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T cell activation by monoclonal antibodies bound to tumor cells by a cell surface displayed single-chain antibody
Journal of Immunological Methods 224 Ž1999. 151–160
T cell activation by monoclonal antibodies bound to tumor cells by a cell surface displayed single-chain antibody Hans-Jurgen Rode a , Ulrich Moebius b, Melvyn Little ¨
a,)
a
Recombinant Antibody Group, Diagnostics and Experimental Therapy Programme, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany b Tumor Gene Therapy Group, Diagnostics and Experimental Therapy Programme, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany Received 27 November 1998; received in revised form 19 January 1999; accepted 25 January 1999
Abstract Tumor cells often lack the costimulatory molecules necessary for T cell activation. However, the transformation of cells with more than one stimulatory molecule is a difficult procedure. We therefore developed a retroviral vector for the expression of a cell membrane anchored single-chain antibody fragment ŽscFv. directed against the hapten 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one ŽphOx.. Proteins and peptides can be readily modified with this hapten, thus, enabling them to be bound to cells with the cell surface displayed anti-phOx scFv. To test combinations of surface-bound stimulatory molecules on T cell activation, SK-Mel63 human melanoma cells expressing the membrane anchored anti-phOx scFv were incubated with phOx-labeled mAbs against CD3, CD28 and CD5. Cells presenting a given mixture of modified anti-CD3 and anti-CD28 molecules stimulated T cell activation better than any single antibody and a given mixture of anti-CD3, anti-CD28 and anti-CD5 provided a stimulatory response higher than the best double combination. However, the relative concentrations are very important and must be carefully chosen. Concentrations of antibodies giving good T cell responses when used alone can block synergistic effects. q 1999 Elsevier Science B.V. All rights reserved. Keywords: T cell activation; Costimulatory molecules; Single-chain antibodies; Cell-surface display
1. Introduction A resting T cell can be activated or inactivated and even deleted after recognising a foreign peptide on an antigen presenting cell depending on the presence or absence of accompanying interactions )
Corresponding author. German Cancer Research Center, Department D0500, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. Tel.: q49-6221-42-3450; Fax: q49-6221-42-3462; E-mail: [email protected]
ŽMueller et al., 1989.. After binding, the ab- or gd-heterodimers of the T cell receptor and the associated CD3 complex send a so-called ‘first signal’ into the cell interior ŽWeiss, 1993.. This signal alone is not sufficient to induce the proliferation and differentiation of T cells according to the ‘two signal hypothesis’ ŽSchwartz, 1990.. A second costimulatory signal is necessary for their full activation, otherwise they become anergic. Many of the costimulatory signals are mediated by molecules that are involved in cell adhesion such as the lymphocyte
0022-1759r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 Ž 9 9 . 0 0 0 1 8 - 6
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function associated antigens ŽLFA. and the intercellular adhesion molecules ŽICAM.. For example, CD11ar18 ŽLFA-1. and CD58 ŽLFA-3. on B cells bind to CD54 ŽICAM-1. and CD2 on T cells, respectively ŽClark and Ledbetter, 1994.. Some of the strongest costimulatory signals are provided by CD80 ŽB7.1. and CD86 ŽB7.2. that both bind to the receptors CD28 and CTLA-4 on T cells ŽYokochi et al., 1982; Freeman et al., 1989; Azuma et al., 1993; Freeman et al., 1993; Linsley and Ledbetter, 1993.. The expression of these costimulatory signals in tumor cells expressing tumor associated antigens can render them immunogenic. A mouse melanoma cell line transformed with CD80 induced a specific T cell mediated immune reaction after being transferred back into a mouse ŽChen et al., 1992; Baskar et al., 1993; Townsend and Allison, 1993.. Not only were the CD80-transformed tumor cells destroyed, but also the parental non-transformed tumor cells were specifically eliminated. Therefore, providing costimulatory molecules for the activation of specific immune responses would seem to be a promising strategy for the treatment of cancer patients. Little is known about the effect of expressing two or more signals on tumor cells deficient in costimulatory molecules, since the transformation of cells with more than one gene and the subsequent selection of the best transformants is a rather time-consuming procedure. However, we recently published a method that would allow any mixture of proteins to be rapidly presented on the cell surface ŽRode et al., 1996.. In this procedure, tumor cells are transformed only once with a retroviral expression vector containing the gene for a single-chain antibody fragment ŽscFv. directed against the small hapten 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one ŽphOx.. The gene contains a signal peptide for protein export and a hydrophobic membrane binding domain to anchor it to the cell surface. Proteins and peptides can then be easily bound to this scFv, since their free amino and sulfhydryl groups can be readily modified with the phOx hapten. In the present study, we investigated the effects of providing a human melanoma cell line with a first signal Žanti-CD3 mAb. combined with costimulatory molecules on T cell proliferation. In addition to an anti-CD28 mAb, we tested the effect of an anti-CD5
mAb that has been shown to promote T cell activation ŽCeuppens and Baroja, 1986; Van de Velde et al., 1991; Luo et al., 1992.. We were able to show that the simultaneous presentation of three antibodies on the surface of the tumor cells had a synergistic effect on the proliferation of T cells, but only at certain relative concentrations one to another.
2. Materials and methods 2.1. Vector construction To construct the retroviral vector pMESV-hB7.1 containing cDNA coding for the costimulatory molecule CD80 ŽFreeman et al., 1989., we excised the CD80 DNA from the plasmid pcEXV-hB7 ŽNorton et al., 1992. using the EcoRI restriction enzyme. The CD80 DNA was then cloned into the EcoRI site of the retroviral vector p50-M-x-neo Žkindly provided by Dr. C. Stocking, University Hospital Eppendorf, Hamburg, Germany., which is based on the murine embryonic stem cell virus ŽMESV. ŽBaum et al., 1995. containing the long terminal repeats ŽLTRs. of the myeloproliferative sarcoma virus ŽMPSV. to obtain pMESV-hB7.1. For expression of the membrane anchored antiphOx single-chain Fv, the retroviral vector pMESVSP-a phOx-Ex-TM was constructed. DNA coding for the complete transmembrane domain ŽTM. of Fc-gReceptor II ŽFc-g-RII. and part of the extracellular domain ŽEX. was amplified by PCR from the vector pcDL-SRa 296-FcgRIIct-ŽStuart et al., 1987. using the oligonucleotides P1 Ž5X-CAAGGATCCGGCACCTTCTCCATCCCACAA GCAAACC-3X . and P2 Ž5X - GCGA ATTCCTCGAGTTACCTGCAGTAGATCAAGGCCACTAC-3X . that introduce the BamHI and EcoRI sites Žunderlined., respectively, for cloning. A total of 10 ng of template DNA was mixed in a standard PCR Ž50 ml final volume. containing 10 pmol of each oligonucleotide, 200 mM dNTP and 2 units of VentR DNA polymerase ŽNew England Biolabs, SchwalbachrTaunus, Germany. in Vent buffer. All the PCRs were carried out with the MiniCyclerTM ŽMJ Research, Watertown, MA, USA.. The samples were denatured for 3 min at 958C followed by 20 PCR cycles of 30 s denatura-
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tion at 958C, 1 min annealing at 608C and 1 min extension at 758C. The PCR ŽEX-TM. products were purified with the QIAquick PCR Purification Kit ŽQiagen, Hilden, Germany.. The gene SP-a phOx coding for the signal sequence of the interleukin-6receptor ŽSP. and the anti-phOx single-chain Fv gene Ž a phOx. was excised from the vector pLOPP2-phOx ŽRode et al., 1996. with the restriction enzymes EcoRI and BamHI and ligated with the purified EX-TM PCR product of Fc-g-RII. The SP-a phOxEx-TM gene was purified from a 1% agarose gel. To increase the amount of ligation product, a PCR was carried out using the primer P3 Ž5X-GAGAATTCCTCGAGCCATGTGGCCGTCGGCT-3X . and P2 under conditions as described above. The amplified SP-a phOx-EX-TM gene was purified with the QIAquick PCR Purification Kit, digested with EcoRI, again purified and cloned into the EcoRI site of the retroviral vector p50-M-x-neo. A diagram of the final expression vector pMESV-SP-a phOx-Ex-TM is shown in Fig. 1. 2.2. Cell culture The melanoma cell line SK-Mel63 Žkindly supplied by Dr. A. Knuth, Nordwest-krankenhaus, FrankfurtrMain, Germany. was cultured at 378C in Dulbecco’s modified Eagle medium ŽDMEM. ŽGibco
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BRL, Eggenstein, Germany. containing 10% fetal calf serum ŽSeromed, Berlin, Germany., penicillin Ž100 Urml.rstreptomycin Ž100 mgrml. ŽSeromed. and 2 mM L-glutamine ŽSeromed.. T lymphocytes were purified from peripheral blood mononuclear cells ŽPBMC, prepared by density centrifugation. as described ŽTraversari et al., 1992.. SK-Mel63 cells were transfected with 20 mg of the retroviral vectors pMESV-hB7.1 or pMESV-SP-aphOx-EX-TM by calcium phosphate precipitation ŽGraham and Van der Eb, 1973.. Fresh medium was supplied after 16 h. Three days later the cells were selected with medium containing 1 mgrml geneticin ŽG-418, Gibco. to establish stable cell lines. 2.3. Coupling of phOx to proteins Purified mouse monoclonal IgGs, anti-CD3, antiCD5 and anti-CD28 were coupled with the hapten 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one ŽphOx; Sigma–Aldrich, Deisenhofen, Germany. as already described ŽRode et al., 1996.. The phOxhapten was coupled at a molar excess of 2.5, 5, 10, 20 and 40 to the anti-CD3 and anti-CD28 and with a molar excess of 10, 25, 40 and 50 to anti-CD5. The anti-CD3 OKT3 Žhybridoma cell line, derived from ATCC, Catalog No. CRL 8001. and the anti-CD28
Fig. 1. Schematic representation of retroviral vector pMESV-SP-a phOx-Ex-TM. Abbreviations: LTR, long terminal repeat of myeloproliferative sarcoma virus; neoR, neomycin resistence gene; s.d., splice donor site; s.a., splice acceptor site; SP, signal peptide of interleukin-6 receptor ŽIL-6-R.; EX, extracellular domain of Fc-g-Receptor II ŽFc-g-RII.; TM, transmembrane domain of Fc-g-RII; VH and VL , heavy and light chain variable domains; y, packaging signal; EcoRI, restriction enzyme site.
