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The Journal of Immunology, 2004, 173: 7647-7653.
Copyright © 2004 by The American Association of Immunologists

T Cell Activation by Antibody-Like Immunoreceptors: Increase in Affinity of the Single-Chain Fragment Domain above Threshold Does Not Increase T Cell Activation against Antigen-Positive Target Cells but Decreases Selectivity1

Markus Chmielewski*, Andreas Hombach*, Claudia Heuser*, Gregory P. Adams{dagger} and Hinrich Abken2,*

* Klinik I für Innere Medizin, Tumorgenetik, Kliniken der Universität zu Köln, and Center for Molecular Medicine Cologne, Köln, Germany; and {dagger} Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, PA 19111


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chimeric TCRs with an Ab-derived binding domain confer predefined specificity and MHC-independent target binding to T cells for use in adoptive immunotherapy. We investigated the impact of receptor binding affinity on the activation of grafted T cells. A series of anti-ErbB2 single-chain fragment binding domains with a Kd ranging from 3.2 x 10–7 to 1.5 x 10–11 M was linked to CD3{zeta}-derived immunoreceptors and expressed in human PBL. Solid phase bound ErbB2 protein triggered activation of receptor-grafted T cells in a dose-dependent manner. The activation threshold inversely correlated with the affinity of the receptor binding domain. The maximum level of cellular activation, however, was the same and independent of the binding affinity. Upon binding to ErbB2+ cells, T cells grafted with immunoreceptors carrying a single-chain fragment of Kd < 10–8 M were activated in a similar fashion against cells with different amounts of ErbB2 on the surface. T cells with a low affinity receptor (Kd > 10–8 M), however, were activated exclusively by cells with high amounts of ErbB2. In conclusion, recombinant immunoreceptors of higher affinity do not necessarily induce a more potent activation of T cells than low affinity immunoreceptors, but the higher affinity immunoreceptors exhibit less discrimination between target cells with high or low Ag expression levels.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Recombinant immunoreceptors containing an Ab-derived binding domain can mediate activation of both CD4+ and CD8+ T cells in a highly efficient, MHC-independent manner upon binding to Ag-expressing target cells (1, 2, 3). The Ag binding domain of this type of immunoreceptor consists of an Ab derived single-chain fragment (scFv)3 linked either directly or via an extracellular spacer to a transmembrane and intracellular signaling moiety that is most frequently derived from either the CD3 {zeta}-chain or the highly homologous Fc{epsilon}RI {gamma}-chain (4, 5). Whereas the impact of the spacer and the intracellular signaling domain on receptor-mediated cellular activation was extensively investigated (6, 7, 8), little is known about the influence of the affinity of the Ab-derived binding domain on the efficiency of target cell binding and activation of grafted effector cells.

Presently, two nonexclusive concepts for T cell activation via the endogenous TCR are discussed: 1) the affinity of the TCR to its MHC-bound peptide ligand is tightly correlated with the efficiency of cellular activation (9); and 2) efficient T cell activation requires serial triggering of the TCR that is generally of low affinity and binds to small numbers of MHC-peptide complexes (10). Cellular activation via chimeric immunoreceptors differs from activation via TCR in so far that the recombinant immunoreceptor harbors an Ab-derived domain of high affinity for binding. The Ag, moreover, is bound by the immunoreceptor independently of the MHC. The impact of the binding affinity on the efficiency of T cell activation by immunoreceptors has not been studied in detail. It is therefore unresolved whether recombinant immunoreceptors with a high affinity binding domain are superior to immunoreceptors with low binding affinities with respect to receptor-mediated cellular activation. To address this issue, we generated a panel of recombinant anti-ErbB2 (Her2/Neu) immunoreceptors with scFv binding domains of different affinities, i.e., ranging from 3.2 x 10–7 to 1.5 x 10–11 M, but specificity for the same epitope of ErbB2. The anti-ErbB2 scFvs of different affinities have been generated by site-directed mutagenesis of the anti-ErbB2 scFv C6.5 (11). By expression of this panel of anti-ErbB2 immunoreceptors in human PBL, we recorded activation of receptor grafted T cells against solid phase bound ErbB2 and against tumor cells with different amounts of ErbB2 on the cell surface, respectively.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and reagents

