radioactivity in medium harvested from a suspension of radiolabeled cells treated with 1% Triton X-100 (Sigma) and of radiolabeled target cells, respectively. The 763.74 scFv fragment was derived from the anti-HMW-MAA MoAb 763.74 by ligation of the VH and VL DNA fragments via a peptide coding linker as described in Materials and Methods. The VH region nucleotide sequence of the scFv763.74 is identical to that of the parental MoAb, whereas the VL region nucleotide sequence of the scFv763.74 is different at two positions without amino acid change (Figure 1). The scFv763.74 DNA (750 bp) was inserted into phagemid vector pCANTAB5E in frame with the M13 gene 3 and the Etag DNA. Phages displaying the scFv763.74-gp3 fusion protein at their surface were enriched by panning on the anti-id MoAb MK2–23, which had been elicited with MoAb 763.74. The scFv763.74 is approximately 34 kDa in size under reducing conditions as detected by western blot analysis using anti-E tag MoAb (Figure 2).Figure 2Immunoblot analysis of scFv763.74.E. coli HB2151 bacteria were transfected with scFv763.74 DNA expression construct that contains an E-tag fused to the 3′ end of the scFv. Proteins from periplasmic extracts of transfected (lanes 1 and 2) and nontransfected (lane 3) E. coli cells were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane. The scFv763.74 protein was detected by anti-E-tag MoAb.View Large Image Figure ViewerDownload (PPT) The scFv763.74 retained the specificity of the parental MoAb 763.74 as it displayed specific reactivity in ELISA with a protein extract from the HMW-MAA bearing SK-MEL-5 melanoma cells and with the anti-id MoAb MK2–23. In addition, scFv763.74 stained SK-MEL-5 cells specifically in IIF (Figure 3). In competition experiments the parental MoAb 763.74 inhibited binding of scFv fragments to anti-id MoAb MK2–23 (Figure 4). As expected, the parental MoAb 763.74 and scFv763.74 compete in binding to melanoma cells Colo 38 (Figure 5). Two lines of evidence indicate that the reciprocal inhibitions are specific. First, parental MoAb 763.74 and scFv763.74 did not inhibit the binding to Colo 38 cells of the unrelated scFv W34 and of anti-HMW-MAA MoAb 225.28, respectively. Second, the binding to Colo 38 cells of parental MoAb 763.74 and of scFv763.74 was not affected by scFv W34 and by anti-HMW-MAA TP41.2, respectively. It is noteworthy that MoAb 225.28 and TP41.2 recognize antigenic determinants spatially distant from that recognized by MoAb 763.74 (Wilson et al., 1981Wilson B.S. Imai K. Natali P.G. Ferrone S. Distribution and molecular characterization of a cell-surface and a cytoplasmic antigen detectable in human melanoma cells with monoclonal antibodies.Int J Cancer. 1981; 28: 293-300Crossref PubMed Scopus (227) Google Scholar).Figure 4Mapping of the idioto
Referência(s)