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15E8 ŽVan Lier et al., 1988. were produced and purified by Dr. Moldenhauer ŽDKFZ, Heidelberg, Germany.. The anti-CD5 Ž9H8. was supplied by Dr. S. Meuer.
shown represent T cell proliferation with subtracted background.
3. Results 2.4. Flow cytometric analysis Cells were detached from tissue culture plates using 0.25% EDTA and washed in PBS. They were then incubated with 1 mgrml mouse IgG or phOxlabeled mouse IgG in a volume of 50 ml for 15 min at 48C, washed three times with PBS and incubated with 5 mgrml FITC-conjugated goat anti-mouse IgG ŽDianova, Hamburg, Germany. for 10 min at 48C. The following antibodies were used: W6r32 ŽantiMHC I, ATCC, Rockville, MD, USA., 12G6 ŽantiMHC II, Dr. S. Meuer, Heidelberg., mAb 104 ŽantiCD80, Pharmingen, Hamburg, Germany., and antiCD3, anti-CD28, anti-CD5 and their respective phOx-coupled derivatives. The fluorescence intensity was determined using a FACScan analyser with Lysis IITM software ŽBecton and Dickinson, Mountain View, CA, USA.. Dead cells stained with 1 mgrml propidium iodide ŽSigma–Aldrich. were excluded.
3.1. Induction of T cell actiÕation after expression of human CD80 in the human melanoma cell line SKMel63 As a positive control for T cell activation, we used the human melanoma cell line SK-Mel63 transfected with the retroviral vector pMESV-hB7.1 containing cDNA coding for the human CD80 gene. Untransfected SK-Mel63 cells expressed MHC I and MHC II, which should be recognised as alloantigens by T lymphocytes, but did not express CD80. The transfected CD80 positive SK-Mel63-pMESV-hB7.1 cells were analysed for their ability to activate the allogeneic peripheral blood T lymphocytes of healthy donors in vitro. They were able to induce a strong proliferation of T cells in contrast to untransfected SK-Mel63 cells Ždata not shown.. 3.2. Cell surface binding of mAbs modified with increasing amounts of phOx
2.5. Proliferation assay Ten thousand irradiated Ž200 Gy. SK-Mel63 cells and their transfected variants were incubated with 10 5 per well peripheral blood T lymphocytes from allogeneic healthy donors in 96-well flat bottomed microtiter plates ŽNUNC. for 5 days. After the addition of 37 kBq 3H-thymidine Ž74.0 GBqrmmol, New England Nuclear, Boston, MA, USA. per well, the cells were incubated for a further 18 h and 3 H-thymidine uptake was measured by liquid scintillation counting and expressed as mean cpm of triplicate wells. Anti-CD3, anti-CD5, and anti-CD28 mAbs conjugated at 20-, 40-, and 10-fold excess with phOx, respectively, were used to provide additional signals for T cell activation. phOx-conjugated mAbs were used at 0.1 mgrml or 1.0 mgrml ŽantiCD3, anti-CD5. and 0.2 mgrml or 2.0 mgrml ŽantiCD28., respectively. T cells were incubated in parallel with untransfected SK-Mel63 cells together with phOx-conjugated mAbs to determine the background proliferation induced by soluble mAbs. The results
To express a single-chain antibody directed against the hapten phOx Ž4-ethoxymethylene-2-phenyl-2oxazoline-5-one. on the surface of tumor cells, we constructed the retroviral vector pMESV-SP-a phOxEx-TM ŽFig. 1.. The signal sequence of the IL-6-receptor was fused to the 5X-end for directing the scFv to the cell surface and a transmembrane domain of the Fc-g-RII-receptor was added to the 3X-end for anchoring the scFv to the cell membrane. In contrast to a previous construct that employed the IL-6-receptor transmembrane domain without a spacer sequence between the scFv and the cell membrane ŽRode et al., 1996., we used the Fc-g-RII-receptor transmembrane domain with a 42 amino acid extension. This receptor does not appear to be shed to any significant extent and the spacer provides more flexibility, which was reflected in better binding of phOx-labeled magnetic beads Ždata not shown.. After transfecting the human melanoma cell line SK-Mel63 with pMESV-SP-a phOx-Ex-TM, we first investigated the optimal amount of modification with
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the phOx-hapten for binding the mAbs used in this study to the membrane anchored anti-phOx singlechain antibody ŽscFv.. The anti-CD3 and anti-CD28 mAbs were coupled with a 2.5-, 5-, 10-, 20- and 40-fold molar excess of phOx to antibody and the anti-CD5 mAb with a 10-, 25-, 40- and 50-fold molar excess of phOx-hapten to antibody. Maximum binding of the anti-CD3-phOx mAb was reached at a molar phOx-excess of 20-fold, for the anti-CD28phOx mAb at a 40-fold molar excess and for the anti-CD5-phOx mAb at a 10-fold molar excess ŽFig. 2D, F and H.. The phOx-modified mAb did not bind non-specifically to untransfected cells ŽFig. 2C, E and G. and unmodified mAbs did not bind to cells expressing the anti-phOx scFv ŽFig. 2B.. The binding of the modified antibodies to the transformed cells seems to be very strong, since cells that had been loaded with the modified anti-CD28-phOxŽ40. mAb Ž40 molar excess of phOx. and then washed showed no significant reduction in their FACScan signal after a 24-h incubation at 48C Ždata not shown.. This indicates that the antibody is probably bound by more than one phOx hapten to the cell surface, thus, increasing the avidity of binding. 3.3. Influence of phOx-coupling on binding of antibodies to their antigens The modification of antibodies with the phOx hapten might disturb the structure of the antigen binding site. We therefore tested the antigen binding capacity of phOx conjugated antibodies to T cells by FACScan. The anti-CD3 mAb coupled with a 2.5-, 5-, 10- and 20-fold molar excess of phOx bound to CD3 q Jurkat cells in amounts similar to that of the original unmodified mAb ŽFig. 3B,C.. In contrast, a 40-fold molar excess of the phOx hapten resulted in a reduced amount of binding ŽFig. 3D.. The binding of the anti-CD28 mAb to CD28 q HPB-ALL cells was hardly affected by a molar phOx excess of up to 40-fold ŽFig. 3G,H. as compared to the amount bound of the original unmodified mAb ŽFig. 3F.. Similarly, there was no significant reduction in binding of the anti-CD5 mAb to CD5 q Jurkat cells after incubation with a 10-, 25-, 40- and 50-fold molar excess of the phOx hapten ŽFig. 3J, K, L.. Results presented in Figs. 2 and 3 imply that anti-CD3,
Fig. 2. Binding of phOx-coupled mAbs to untransfected SK-Mel63 cells or cells expressing the membrane anchored anti-phOx scFv ŽSK-Mel63-pMESV-SP-a phOx-Ex-TM.. Cells were incubated with phOx-labeled CD3, CD28, CD5 mAbs conjugated at 20-, 40and 10-fold excess of phOx-hapten to antibody, respectively, ŽC–H. or medium ŽA, B.. Afterwards cells were washed and stained with anti-mouse FITC-labeled IgG. Fluorescence was determined by flow cytometry.