293T cells are human embryonic kidney cells that express the SV40 large T Ag (12). SK-OV-3 (ATCC HTB77; American Type Culture Collection, Manassas, VA), MCF-7 (ATCC HTB22) Colo201 (ATCC CCL 224), and Colo320 (ATCC CCL 220.1) were derived from various adenocarcinomas expressing different amounts of ErbB2. HaCat is a transformed human keratinocyte cell line (13) and was a gift from Dr. N. Fusenig (German Cancer Research Center, Heidelberg, Germany). Primary human keratinocytes were prepared and maintained as described previously (14). OKT3 (ATCC CRL 8001) is a hybridoma cell line that produces the anti-CD3 mAb OKT3. 293T cells were cultured in DMEM supplemented with 10% (v/v) FCS, all other cell lines were cultured in RPMI 1640 medium and 10% (v/v) FCS (all from Invitrogen Life Technologies, Paisley, U.K.). OKT3 mAb was affinity-purified from hybridoma supernatants using goat anti-mouse IgG2a Abs (Southern Biotechnology Associates, Birmingham, AL) that were immobilized on N-hydroxy-succinimide-ester-activated Sepharose as recommended by the manufacturer (Amersham Biosciences, Freiburg, Germany). Human IgG1 Abs and the PE- and FITC-conjugated anti-CD3 mAb UCHT1 were purchased from DakoCytomation (Hamburg, Germany). The goat anti-human IgG Ab and its FITC- and PE-conjugated F(ab')2 derivatives were purchased from Southern Biotechnology Associates. The anti-ErbB2 mAb Ab5 was purchased from Oncogene (Cambridge, MA), and the recombinant ErbB2-IgG Fc fusion protein was purchased from R&D Systems (Minneapolis, MN). The anti-human IFN-{gamma} mAb NIB42 and the biotinylated anti-human IFN-{gamma} mAb 4S.B3 were purchased from BD Biosciences (San Diego, CA). Generation of the anti-ErbB2 scFvs was described in detail previously (11). The binding parameters of the anti-ErbB2 scFv are summarized in Table I.


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  		Table I. Characteristics of the anti-ErbB2 scFvs used in this study and of recombinant immunoreceptors derived thereof

 
Generation of recombinant immunoreceptors

To generate the retroviral expression cassettes for ErbB2-specific recombinant immunoreceptors, the DNA coding for C6.5 scFv and its derivatives (gift from Dr. J. Marks, University of California, San Francisco, CA; Table I) were amplified by PCR and flanked by NcoI (5') and BamHI (3') restriction sites, respectively, using the following set of primer oligonucleotides: 5'-CGTACCATGGATTTTGAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAATGTCTAGACCGGCGATGGCCCACCAGGTG-3' (sense) and 5'-TTCTGGATCCGCACCTAGGACGGTGACCTT-3' (antisense; restriction sites are underlined). The BW431/26-scFv DNA of the anti-CEA receptor BW431/26-scFv-Fc{zeta} receptors in pBullet (15, 16) was cleaved out by NcoI and BamHI and replaced by the digested anti-ErbB2 scFv PCR products.

Expression of recombinant immunoreceptors

To express recombinant receptors in T cells from the peripheral blood, the expression cassettes were inserted into the retroviral vector pBullet (15, 17) as recently described (18). Retroviral transduction of T cells with recombinant receptors was described in detail previously (12, 15, 16, 17, 19), and receptor expression was monitored by flow cytometric analysis. Recombinant receptors were also expressed in 293T cells after transfection of the vector DNA by calcium phosphate coprecipitation (20 µg of DNA/2 x 106 cells). Cells were harvested after 48 h and subjected to analysis.

Immunofluorescence analysis

ErbB2 expression was determined by flow cytometry using the anti-ErbB2 mAb Ab5 and an isotype-matched control mAb (BD Biosciences; each 10 µg/ml). Bound Abs were detected by an FITC-conjugated F(ab)2-anti-mouse IgG Ab (5 µg/ml; Southern Biotechnology Associates), and mean fluorescence was determined. Recombinant receptor-grafted T cells were identified by two-color immunofluorescence using a PE- or FITC-conjugated F(ab')2 anti-human IgG1 Ab (1 µg/ml) and an FITC- or PE-conjugated anti-CD3 mAb (UCHT-1; 1/20). Immunofluorescence was analyzed using a FACScan cytofluorometer equipped with CellQuest research software (BD Biosciences, Mountain View, CA). To identify T cells with recombinant receptor expression, we set markers with 99% of nontransduced T cells beyond.