anti-CD5 and anti-CD28 mAbs coupled at 20-, 10and 40-fold molar excess, respectively, are optimal for stimulation of T cells.
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Fig. 3. Influence of phOx-labeling on antigen binding. T cells ŽJurkat or HPB-ALL. were incubated with 1 mgrml anti-CD3, anti-CD28 or anti-CD5 antibodies ŽB, F and J. or their derivatives labeled at 10-, 20-, 40- or 50-fold excess of phOx-hapten ŽC, D, G, H, K and L. or medium alone ŽA, E and I.. Following washing and staining with anti-mouse FITC-labeled IgG, fluorescence was determined by flow cytometry.
3.4. Effect of modified anti-CD3, anti-CD28 and anti-CD5 mAbs on T cell proliferation The effect of modified anti-CD3, anti-CD28 and anti-CD5 mAbs on the proliferation of allogenic T lymphocytes was compared at two different concentrations. The modified anti-CD5-phOxŽ10. mAb Ž10 = phOx. had no significant effect on the thymidine
uptake of the T cells at comparable concentrations of 0.1 and 1.0 mgrml ŽFig. 4, lanes B and C.. The anti-CD3-phOxŽ20. mAb Ž20 = phOx. was added to SK-Mel63 cells expressing the anti-phOx scFv in concentrations of 0.1 or 1.0 mgrml. After incubation with T cells, a significant uptake of thymidine was observed at both concentrations, whereby the best stimulation was obtained at the lower concentration
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Fig. 4. Induction of T cell proliferation by SK-Mel63-pMESV-SP-a phOx-Ex-TM cells expressing the cell surface displayed anti-phOx scFv in combinations with phOx-conjugated CD3, CD5, and CD28 mAbs mAbs. A total of 10 5 T cells was incubated with 10 4 Sk-Mel63 melanoma cells transfected with the gene for the anti-phOx scFv and two different concentrations of phOx-conjugated CD3 and CD5 Ž0.1 and 1.0 mgrml. and CD28 Ž0.2 and 2.0 mgrml. mAbs, respectively. Stimulation was performed in the presence of one Žlanes B–G., two Žlanes H–S. or three Žlanes T–V. mAbs. T cell proliferation was determined by thymidine incorporation on day 5.
of the modified anti-CD3 mAb ŽFig. 4, lanes D and E.. The unmodified anti-CD3 mAb showed no significant stimulation of T cell proliferation Ždata not shown.. Similar incubations using 0.2 mgrml of the modified anti-CD28-phOxŽ40. mAb Ž40 = phOx. gave no stimulation of thymidine uptake, whereas a concentration of 2.0 mgrml induced a significant T cell response ŽFig. 4F and G., which was significantly larger than that obtained using the anti-CD3phOxŽ20. mAb ŽFig. 4, lane D.. The effect was similar whether the cells loaded with the anti-CD28phOxŽ40. mAb were washed or not Ždata not shown.. This supports the data mentioned above on the apparent tight binding of the modified mAbs to the cell surface. 3.5. Synergistic effect of stimulatory mAbs on T cell proliferation The combined effect of two and three stimulatory antibodies, respectively, on T cell proliferation was
investigated. We tested all combinations of two simultaneously incubated phOx-coupled antibodies in both concentrations ŽFig. 4, lanes H–S.. We found that the strongest stimulation of T cell proliferation was achieved using 0.1 mgrml or 1 mgrml of the anti-CD3-phOxŽ20. mAb together with 0.2 mgrml of the anti-CD28-phOxŽ40. antibody ŽFig. 4, lanes S and R., although this concentration of anti-CD28phOxŽ40. mAb alone had very little effect on thymidine uptake ŽFig. 4, lane F.. However, in combination with the anti-CD3-phOxŽ20. antibody, the thymidine uptake was higher than that achieved with the anti-CD3-phOxŽ20. alone ŽFig. 4, lanes D and E.. The stimulation seen using 2 mgrml of the anti-CD28-phOxŽ40. mAb was still appreciable but significantly reduced ŽFig. 4, lanes P and Q., even lower than the stimulation obtained using antiCD28-phOxŽ40. alone ŽFig. 4, lane G.. The antiCD5-phOxŽ10. mAb showed no significant stimulatory effects on T cell proliferation at concentrations
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of either 0.1 or 1 mgrml in combination with either the anti-CD3-phOxŽ20. or anti-CD28-phOxŽ40. mAbs ŽFig. 4, lanes H–O.. Indeed, at a concentration of 1 mgrml anti-CD5-phOxŽ10. mAb together with 2 mgrml anti-CD28-phOxŽ40. mAb, the thymidine uptake was considerably lower than that obtained for the anti-CD28-phOxŽ40. mAb alone ŽFig. 4, lanes I with G.. A similar reduction in T cell activation was seen combining the anti-CD5-phOx Ž10. mAb at 1 mgrml with the anti-CD3-phOxŽ20. mAb at 0.1 mgrml ŽFig. 4, lanes L and D.. These effects are most likely due to competition for the available binding sites on the cell surface represented by the number of anti-phOx scFv. The best stimulation of T cell proliferation was achieved using a combination of the three modified antibodies at concentrations of 0.1 mgrml for antiCD3 and anti-CD5 and 0.2 mgrml for anti-CD28 ŽFig. 4, lane V.. The anti-CD5-phOxŽ10., which alone or in combination with anti-CD3-phOxŽ20. or anti-CD28-phOxŽ40. did not result in any significant increase in T cell proliferation ŽFig. 4, lanes H–O., induced a marked response when applied together with both the anti-CD3-phOxŽ20. and anti-CD28phOxŽ40. ŽFig. 4, lane V.. The synergistic effect of the anti-CD5-phOxŽ10. mAb at 0.1 mgrml was also observed using anti-CD28-phOxŽ40. mAb at 2 mgrml ŽFig. 4, lanes U and Q.. At the higher concentration of 1 mgrml for the anti-CD5-phOxŽ10. mAb, a slight reduction of the T cell activation was observed in the triple combination ŽFig. 4, lanes T and P.. This effect was also noted for combinations with only one other mAb Žsee above. and is again probably due to competition for the available binding sites. These results demonstrate that the T cell response can be amplified by the synergistic effect of three different surface-bound antibodies, with the effect highly dependent on their relative concentrations.