Receptor-mediated activation of grafted T cells

T cells were grafted with recombinant anti-ErbB2-scFv-Fc-{zeta} receptors and cultivated in microtiter plates (1 x 104 receptor-grafted T cells/well) that were precoated with different amounts of recombinant ErbB2-Fc fusion protein, human IgG1 Abs for control (each 0.02–12 µg/ml), or an anti-human IgG1 Fc Ab (0.16–20 µg/ml; Southern Biotechnology Associates). After 48 h, supernatants were removed and analyzed by ELISA for IFN-{gamma} as described below. In a second set of experiments, anti-ErbB2 receptor-grafted T cells (1.25 x 103 to 10 x 103 cell/well) were cocultivated for 48 h in 96-well, round-bottom plates with tumor cells that express different amounts of ErbB2 (each 5 x 104 cells/well). The culture supernatants were harvested and analyzed for IFN-{gamma} by ELISA. Briefly, IFN-{gamma} was bound to the solid phase anti-human IFN-{gamma} mAb NIB42 (1 µg/ml) and detected by the biotinylated anti-human IFN-{gamma} mAb 4S.B3 (0.5 µg/ml). The reaction product was visualized by a peroxidase-streptavidin conjugate (1/10,000) and ABTS. The specific cytotoxicity of receptor-grafted T cells against target cells was monitored by a 2,3-bis(2-methoxy-4-nitro-5sulphonyl)-5[(phenyl-amino)carbonyl]-2H-tetrazolium hydroxide (XTT)-based colorimetric assay according to Jost et al. (20). Briefly, receptor-grafted and nontransduced T cells were cocultivated with ErbB2+ or ErbB2– tumor cells as described above. After 48 h, XTT reagent (1 mg/ml; Cell Proliferation Kit II; Roche, Mannheim, Germany) was added to the cells and incubated for 30–90 min at 37°C. Reduction of XTT to formazan by viable tumor cells was monitored colorimetrically at an adsorbance wavelength of 450 nm and a reference wavelength of 650 nm. Maximal reduction of XTT was determined as the mean of six wells containing tumor cells only, and the background as the mean of six wells containing RPMI 1640 medium and 10% (v/v) FCS. The nonspecific formation of formazan due to the presence of effector cells was determined from triplicate wells containing effector cells in the same number as in the corresponding experimental wells. The number of viable tumor cells was calculated as follows: % viability = (OD(exp. wells – corresponding number of effector cells)/OD(tumor cells without effectors – medium)) x 100.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To monitor the impact of the affinity of the scFv domain on receptor-mediated T cell activation, we generated expression cassettes for the anti-ErbB2 immunoreceptors and inserted them into the retroviral vector pBullet (15, 17) as described in Materials and Methods. These anti-ErbB2 scFv-Fc-CD3{zeta} immunoreceptors harbor the same intracellular signaling, transmembrane, and extracellular spacer domains, but an anti-ErbB2 scFv of different affinity, i.e., Kd = 3.2 x 10–7 to 1.5 x 10–11 M. The anti-ErbB2 scFv domains of various affinities were derived from the anti-ErbB2 scFv C6.5 by site-directed mutagenesis (21) (Table I). We grafted peripheral blood T cells from healthy donors with the recombinant immunoreceptors by retroviral gene transfer. FACS analyses revealed high expression of all anti-ErbB2 immunoreceptors with nearly the same density on the cell surface (Fig. 1). C6.5-scFv-Fc-{zeta} immunoreceptor-grafted T cells lysed ErbB2-expressing SK-OV-3 and MCF-7 target cells with high efficiency, whereas anti-CEA BW431/26-scFv-Fc-{zeta} immunoreceptor-grafted T cells lysed solely MCF-7 tumor cells expressing both Ags. As the control, nonmodified lymphocytes did not lyse the tumor cells, demonstrating the specificity of recombinant immunoreceptor-mediated T cell activation (Fig. 2, A and B). We obtained similar results monitoring IFN-{gamma} secretion of anti-ErbB2 and anti-CEA receptor-grafted T cells upon cocultivation with tumor cells (Fig. 2, C and D).