4. Discussion There is increasing evidence that tumor cells express tumor associated antigens that can stimulate an immune response under appropriate conditions. These antigens, in the form of processed peptides bound to MHC molecules, can be derived from viruses, mu-
tated genes or proteins that are overexpressed in the tumor cells ŽVan der Bruggen et al., 1991; Altmann et al., 1992; Gedde-Dahl et al., 1993.. Several melanoma-associated peptides have been characterized in detail ŽTraversari et al., 1992; Brichard et al., 1993; Kawakami et al., 1994.. Twenty-four overexpressed antigens from the tumors of patients with melanomas, renal carcinomas, astrocytomas and Hodgkin lymphomas were found to have elicited a B cell response ŽSahin et al., 1995.. Vaccination of tumor patients with tumor associated peptides and proteins may eventually provide a useful method of inducing an immune response in an adjuvant setting. However, until more is known about the expression and range of antigens present in the largely heterogeneous tumors, the use of tumor cells has the advantage that the products of multiple altered genes can be used as a vaccine even though they have not been identified. In one approach, costimulatory molecules are being used for T lymphocyte activation in order to strengthen the relatively weak immunogenicity of the tumor cells. For example, the application of genetically modified tumor cells, either autologous or allogeneic with shared HLA-alleles, has been initiated in clinical trials with patients suffering from malignant melanoma and renal cell cancer ŽHabicht et al., 1995; Human Gene MarkerrTherapy Clinical Protocols, 1996.. In contrast, however, to the successful induction of a cytotoxic T cell response by a human melanoma cell line transfected with CD80 ŽGraham and Van der Eb, 1973., the transfection of two colorectal cell lines with CD80 did not result in T cell activation, in spite of an adequate expression of class I MHC molecules ŽHabicht et al., 1995.. One solution to this problem might be to express other or additional costimulatory molecules on the tumor cell surface. The transfection of tumor cells with genes coding for several costimulatory molecules is time-consuming and not very efficient. We have therefore developed a method for the simultaneous expression of several proteins or peptides on the cell surface requiring only one cellular transformation with a gene coding for a cell membrane anchored scFv against the phOx antigen. Proteins and peptides can be readily modified with this antigen and then bound to the cell surface through the anti-phOx scFv ŽRode et al., 1996..
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To investigate the effect of simultaneously expressing first and second stimulatory signals bound to the surface of a human melanoma tumor cell line on the activation of T lymphocytes, we modified monoclonal antibodies against the anti-CD3, antiCD28 and anti-CD5 antigens with various molar ratios of the phOx antigen. The anti-CD28 and antiCD5 antibodies could be incubated with relatively high concentrations of the phOx antigen without a significant loss of antigen binding ability. In contrast, the OKT3 anti-CD3 showed a sharp decline in antigen binding at molar ratios of phOx to antibody higher than 20:1. One possible explanation is the presence of a free sulfhydryl group at position 100 A in the center of the third complementarity determining region of the heavy chain variable domain ŽAdair et al., 1994.. Any modifications in this region, which is crucial for antigen binding, would be expected to have a serious effect on the antibody’s affinity. This could also account for the fact that on raising the concentration of the anti-CD3-phOxŽ40. from 0.1 to 1 mgrml, the activation of T cell proliferation was significantly decreased. A population of more highly modified but less active antibodies might be preferentially bound to the tumor cell surface. Higher concentrations of antibody under saturating conditions would then result in a lower proportion of bound functional antibodies. In contrast, the anti-CD28-phOxŽ20. antibody had little effect on T cell activation at a concentration of 0.2 mgrml but a very marked effect at 2 mgrml. However, in combination with anti-CD3-phOxŽ20. or with anti-CD3-phOxŽ20. plus anti-CD5-phOxŽ10., the lower concentration of anti-CD28-phOxŽ40. yielded a significantly higher T cell activation. The most likely explanation is that due to the competition of the modified antibodies for binding to the cell membrane anchored anti-phOx scFv, only very little of the anti-CD3-phOxŽ20. mAb can be bound at the higher concentrations of the anti-CD28-phOxŽ40. mAb. Similarly, higher concentrations of the antiCD5-phOxŽ10. mAb resulted in a decreased T cell activation of either the anti-CD3-phOxŽ20. or the anti-CD28-phOxŽ40. mAbs. The highest T cell activation was achieved by a combination of all three modified antibodies at the lower of the two given concentrations. Similar ratios of these stimulatory antibodies could provide a means to induce a T cell
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response against the refractory colorectal carcinoma cells described above. We therefore intend to test this concept using such refractory model systems in future investigations.
Acknowledgements We would like to thank Dr. K. Khazaie for his help in establishing the retroviral expression system and Dr. G. Moldenhauer for providing purified polyclonal mouse IgG. We are also particularly grateful to Tobias Schurrle for carrying out T cell prolifera¨ tion assays, Dr. G. Winter and colleagues for the original VH - and VL-DNA of the clone phOx31E and Dr. C. Stocking and Dr. W. Ostertag for the retroviral vector p50-M-x-neo.
References Adair, J.R., Athwal, D.S., Bodmer, M.W., Bright, S.M., Collins, A.M., Pulito, V.L., Rao, P.E., Reedman, R., Rothermel, A.L., Xu, D., Zivin, R.A., Jolliffe, K., 1994. Humanization of the murine anti-human CD3 monoclonal antibody OKT3. Hum. Antibodies Hybridomas 5, 41. Altmann, A., Jochmus-Kudielka, I., Frank, R., Gausepohl, H., Moebius, U., Gissmann, L., Meuer, S.C., 1992. Definition of immunogenic determinants of the human papillomavirus type 16 nucleoprotein E7. Eur. J. Cancer 28, 326. Azuma, M., Ito, D., Yagita, H., Okumura, K., Phillips, J.H., Lanier, L.L., Somoza, C., 1993. B70 antigen is a second ligand for CTLA-4 and CD28. Nature 366, 76. Baskar, S., Ostrand-Rosenberg, S., Nabavi, N., Nadler, L.M., Freeman, G.J., Glimcher, L.H., 1993. Constitutive expression of B7 restores immunogenicity of tumor cells expressing truncated major-histocompatibility-complex-class-II molecules. Proc. Natl. Acad. Sci. USA 90, 5687. Baum, C., Hegewisch-Becker, S., Eckert, H.G., Stocking, C., Ostertag, W., 1995. Novel retroviral vectors for efficient expression of the multidrug resistance Ž mdr-1. gene in early hematopoietic cells. J. Virol. 69, 7541. Brichard, V., Van Pel, A., Wolfel, T., Wolfel, C., De plaen, E., ¨ ¨ Lethe, B., Coulie, P., Boon, T., 1993. The tyrosinase gene codes for an antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas. J. Exp. Med. 178, 489. Ceuppens, J.L., Baroja, M.L., 1986. Monoclonal antibodies to the CD5 antigen can provide the necessary second signal for activation of isolated resting T cells by solid-phase-bound OKT3. J. Immunol. 137, 1816. Chen, L., Ashe, S., Brady, W.A., Hellstrom, ¨ I., Hellstrom, ¨ K.E., Ledbetter, J.A., McGowan, P., Linsley, P.S., 1992. Costimulation of anti-tumor immunity by the B7 counter-receptor for the lymphocyte molecules CD28 and CTLA-4. Cell 71, 1093.