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  		FIGURE 1. Two-color immunofluorescence of anti-ErbB2 receptor-grafted peripheral blood T cells. Nontransduced peripheral blood T cells and T cells grafted with anti-ErbB2 immunoreceptors with different affinities of the binding domain (no. 1–5) were simultaneously incubated with a PE-conjugated anti-CD3 mAb and an FITC-conjugated anti-human IgG1 Ab and analyzed by flow cytometry.

 


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  		FIGURE 2. Ag-specific activation of receptor-grafted T cells. Peripheral blood T cells were grafted with the anti-ErbB2 immunoreceptor C6.5-scFv-Fc-{zeta} and the anti-CEA immunoreceptor BW431/26-scFv-Fc-{zeta}, respectively, and cocultivated for 48 h (0.078–10 x 104 receptor-grafted T cells/well) with target cells (5 x 104 cells/well) expressing only ErbB2 (SK-OV-3) or both ErbB2 and CEA (MCF-7) on the cell surface. A and B, The viability of target cells was determined colorimetrically by a tetrazolium salt-based XTT assay, as described in Materials and Methods. C and D, IFN-{gamma} secreted by receptor-grafted T cells into the supernatant was determined by ELISA.

 
We incubated the same number of T lymphocytes that was grafted with anti-ErbB2 receptors harboring binding domains with different affinities in the presence of serial dilutions of solid bound ErbB2 protein and recorded the secretion of IFN-{gamma} indicating T cell activation. As demonstrated in Fig. 3A, anti-ErbB2 receptor-grafted T cells were activated by solid phase bound Ag in a dose-dependent fashion. The amount of ErbB2 required to induce grafted T cells to half-maximal IFN-{gamma} secretion depended on the affinity of the scFv domain of the receptor (Fig. 3A). Maximum amounts of secreted IFN-{gamma} were nearly the same for T cells grafted with immunoreceptors with an scFv Kd <10–8 Mn, whereas upon binding of the anti-ErbB2 immunoreceptor with an scFv Kd of 3.2 x 10–7 M, induction of IFN-{gamma} secretion was extremely low, even after binding to the highest concentration of ErbB2 in the assay (Fig. 3A). As the control, receptor-grafted T cells were activated by cross-linking the extracellular IgG1 CH2CH3 spacer domain with a solid bound anti-human IgG Fc Ab that results in secretion of the same amounts of IFN-{gamma} for all receptors (Fig. 3B), indicating identical activation properties of the signaling domains despite different scFv binding domains of the immunoreceptors.



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  		FIGURE 3. Activation of anti-ErbB2 receptor-grafted T cells by solid phase bound Ag. Serial dilutions of recombinant ErbB2-Fc fusion protein (A) or of an anti-human IgG-Fc Ab (B) were coated onto microtiter plates. Peripheral blood T cells were grafted with anti-ErbB2 immunoreceptors with different Kd values of the anti-ErbB2 scFv domain and incubated for 48 h (1 x 104 receptor-grafted T cells/well). IFN-{gamma} in the supernatant was recorded by ELISA.