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Clark, E.A., Ledbetter, J.A., 1994. How B and T cells talk to each other. Nature 367, 425. Freeman, G.J., Freedman, A.S., Segil, J.M., Lee, G., Whitman, J.F., Nadler, L.M., 1989. B7, a new member of the Ig superfamily with unique expression on activated and neoplastic B cells. J. Immunol. 143, 2714. Freeman, G.J., Gribben, J.G., Boussiotis, V.A., Ng, J.W., Restivo, V.A. Jr., Lombard, L.A., Gray, G.S., Nadler, L.M., 1993. Cloning of B7-2: a CTLA-4 counter-receptor that costimulates human T cell proliferation. Science 262, 909. Gedde-Dahl, T., Fossum, B., Eriksen, J.A., Thorsby, E., Gaudernack, G., 1993. T cell clones specific for p21 ras-derived peptides: characterization of their fine specificity and HLA restriction. Eur. J. Immunol. 23, 754. Graham, F.L., Van der Eb, A.J., 1973. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456. Habicht, A., Lindauer, M., Galmbacher, P., Rudy, W., Gebert, J., Schackert, H.K., Meuer, S.C., Moebius, U., 1995. Development of immunogenic colorectal cancer cell lines for vaccination: expression of CD80 ŽB7.1. is not sufficient to restore impaired primary T cell activation in vitro. Eur. J. Cancer A 31, 2396. Human Gene MarkerrTherapy Clinical Protocols, 1996. Human Gene Therapy 7, p. 567. Kawakami, Y., Eliyahu, S., Delgado, C.H., Robbins, P.F., Rivoltini, L., Topalian, S.L., Miki, T., Rosenberg, S.A., 1994. Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor. Proc. Natl. Acad. Sci. USA 91, 3515. Linsley, P.S., Ledbetter, J.A., 1993. The role of the CD28 receptor during T cell responses to antigen. Annu. Rev. Immunol. 11, 191. Luo, W., Van de Velde, H., Von Hoegen, I., Parnes, J.R., Thielemans, K., 1992. Ly-1 ŽCD5., a membrane glycoprotein of mouse T lymphocytes and a subset of B cells, is a natural ligand of the B cell surface protein Lyb-2 ŽCD72.. J. Immunol. 148, 1630. Mueller, D.L., Jenkins, M.K., Schwartz, R.H., 1989. Clonal expansion versus functional clonal inactivation: a costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy. Ann. Rev. Immunol. 7, 445. Norton, S.D., Zuckerman, L., Urdahl, K.B., Shefner, R., Miller, J., Jenkins, M.K., 1992. The CD28 ligand, B7, enhances IL-2
production by providing a costimulatory signal to T cells. J. Immunol. 149, 1556. Rode, H.J., Little, M., Fuchs, P., Dorsam, H., Schooltink, H., de ¨ Ines, S., Breitling, F., 1996. Cell surface display of ´ C., Dubel, ¨ a single-chain antibody for attaching polypeptides. BioTechniques 21, 650. ¨ Schmitt, H., Cochlovius, B., Johannes, T., Sahin, U., Tureci, O., ¨ Schmits, R., Stenner, F., Luo, G., Schobert, I., Pfreundschuh, M., 1995. Human neoplasms elicit multiple specific immune responses in the autologous host. Proc. Natl. Acad. Sci. USA 92, 11810. Schwartz, R.H., 1990. A cell culture model for T lymphocyte clonal anergy. Science 248, 1349. Stuart, S.G., Trounstine, M.L., Vaux, D.J., Koch, T., Martens, C.L., Mellman, I., Moore, K.W., 1987. Isolation and expression of cDNA clones encoding a human receptor for IgG ŽFc gRII.. J. Exp. Med. 166, 1668. Townsend, S.E., Allison, J.P., 1993. Tumor rejection after direct costimulation of CD8q T cells by B7-transfected melanoma cells. Science 259, 368. Traversari, C., Van-der-Bruggen, P., Luescher, I.F., Lurquin, C., Chomez, P., Van-Pel, A., De-Plaen, E., Amar-Costesec, A., Boon, T., 1992. A nonapeptide encoded by human gene MAGE-1 is recognized on HLA-A1 by cytolytic T lymphocytes directed against tumor antigen MZ2-E. J. Exp. Med. 176, 1453. Van der Bruggen, P., Traversari, C., Chomez, P., Lurquin, C., De Plaen, E., Van den Eynde, B., Knuth, A., Boon, T., 1991. A gene encoding an antigen recognized by cytotoxic T lymphocytes on a human melanoma. Science 254, 1643. Van de Velde, H., Von Hoegen, I., Luo, W., Parnes, J.R., Thielemans, K., 1991. The B cell surface protein CD72rLyb-2 is the ligand for CD5. Nature 351, 662. Van Lier, R.A., Brouwer, M., Aarden, L.A., 1988. Signals involved in T cell activation. T cell proliferation induced through the synergistic action of anti-CD28 and anti-CD2 monoclonal antibodies. Eur. J. Immunol. 18, 167. Weiss, A., 1993. T cell antigen receptor signal transduction: a tale of tails and cytoplasmic protein-tyrosine kinases. Cell 73, 209. Yokochi, T., Holly, R.D., Clark, E.A., 1982. B lymphoblast antigen ŽBB-1. expressed on Epstein–Barr virus-activated B cell blasts, B lymphoblastoid cell lines, and Burkitt’s lymphomas. J. Immunol. 128, 823.
T cell activation by monoclonal antibodies bound to tumor cells by a cell surface displayed single-chain antibody Hans-Jurgen Rode a , Ulrich Moebius b, Melvyn Little ¨
a,)
a
Recombinant Antibody Group, Diagnostics and Experimental Therapy Programme, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany b Tumor Gene Therapy Group, Diagnostics and Experimental Therapy Programme, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany Received 27 November 1998; received in revised form 19 January 1999; accepted 25 January 1999
Abstract Tumor cells often lack the costimulatory molecules necessary for T cell activation. However, the transformation of cells with more than one stimulatory molecule is a difficult procedure. We therefore developed a retroviral vector for the expression of a cell membrane anchored single-chain antibody fragment ŽscFv. directed against the hapten 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one ŽphOx.. Proteins and peptides can be readily modified with this hapten, thus, enabling them to be bound to cells with the cell surface displayed anti-phOx scFv. To test combinations of surface-bound stimulatory molecules on T cell activation, SK-Mel63 human melanoma cells expressing the membrane anchored anti-phOx scFv were incubated with phOx-labeled mAbs against CD3, CD28 and CD5. Cells presenting a given mixture of modified anti-CD3 and anti-CD28 molecules stimulated T cell activation better than any single antibody and a given mixture of anti-CD3, anti-CD28 and anti-CD5 provided a stimulatory response higher than the best double combination. However, the relative concentrations are very important and must be carefully chosen. Concentrations of antibodies giving good T cell responses when used alone can block synergistic effects. q 1999 Elsevier Science B.V. All rights reserved. Keywords: T cell activation; Costimulatory molecules; Single-chain antibodies; Cell-surface display
1. Introduction A resting T cell can be activated or inactivated and even deleted after recognising a foreign peptide on an antigen presenting cell depending on the presence or absence of accompanying interactions )
Corresponding author. German Cancer Research Center, Department D0500, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. Tel.: q49-6221-42-3450; Fax: q49-6221-42-3462; E-mail: [email protected]
ŽMueller et al., 1989.. After binding, the ab- or gd-heterodimers of the T cell receptor and the associated CD3 complex send a so-called ‘first signal’ into the cell interior ŽWeiss, 1993.. This signal alone is not sufficient to induce the proliferation and differentiation of T cells according to the ‘two signal hypothesis’ ŽSchwartz, 1990.. A second costimulatory signal is necessary for their full activation, otherwise they become anergic. Many of the costimulatory signals are mediated by molecules that are involved in cell adhesion such as the lymphocyte
0022-1759r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 Ž 9 9 . 0 0 0 1 8 - 6
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function associated antigens ŽLFA. and the intercellular adhesion molecules ŽICAM.. For example, CD11ar18 ŽLFA-1. and CD58 ŽLFA-3. on B cells bind to CD54 ŽICAM-1. and CD2 on T cells, respectively ŽClark and Ledbetter, 1994.. Some of the strongest costimulatory signals are provided by CD80 ŽB7.1. and CD86 ŽB7.2. that both bind to the receptors CD28 and CTLA-4 on T cells ŽYokochi et al., 1982; Freeman et al., 1989; Azuma et al., 1993; Freeman et al., 1993; Linsley and Ledbetter, 1993.. The expression of these costimulatory signals in tumor cells expressing tumor associated antigens can render them immunogenic. A mouse melanoma cell line transformed with CD80 induced a specific T cell mediated immune reaction after being transferred back into a mouse ŽChen et al., 1992; Baskar et al., 1993; Townsend and Allison, 1993.. Not only were the CD80-transformed tumor cells destroyed, but also the parental non-transformed tumor cells were specifically eliminated. Therefore, providing costimulatory molecules for the activation of specific immune responses would seem to be a promising strategy for the treatment of cancer patients. Little is known about the effect of expressing two or more signals on tumor cells deficient in costimulatory molecules, since the transformation of cells with more than one gene and the subsequent selection of the best transformants is a rather time-consuming procedure. However, we recently published a method that would allow any mixture of proteins to be rapidly presented on the cell surface ŽRode et al., 1996.. In this procedure, tumor cells are transformed only once with a retroviral expression vector containing the gene for a single-chain antibody fragment ŽscFv. directed against the small hapten 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one ŽphOx.. The gene contains a signal peptide for protein export and a hydrophobic membrane binding domain to anchor it to the cell surface. Proteins and peptides can then be easily bound to this scFv, since their free amino and sulfhydryl groups can be readily modified with the phOx hapten. In the present study, we investigated the effects of providing a human melanoma cell line with a first signal Žanti-CD3 mAb. combined with costimulatory molecules on T cell proliferation. In addition to an anti-CD28 mAb, we tested the effect of an anti-CD5
mAb that has been shown to promote T cell activation ŽCeuppens and Baroja, 1986; Van de Velde et al., 1991; Luo et al., 1992.. We were able to show that the simultaneous presentation of three antibodies on the surface of the tumor cells had a synergistic effect on the proliferation of T cells, but only at certain relative concentrations one to another.
2. Materials and methods 2.1. Vector construction To construct the retroviral vector pMESV-hB7.1 containing cDNA coding for the costimulatory molecule CD80 ŽFreeman et al., 1989., we excised the CD80 DNA from the plasmid pcEXV-hB7 ŽNorton et al., 1992. using the EcoRI restriction enzyme. The CD80 DNA was then cloned into the EcoRI site of the retroviral vector p50-M-x-neo Žkindly provided by Dr. C. Stocking, University Hospital Eppendorf, Hamburg, Germany., which is based on the murine embryonic stem cell virus ŽMESV. ŽBaum et al., 1995. containing the long terminal repeats ŽLTRs. of the myeloproliferative sarcoma virus ŽMPSV. to obtain pMESV-hB7.1. For expression of the membrane anchored antiphOx single-chain Fv, the retroviral vector pMESVSP-a phOx-Ex-TM was constructed. DNA coding for the complete transmembrane domain ŽTM. of Fc-gReceptor II ŽFc-g-RII. and part of the extracellular domain ŽEX. was amplified by PCR from the vector pcDL-SRa 296-FcgRIIct-ŽStuart et al., 1987. using the oligonucleotides P1 Ž5X-CAAGGATCCGGCACCTTCTCCATCCCACAA GCAAACC-3X . and P2 Ž5X - GCGA ATTCCTCGAGTTACCTGCAGTAGATCAAGGCCACTAC-3X . that introduce the BamHI and EcoRI sites Žunderlined., respectively, for cloning. A total of 10 ng of template DNA was mixed in a standard PCR Ž50 ml final volume. containing 10 pmol of each oligonucleotide, 200 mM dNTP and 2 units of VentR DNA polymerase ŽNew England Biolabs, SchwalbachrTaunus, Germany. in Vent buffer. All the PCRs were carried out with the MiniCyclerTM ŽMJ Research, Watertown, MA, USA.. The samples were denatured for 3 min at 958C followed by 20 PCR cycles of 30 s denatura-
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tion at 958C, 1 min annealing at 608C and 1 min extension at 758C. The PCR ŽEX-TM. products were purified with the QIAquick PCR Purification Kit ŽQiagen, Hilden, Germany.. The gene SP-a phOx coding for the signal sequence of the interleukin-6receptor ŽSP. and the anti-phOx single-chain Fv gene Ž a phOx. was excised from the vector pLOPP2-phOx ŽRode et al., 1996. with the restriction enzymes EcoRI and BamHI and ligated with the purified EX-TM PCR product of Fc-g-RII. The SP-a phOxEx-TM gene was purified from a 1% agarose gel. To increase the amount of ligation product, a PCR was carried out using the primer P3 Ž5X-GAGAATTCCTCGAGCCATGTGGCCGTCGGCT-3X . and P2 under conditions as described above. The amplified SP-a phOx-EX-TM gene was purified with the QIAquick PCR Purification Kit, digested with EcoRI, again purified and cloned into the EcoRI site of the retroviral vector p50-M-x-neo. A diagram of the final expression vector pMESV-SP-a phOx-Ex-TM is shown in Fig. 1. 2.2. Cell culture The melanoma cell line SK-Mel63 Žkindly supplied by Dr. A. Knuth, Nordwest-krankenhaus, FrankfurtrMain, Germany. was cultured at 378C in Dulbecco’s modified Eagle medium ŽDMEM. ŽGibco
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BRL, Eggenstein, Germany. containing 10% fetal calf serum ŽSeromed, Berlin, Germany., penicillin Ž100 Urml.rstreptomycin Ž100 mgrml. ŽSeromed. and 2 mM L-glutamine ŽSeromed.. T lymphocytes were purified from peripheral blood mononuclear cells ŽPBMC, prepared by density centrifugation. as described ŽTraversari et al., 1992.. SK-Mel63 cells were transfected with 20 mg of the retroviral vectors pMESV-hB7.1 or pMESV-SP-aphOx-EX-TM by calcium phosphate precipitation ŽGraham and Van der Eb, 1973.. Fresh medium was supplied after 16 h. Three days later the cells were selected with medium containing 1 mgrml geneticin ŽG-418, Gibco. to establish stable cell lines. 2.3. Coupling of phOx to proteins Purified mouse monoclonal IgGs, anti-CD3, antiCD5 and anti-CD28 were coupled with the hapten 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one ŽphOx; Sigma–Aldrich, Deisenhofen, Germany. as already described ŽRode et al., 1996.. The phOxhapten was coupled at a molar excess of 2.5, 5, 10, 20 and 40 to the anti-CD3 and anti-CD28 and with a molar excess of 10, 25, 40 and 50 to anti-CD5. The anti-CD3 OKT3 Žhybridoma cell line, derived from ATCC, Catalog No. CRL 8001. and the anti-CD28
Fig. 1. Schematic representation of retroviral vector pMESV-SP-a phOx-Ex-TM. Abbreviations: LTR, long terminal repeat of myeloproliferative sarcoma virus; neoR, neomycin resistence gene; s.d., splice donor site; s.a., splice acceptor site; SP, signal peptide of interleukin-6 receptor ŽIL-6-R.; EX, extracellular domain of Fc-g-Receptor II ŽFc-g-RII.; TM, transmembrane domain of Fc-g-RII; VH and VL , heavy and light chain variable domains; y, packaging signal; EcoRI, restriction enzyme site.
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15E8 ŽVan Lier et al., 1988. were produced and purified by Dr. Moldenhauer ŽDKFZ, Heidelberg, Germany.. The anti-CD5 Ž9H8. was supplied by Dr. S. Meuer.
shown represent T cell proliferation with subtracted background.
3. Results 2.4. Flow cytometric analysis Cells were detached from tissue culture plates using 0.25% EDTA and washed in PBS. They were then incubated with 1 mgrml mouse IgG or phOxlabeled mouse IgG in a volume of 50 ml for 15 min at 48C, washed three times with PBS and incubated with 5 mgrml FITC-conjugated goat anti-mouse IgG ŽDianova, Hamburg, Germany. for 10 min at 48C. The following antibodies were used: W6r32 ŽantiMHC I, ATCC, Rockville, MD, USA., 12G6 ŽantiMHC II, Dr. S. Meuer, Heidelberg., mAb 104 ŽantiCD80, Pharmingen, Hamburg, Germany., and antiCD3, anti-CD28, anti-CD5 and their respective phOx-coupled derivatives. The fluorescence intensity was determined using a FACScan analyser with Lysis IITM software ŽBecton and Dickinson, Mountain View, CA, USA.. Dead cells stained with 1 mgrml propidium iodide ŽSigma–Aldrich. were excluded.