 
We asked whether the receptor affinity has an impact on activation of receptor-grafted T cells upon binding to tumor cells that express ErbB2 in different amounts on the cell surface. For this purpose we estimated the ErbB2 expression of several cell lines and primary keratinocytes, respectively, by flow cytometry (Fig. 4). Cells with a mean fluorescence intensity (MFI) >100 arbitrary units were regarded as highly positive (ErbB2+++), cells with an MFI >10 arbitrary units were regarded as medium positive (ErbB2++), and cells with <10 arbitrary MFI units were regarded as weakly positive or negative (ErbB2+/–). Anti-ErbB2 receptor-grafted T cells were coincubated with each of these cells at different T cell:tumor cell ratios, and specific cytolysis and induction of IFN-{gamma} secretion by grafted T cells were recorded. As summarized in Fig. 5, T cells equipped with immunoreceptors with an scFv Kd of <10–8 M lysed SK-OV-3 (ErbB2+++), Colo 201 (ErbB2++), and MCF-7 (ErbB2++) cells, respectively, with similar efficiencies, whereas T cells engrafted with immunoreceptor 5 (C6.5G98A-scFv-Fc-{zeta}), which harbors an scFv with a Kd >10–8 M, were much less efficient. As a control, nonmodified lymphocytes did not lyse tumor cells. The efficiency of target cell lysis was dependent on the amount of ErbB2 on the surface of the target cells, but was only partially dependent on the affinity of the scFv binding domain. Increasing the affinity of the scFv domain, i.e., Kd <10–8 M, did not increase the efficiency of cytolysis of target cells with high and medium ErbB2 expressions. In contrast, T cells expressing the low affinity anti-ErbB2 immunoreceptor 5 (scFv Kd = 3.2 x 10–7 M) required a higher E:T cell ratio to result in the same efficiency of cytolysis compared with T cells with high affinity receptors. In contrast, anti-ErbB2 receptor-grafted T cells of any affinity did not lyse Colo320 tumor cells or transformed (HaCat) and primary keratinocytes, respectively, that express only low amounts of ErbB2.



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  		FIGURE 4. ErbB2 expression of target cells. Cells of ErbB2-expressing lines (SK-OV-3, Colo201, MCF-7, Colo320, and HaCat) and primary cells (PBL and keratinocytes) for the control (each 1 x 106 cells) were incubated with an anti-ErbB2 mAb or an isotype-matched control mAb. Bound Abs were detected by an FITC-conjugated anti-mouse IgG Ab, and cells were analyzed by flow cytometry. The MFI of bound Abs was determined and is expressed as arbitrary units. Due to their immunoreactivity, the cells were characterized as high (MFI, >100), medium (MFI, >10), and low (MFI, <10) ErbB2-positive or ErbB2-negative cells, respectively.

 


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  		FIGURE 5. Ag-specific lysis of ErbB2-expressing target cells by receptor-grafted T cells. Peripheral blood T cells were grafted with anti-ErbB2 immunoreceptors with different Kd values of the respective scFv domain and cocultivated for 48 h (0.078–10 x 104 receptor-grafted T cells/well) with target cells (5 x 104 cells/well) expressing different amounts of ErbB2 on the cell surface. The viability of ErbB2-positive target cells was determined colorimetrically by a tetrazolium salt-based XTT assay, as described in Materials and Methods.

 
We obtained corresponding results when monitoring IFN-{gamma} secretion of receptor-grafted T cells (Fig. 6). Coincubation of ErbB2+++ (SK-OV-3) tumor cells with T cells expressing anti-ErbB2 immunoreceptors with an scFv of Kd <10–8 M resulted in secretion of similar amounts of IFN-{gamma} that was dependent on the ErbB2 expression level on the tumor cells rather than on the affinity of the recombinant immunoreceptor. In contrast, T cells equipped with low affinity anti-ErbB2 immunoreceptor 5 (C6.5G98A-scFv-Fc-{zeta}) were only induced to IFN-{gamma} secretion by incubation with SK-OV-3 cells that express high amounts of ErbB2. Colo320 cells or transformed (HaCat) or primary keratinocytes that express only low amounts of ErbB2 on the cell surface activated neither high nor low affinity immunoreceptor-grafted T cells to IFN-{gamma} secretion. Notably, in medium, high ErbB2-expressing Colo201 cells did not induce substantial IFN-{gamma} secretion in anti-ErbB2 receptor-grafted T cells, whereas high affinity receptor-grafted T cells lysed these cells efficiently (cf., Fig. 5). This may be due to IL-10 secretion of Colo201 tumor cells (data not shown), which was demonstrated to suppress IFN-{gamma} secretion of activated T cells (22).