3.1. Induction of T cell actiÕation after expression of human CD80 in the human melanoma cell line SKMel63 As a positive control for T cell activation, we used the human melanoma cell line SK-Mel63 transfected with the retroviral vector pMESV-hB7.1 containing cDNA coding for the human CD80 gene. Untransfected SK-Mel63 cells expressed MHC I and MHC II, which should be recognised as alloantigens by T lymphocytes, but did not express CD80. The transfected CD80 positive SK-Mel63-pMESV-hB7.1 cells were analysed for their ability to activate the allogeneic peripheral blood T lymphocytes of healthy donors in vitro. They were able to induce a strong proliferation of T cells in contrast to untransfected SK-Mel63 cells Ždata not shown.. 3.2. Cell surface binding of mAbs modified with increasing amounts of phOx
2.5. Proliferation assay Ten thousand irradiated Ž200 Gy. SK-Mel63 cells and their transfected variants were incubated with 10 5 per well peripheral blood T lymphocytes from allogeneic healthy donors in 96-well flat bottomed microtiter plates ŽNUNC. for 5 days. After the addition of 37 kBq 3H-thymidine Ž74.0 GBqrmmol, New England Nuclear, Boston, MA, USA. per well, the cells were incubated for a further 18 h and 3 H-thymidine uptake was measured by liquid scintillation counting and expressed as mean cpm of triplicate wells. Anti-CD3, anti-CD5, and anti-CD28 mAbs conjugated at 20-, 40-, and 10-fold excess with phOx, respectively, were used to provide additional signals for T cell activation. phOx-conjugated mAbs were used at 0.1 mgrml or 1.0 mgrml ŽantiCD3, anti-CD5. and 0.2 mgrml or 2.0 mgrml ŽantiCD28., respectively. T cells were incubated in parallel with untransfected SK-Mel63 cells together with phOx-conjugated mAbs to determine the background proliferation induced by soluble mAbs. The results
To express a single-chain antibody directed against the hapten phOx Ž4-ethoxymethylene-2-phenyl-2oxazoline-5-one. on the surface of tumor cells, we constructed the retroviral vector pMESV-SP-a phOxEx-TM ŽFig. 1.. The signal sequence of the IL-6-receptor was fused to the 5X-end for directing the scFv to the cell surface and a transmembrane domain of the Fc-g-RII-receptor was added to the 3X-end for anchoring the scFv to the cell membrane. In contrast to a previous construct that employed the IL-6-receptor transmembrane domain without a spacer sequence between the scFv and the cell membrane ŽRode et al., 1996., we used the Fc-g-RII-receptor transmembrane domain with a 42 amino acid extension. This receptor does not appear to be shed to any significant extent and the spacer provides more flexibility, which was reflected in better binding of phOx-labeled magnetic beads Ždata not shown.. After transfecting the human melanoma cell line SK-Mel63 with pMESV-SP-a phOx-Ex-TM, we first investigated the optimal amount of modification with
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the phOx-hapten for binding the mAbs used in this study to the membrane anchored anti-phOx singlechain antibody ŽscFv.. The anti-CD3 and anti-CD28 mAbs were coupled with a 2.5-, 5-, 10-, 20- and 40-fold molar excess of phOx to antibody and the anti-CD5 mAb with a 10-, 25-, 40- and 50-fold molar excess of phOx-hapten to antibody. Maximum binding of the anti-CD3-phOx mAb was reached at a molar phOx-excess of 20-fold, for the anti-CD28phOx mAb at a 40-fold molar excess and for the anti-CD5-phOx mAb at a 10-fold molar excess ŽFig. 2D, F and H.. The phOx-modified mAb did not bind non-specifically to untransfected cells ŽFig. 2C, E and G. and unmodified mAbs did not bind to cells expressing the anti-phOx scFv ŽFig. 2B.. The binding of the modified antibodies to the transformed cells seems to be very strong, since cells that had been loaded with the modified anti-CD28-phOxŽ40. mAb Ž40 molar excess of phOx. and then washed showed no significant reduction in their FACScan signal after a 24-h incubation at 48C Ždata not shown.. This indicates that the antibody is probably bound by more than one phOx hapten to the cell surface, thus, increasing the avidity of binding. 3.3. Influence of phOx-coupling on binding of antibodies to their antigens The modification of antibodies with the phOx hapten might disturb the structure of the antigen binding site. We therefore tested the antigen binding capacity of phOx conjugated antibodies to T cells by FACScan. The anti-CD3 mAb coupled with a 2.5-, 5-, 10- and 20-fold molar excess of phOx bound to CD3 q Jurkat cells in amounts similar to that of the original unmodified mAb ŽFig. 3B,C.. In contrast, a 40-fold molar excess of the phOx hapten resulted in a reduced amount of binding ŽFig. 3D.. The binding of the anti-CD28 mAb to CD28 q HPB-ALL cells was hardly affected by a molar phOx excess of up to 40-fold ŽFig. 3G,H. as compared to the amount bound of the original unmodified mAb ŽFig. 3F.. Similarly, there was no significant reduction in binding of the anti-CD5 mAb to CD5 q Jurkat cells after incubation with a 10-, 25-, 40- and 50-fold molar excess of the phOx hapten ŽFig. 3J, K, L.. Results presented in Figs. 2 and 3 imply that anti-CD3,
Fig. 2. Binding of phOx-coupled mAbs to untransfected SK-Mel63 cells or cells expressing the membrane anchored anti-phOx scFv ŽSK-Mel63-pMESV-SP-a phOx-Ex-TM.. Cells were incubated with phOx-labeled CD3, CD28, CD5 mAbs conjugated at 20-, 40and 10-fold excess of phOx-hapten to antibody, respectively, ŽC–H. or medium ŽA, B.. Afterwards cells were washed and stained with anti-mouse FITC-labeled IgG. Fluorescence was determined by flow cytometry.
anti-CD5 and anti-CD28 mAbs coupled at 20-, 10and 40-fold molar excess, respectively, are optimal for stimulation of T cells.
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Fig. 3. Influence of phOx-labeling on antigen binding. T cells ŽJurkat or HPB-ALL. were incubated with 1 mgrml anti-CD3, anti-CD28 or anti-CD5 antibodies ŽB, F and J. or their derivatives labeled at 10-, 20-, 40- or 50-fold excess of phOx-hapten ŽC, D, G, H, K and L. or medium alone ŽA, E and I.. Following washing and staining with anti-mouse FITC-labeled IgG, fluorescence was determined by flow cytometry.
3.4. Effect of modified anti-CD3, anti-CD28 and anti-CD5 mAbs on T cell proliferation The effect of modified anti-CD3, anti-CD28 and anti-CD5 mAbs on the proliferation of allogenic T lymphocytes was compared at two different concentrations. The modified anti-CD5-phOxŽ10. mAb Ž10 = phOx. had no significant effect on the thymidine
uptake of the T cells at comparable concentrations of 0.1 and 1.0 mgrml ŽFig. 4, lanes B and C.. The anti-CD3-phOxŽ20. mAb Ž20 = phOx. was added to SK-Mel63 cells expressing the anti-phOx scFv in concentrations of 0.1 or 1.0 mgrml. After incubation with T cells, a significant uptake of thymidine was observed at both concentrations, whereby the best stimulation was obtained at the lower concentration
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Fig. 4. Induction of T cell proliferation by SK-Mel63-pMESV-SP-a phOx-Ex-TM cells expressing the cell surface displayed anti-phOx scFv in combinations with phOx-conjugated CD3, CD5, and CD28 mAbs mAbs. A total of 10 5 T cells was incubated with 10 4 Sk-Mel63 melanoma cells transfected with the gene for the anti-phOx scFv and two different concentrations of phOx-conjugated CD3 and CD5 Ž0.1 and 1.0 mgrml. and CD28 Ž0.2 and 2.0 mgrml. mAbs, respectively. Stimulation was performed in the presence of one Žlanes B–G., two Žlanes H–S. or three Žlanes T–V. mAbs. T cell proliferation was determined by thymidine incorporation on day 5.
of the modified anti-CD3 mAb ŽFig. 4, lanes D and E.. The unmodified anti-CD3 mAb showed no significant stimulation of T cell proliferation Ždata not shown.. Similar incubations using 0.2 mgrml of the modified anti-CD28-phOxŽ40. mAb Ž40 = phOx. gave no stimulation of thymidine uptake, whereas a concentration of 2.0 mgrml induced a significant T cell response ŽFig. 4F and G., which was significantly larger than that obtained using the anti-CD3phOxŽ20. mAb ŽFig. 4, lane D.. The effect was similar whether the cells loaded with the anti-CD28phOxŽ40. mAb were washed or not Ždata not shown.. This supports the data mentioned above on the apparent tight binding of the modified mAbs to the cell surface. 3.5. Synergistic effect of stimulatory mAbs on T cell proliferation The combined effect of two and three stimulatory antibodies, respectively, on T cell proliferation was
investigated. We tested all combinations of two simultaneously incubated phOx-coupled antibodies in both concentrations ŽFig. 4, lanes H–S.. We found that the strongest stimulation of T cell proliferation was achieved using 0.1 mgrml or 1 mgrml of the anti-CD3-phOxŽ20. mAb together with 0.2 mgrml of the anti-CD28-phOxŽ40. antibody ŽFig. 4, lanes S and R., although this concentration of anti-CD28phOxŽ40. mAb alone had very little effect on thymidine uptake ŽFig. 4, lane F.. However, in combination with the anti-CD3-phOxŽ20. antibody, the thymidine uptake was higher than that achieved with the anti-CD3-phOxŽ20. alone ŽFig. 4, lanes D and E.. The stimulation seen using 2 mgrml of the anti-CD28-phOxŽ40. mAb was still appreciable but significantly reduced ŽFig. 4, lanes P and Q., even lower than the stimulation obtained using antiCD28-phOxŽ40. alone ŽFig. 4, lane G.. The antiCD5-phOxŽ10. mAb showed no significant stimulatory effects on T cell proliferation at concentrations
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of either 0.1 or 1 mgrml in combination with either the anti-CD3-phOxŽ20. or anti-CD28-phOxŽ40. mAbs ŽFig. 4, lanes H–O.. Indeed, at a concentration of 1 mgrml anti-CD5-phOxŽ10. mAb together with 2 mgrml anti-CD28-phOxŽ40. mAb, the thymidine uptake was considerably lower than that obtained for the anti-CD28-phOxŽ40. mAb alone ŽFig. 4, lanes I with G.. A similar reduction in T cell activation was seen combining the anti-CD5-phOx Ž10. mAb at 1 mgrml with the anti-CD3-phOxŽ20. mAb at 0.1 mgrml ŽFig. 4, lanes L and D.. These effects are most likely due to competition for the available binding sites on the cell surface represented by the number of anti-phOx scFv. The best stimulation of T cell proliferation was achieved using a combination of the three modified antibodies at concentrations of 0.1 mgrml for antiCD3 and anti-CD5 and 0.2 mgrml for anti-CD28 ŽFig. 4, lane V.. The anti-CD5-phOxŽ10., which alone or in combination with anti-CD3-phOxŽ20. or anti-CD28-phOxŽ40. did not result in any significant increase in T cell proliferation ŽFig. 4, lanes H–O., induced a marked response when applied together with both the anti-CD3-phOxŽ20. and anti-CD28phOxŽ40. ŽFig. 4, lane V.. The synergistic effect of the anti-CD5-phOxŽ10. mAb at 0.1 mgrml was also observed using anti-CD28-phOxŽ40. mAb at 2 mgrml ŽFig. 4, lanes U and Q.. At the higher concentration of 1 mgrml for the anti-CD5-phOxŽ10. mAb, a slight reduction of the T cell activation was observed in the triple combination ŽFig. 4, lanes T and P.. This effect was also noted for combinations with only one other mAb Žsee above. and is again probably due to competition for the available binding sites. These results demonstrate that the T cell response can be amplified by the synergistic effect of three different surface-bound antibodies, with the effect highly dependent on their relative concentrations.
4. Discussion There is increasing evidence that tumor cells express tumor associated antigens that can stimulate an immune response under appropriate conditions. These antigens, in the form of processed peptides bound to MHC molecules, can be derived from viruses, mu-
tated genes or proteins that are overexpressed in the tumor cells ŽVan der Bruggen et al., 1991; Altmann et al., 1992; Gedde-Dahl et al., 1993.. Several melanoma-associated peptides have been characterized in detail ŽTraversari et al., 1992; Brichard et al., 1993; Kawakami et al., 1994.. Twenty-four overexpressed antigens from the tumors of patients with melanomas, renal carcinomas, astrocytomas and Hodgkin lymphomas were found to have elicited a B cell response ŽSahin et al., 1995.. Vaccination of tumor patients with tumor associated peptides and proteins may eventually provide a useful method of inducing an immune response in an adjuvant setting. However, until more is known about the expression and range of antigens present in the largely heterogeneous tumors, the use of tumor cells has the advantage that the products of multiple altered genes can be used as a vaccine even though they have not been identified. In one approach, costimulatory molecules are being used for T lymphocyte activation in order to strengthen the relatively weak immunogenicity of the tumor cells. For example, the application of genetically modified tumor cells, either autologous or allogeneic with shared HLA-alleles, has been initiated in clinical trials with patients suffering from malignant melanoma and renal cell cancer ŽHabicht et al., 1995; Human Gene MarkerrTherapy Clinical Protocols, 1996.. In contrast, however, to the successful induction of a cytotoxic T cell response by a human melanoma cell line transfected with CD80 ŽGraham and Van der Eb, 1973., the transfection of two colorectal cell lines with CD80 did not result in T cell activation, in spite of an adequate expression of class I MHC molecules ŽHabicht et al., 1995.. One solution to this problem might be to express other or additional costimulatory molecules on the tumor cell surface. The transfection of tumor cells with genes coding for several costimulatory molecules is time-consuming and not very efficient. We have therefore developed a method for the simultaneous expression of several proteins or peptides on the cell surface requiring only one cellular transformation with a gene coding for a cell membrane anchored scFv against the phOx antigen. Proteins and peptides can be readily modified with this antigen and then bound to the cell surface through the anti-phOx scFv ŽRode et al., 1996..
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To investigate the effect of simultaneously expressing first and second stimulatory signals bound to the surface of a human melanoma tumor cell line on the activation of T lymphocytes, we modified monoclonal antibodies against the anti-CD3, antiCD28 and anti-CD5 antigens with various molar ratios of the phOx antigen. The anti-CD28 and antiCD5 antibodies could be incubated with relatively high concentrations of the phOx antigen without a significant loss of antigen binding ability. In contrast, the OKT3 anti-CD3 showed a sharp decline in antigen binding at molar ratios of phOx to antibody higher than 20:1. One possible explanation is the presence of a free sulfhydryl group at position 100 A in the center of the third complementarity determining region of the heavy chain variable domain ŽAdair et al., 1994.. Any modifications in this region, which is crucial for antigen binding, would be expected to have a serious effect on the antibody’s affinity. This could also account for the fact that on raising the concentration of the anti-CD3-phOxŽ40. from 0.1 to 1 mgrml, the activation of T cell proliferation was significantly decreased. A population of more highly modified but less active antibodies might be preferentially bound to the tumor cell surface. Higher concentrations of antibody under saturating conditions would then result in a lower proportion of bound functional antibodies. In contrast, the anti-CD28-phOxŽ20. antibody had little effect on T cell activation at a concentration of 0.2 mgrml but a very marked effect at 2 mgrml. However, in combination with anti-CD3-phOxŽ20. or with anti-CD3-phOxŽ20. plus anti-CD5-phOxŽ10., the lower concentration of anti-CD28-phOxŽ40. yielded a significantly higher T cell activation. The most likely explanation is that due to the competition of the modified antibodies for binding to the cell membrane anchored anti-phOx scFv, only very little of the anti-CD3-phOxŽ20. mAb can be bound at the higher concentrations of the anti-CD28-phOxŽ40. mAb. Similarly, higher concentrations of the antiCD5-phOxŽ10. mAb resulted in a decreased T cell activation of either the anti-CD3-phOxŽ20. or the anti-CD28-phOxŽ40. mAbs. The highest T cell activation was achieved by a combination of all three modified antibodies at the lower of the two given concentrations. Similar ratios of these stimulatory antibodies could provide a means to induce a T cell
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response against the refractory colorectal carcinoma cells described above. We therefore intend to test this concept using such refractory model systems in future investigations.
Acknowledgements We would like to thank Dr. K. Khazaie for his help in establishing the retroviral expression system and Dr. G. Moldenhauer for providing purified polyclonal mouse IgG. We are also particularly grateful to Tobias Schurrle for carrying out T cell prolifera¨ tion assays, Dr. G. Winter and colleagues for the original VH - and VL-DNA of the clone phOx31E and Dr. C. Stocking and Dr. W. Ostertag for the retroviral vector p50-M-x-neo.
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