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  		FIGURE 6. Ag-specific IFN-{gamma} secretion by receptor-grafted T cells upon cocultivation with ErbB2-expressing target cells. Peripheral blood T cells were grafted with anti-ErbB2 receptors with different Kd values of the scFv binding domain and cocultivated for 48 h (0.078–10 x 104 receptor-grafted T cells/well) with target cells (5 x 104 cells/well) expressing different amounts of ErbB2 on the cell surface. IFN-{gamma} secreted by receptor-grafted T cells into the supernatant was determined by ELISA.

 
Because the affinity of the scFv binding domain with a Kd <10–8 M has no obvious impact on receptor-mediated cellular activation by coincubation with ErbB2+++ cells, we asked whether the affinity of the binding domain may affect the kinetics of target cell lysis and induction of IFN-{gamma} secretion. We coincubated T cells equipped with anti-ErbB2 receptor with various ErbB2-expressing tumor cells and monitored tumor cell lysis and induction of IFN-{gamma} secretion at different time points. As summarized in Fig. 7, T cells grafted with immunoreceptors of high and medium affinity lysed ErbB2-positive tumor cells with similar kinetics, whereas T cells equipped with the low affinity immunoreceptor 5 (C6.5G98A-scFv-Fc-{zeta}) lysed only ErbB2+++ SK-OV-3 cells, but substantially slower than T cells equipped with one of the high affinity receptors (Fig. 7, A, C, and E). Analysis of IFN-{gamma} secretion over time revealed similar results; T cells equipped with immunoreceptors of high affinity (scFv Kd, <10–8 M) secrete IFN-{gamma} in a similar fashion, but T cells with the low affinity receptor did not secrete detectable amounts of IFN-{gamma} in the presence of SK-OV-3 cells in this experiment (Fig. 7, B, D, and F).



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  		FIGURE 7. Time course of Ag-specific activation of receptor-grafted T cells upon cocultivation with ErbB2-expressing target cells. Peripheral blood T cells were grafted with anti-ErbB2 receptors with different Kd values of the scFv binding domain and cocultivated (5 x 103 receptor-grafted T cells/well) with target cells (3 x 104 cells/well) that express different amounts of ErbB2 on the cell surface. IFN-{gamma} secreted into the culture medium and target cell lysis were monitored at different time points. A, C, and E, Viability of ErbB2-positive target cells was determined colorimetrically by a tetrazolium salt-based XTT assay. B, D, and F, IFN-{gamma} secreted by receptor-grafted T cells into the culture supernatant was determined by ELISA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have generated five nearly identical recombinant immunoreceptors that bind to the same epitope of ErbB2, but with different affinities. The Kd of the scFvs used to bind ErbB2 range from 3.2 x 10–7 to 1.5 x 10–11 M (Table I). All immunoreceptors are equally capable of mediating cellular activation to the same extent, as demonstrated by cross-linking the extracellular spacer domain with an Ab. In contrast, the amount of solid phase bound ErbB2 required for half-maximal induction of IFN-{gamma} secretion by grafted T cells depends on the affinity of the scFv domain of the receptor (Fig. 3). Thus, within this range, the threshold of immobilized Ag required for cellular activation depends strictly on the affinity of the scFv domain of the immunoreceptor. The level of maximum amounts of secreted IFN-{gamma} by activated grafted T cells, however, is independent of the binding affinity of the immunoreceptor.

In the situation of cellular activation upon binding to ErbB2-positive target cells, the correlation between the affinity of the binding domain and cellular activation is not as obvious. T cells grafted with medium and high affinity anti-ErbB2 immunoreceptors (Kd, <10–8 M), respectively, are activated in a similar fashion, whereas T cells expressing an immunoreceptor with a low affinity binding domain (Kd, 3.2 x 10–7 M) are activated with substantially lower efficiency. Accordingly, for T cell activation via medium and high affinity immunoreceptors, the expression level of the target Ag on the cell surface, rather than the affinity of the binding domain, determines the efficiency of T cell activation with respect to target cell lysis and cytokine secretion. As a consequence, an increase in the affinity of the binding domain above the level of Kd < 10–8 M does not additionally improve the T cell activation properties of the recombinant receptor. In contrast, an immunoreceptor with a low affinity anti-ErbB2 binding domain (Kd, > 10–8 M) efficiently activates grafted T cells only against cells with high densities of ErbB2 on the cell surface.

Our data obtained from cell activation experiments using solid phase bound receptor ligand support the concept that the affinity of this type of recombinant TCR is directly correlated to the number of Ag-bound receptor molecules that are required for cellular activation. This conclusion favors the concept of T cell activation by an increasing number of receptor ligands (9), rather than the concept of serial triggering of few receptor molecules (10) to generate efficient T cell activation. As a cautionary note, the immunoreceptors used in this study have Ab-derived binding domains for MHC-independent recognition, which bind to Ag with higher affinity than TCRs whose affinity for MHC-bound peptide ligands is generally several orders of magnitude lower. Even the lowest binding affinity analyzed (Kd, 3.2 x 10–7 M) is much higher than the average affinity of the TCR for binding MHC-peptide complexes (23). Because the concept of serial TCR triggering requires low, rather than high, affinity binding, it is unlikely that serial receptor triggering significantly contributes to T cell activation via the MHC-independent, Ab-derived immunoreceptors used in this study.

Moreover, our results indicate that from the practical point of view the efficiency of immunoreceptor-mediated target cell lysis depends on the affinity of the binding domain. Below a Kd of ~10–8 M of the scFv domain, the immunoreceptors activate grafted T cells with similar efficiency against Ag-positive target cells with different levels of ErbB2. Chames et al. (24) recently reported that recombinant immunoreceptors with specificity for HLA-A1-bound, MAGE1-A1-derived peptide mediate target cell lysis in an affinity-dependent fashion. In this study two Ab-derived binding domains with Kd of 2.5 x 10–7 and 1.4 x 10–8 M, respectively, were inserted into the recombinant receptor molecule. This is in accordance with our data showing that for scFvs with Kd values between 10–7 and 10–8 M, the affinity correlates with the efficiency of target cell lysis. Systematical analysis, however, revealed that raising the scFv affinity above this value did not result in improved cellular activation and target cell lysis, not even toward tumor cells with low Ag expression (Figs. 4 and 5). These data are in striking contrast with cellular activation of grafted T cells via solid phase bound ligands (Fig. 3), implying that Ag binding and receptor clustering may occur in a different fashion.

In contrast, a low affinity immunoreceptor is capable, at least in part, of discriminating between target cells with high and low ErbB2 expression levels. This property will be helpful for the targeting of cells with Ags, such as ErbB2, that are overexpressed in tumor tissues, but are also found in low densities on normal cells. Although keratinocytes with low ErbB2 expression were not lysed by both low and high affinity anti-ErbB2 immunoreceptors in our in vitro system, there may be the risk for autoaggression using a recombinant receptor with high affinity for clinical application. In summary, raising the affinity of the scFv domain with MHC-independent ErbB2 binding properties could be ambiguous on adoptive immunotherapy: 1) immunoreceptors with high affinity (Kd, <10–8 M) are more efficient in cellular activation and target cell lysis than low affinity receptors, but increases in affinity beyond 10–8 M do not result in additional increases in receptor-mediated cellular activation; and 2) in T cell activation, immunoreceptors with high affinity scFv domains discriminate less between high and low Ag-expressing target cells than do immunoreceptors with low affinity scFv domains.


   Acknowledgments
 
We thank Dr. James D. Marks (University of California, San Francisco, CA) for providing us with the anti-ErbB2 scFv C6.5 and derivatives thereof.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Deutsche Forschungsgemeinschaft, Bonn (Ab58/7-1); the Deutsche Krebshilfe, Bonn (70-2235-Ab1); the Center for Molecular Medicine, Cologne (TP70); and the Koln Fortune Program. Back

2 Address correspondence and reprint requests to Dr. Hinrich Abken, Klinik I für Innere Medizin, Labor für Tumorgenetik, Universität zu Köln, Josef Stelzmann Strasse 9, D-50924 Köln, Germany. E-mail address: hinrich.abken{at}medizin.uni-koeln.de Back

3 Abbreviations used in this paper: scFv, single-chain fragment; MFI, mean fluorescence intensity; XTT, 2,3-bis(2-methoxy-4-nitro-5-sulphonyl)-5[(phenyl-amino)carbonyl]-2H-tetrazolium hydroxide. Back

Received for publication March 8, 2004. Accepted for publication October 11, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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