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The Journal of Immunology, 2006, 176: 3223-3232.
Copyright © 2006 by The American Association of Immunologists

Visualization of p53264–272/HLA-A*0201 Complexes Naturally Presented on Tumor Cell Surface by a Multimeric Soluble Single-Chain T Cell Receptor1

Xiaoyun Zhu*, Heather J. Belmont*, Shari Price-Schiavi{dagger}, Bai Liu*, Hyung-il Lee*, Marilyn Fernandez*, Richard L. Wong{ddagger}, Janette Builes*, Peter R. Rhode* and Hing C. Wong2,*

* Altor BioScience Corp., Miramar, FL 33025; {dagger} Pathology Associates Inc., Frederick, MD 21701; and {ddagger} University of Florida, Gainesville, FL 32612


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Intracellular Ags are processed into small peptides that are presented on cell surfaces in the context of HLA class I molecules. These peptides are recognized by TCRs displayed by CD8+ T lymphocytes (T cells). To date, direct identification and quantitation of these peptides has relied primarily on mass spectrometry analysis, which is expensive and requires large quantities of diseased tissues to obtain useful results. Here we demonstrate that multimerization of a soluble single-chain TCR (scTCR), recognizing a peptide from p53 presented in the context of HLA-A2.1, could be used to directly visualize and quantitate peptide/MHC complexes on unmanipulated human tumor cells. Tumor cells displaying as few as 500 peptide/MHC complexes were readily detectable by flow cytometry. The scTCR/multimers exhibited exquisite recognition capability and could distinguish peptides differing in as little as a single amino acid. We also demonstrate that scTCR/multimers could specifically stain human tumors generated in mice, as well as tumors obtained from patient biopsies. Thus, scTCR/multimers represent a novel class of immunostaining reagents that could be used to validate, quantitate, or monitor epitope presentation by cancer cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T cells play a key role in tumor surveillance and viral immunity through their exquisite peptide specificity. The success of viral vaccination strategies demonstrates the enormous potential of T cell-based approaches, as vaccination has greatly reduced or even eradicated infectious diseases caused by a number of different viruses. T cell-based approaches are now being explored as strategies for cancer therapy (1, 2, 3, 4, 5, 6, 7).

A major consideration for T cell-based tumor immunotherapy is whether or not the appropriate target Ag has been chosen. There are currently ~70 known HLA class I and class II associated tumor Ags (8). A valid T cell target tumor Ag must be specifically expressed by the tumor and/or substantially overexpressed by the tumor relative to other normal cell types so that the given therapy preferentially targets the tumor rather than normal tissues. In addition, the Ag must be processed and presented in the context of HLA molecules (8). The chances that large amounts of any given peptide will be presented in the context of HLA on the tumor cell surface are very low. In addition, the expression of a candidate Ag by a tumor does not necessarily mean that a target peptide Ag of interest will be presented in the context of HLA. Disease stage-specific or heterogeneous protein expression may also limit the effectiveness of Ag targeting approaches. It is unknown how many of the identified tumor Ags are suitable targets for T cell-based tumor immunotherapy (8). Thus, it would be useful to have a means to identify and validate suitable target Ags for T cell-based immunotherapy.

In addition to target Ag identification and validation, analysis of Ag expression can serve as an important criterion for identifying which patients are most likely to benefit from targeted therapeutic approaches. In the case of Herceptin therapy for breast cancer, the degree of ErbB-2 (HER2/neu) overexpression correlates with treatment effect of the Ab (9, 10). As a result, a number of techniques, including immunohistochemistry to assess ErbB-2 protein overexpression and fluorescence in situ hybridization to assess gene amplification, have been developed and approved for use in evaluating ErbB-2 status before therapeutic intervention (10). Similarly, other FDA-approved diagnostic assays are being used to help determine patient eligibility for treatment with therapeutics specific to c-Kit and epidermal growth factor receptor (11, 12). Optimally, for any targeted cancer therapy, methods should be designed to assess the expression of the target Ag before treatment. In the case of a T cell or TCR-based therapy, this analysis should exhibit specificity and sensitivity sufficient to measure the level of a given peptide presented in the context of the appropriate MHC complex.

We have recently described the construction and characterization of soluble single-chain TCR (scTCR)3-based fusion proteins wherein the TCR portion recognizes a wild type human p53 peptide (aa 264–272) presented in the context of HLA-A2.1 (264scTCR) (6, 7). These 264scTCR fusion proteins retain MHC-restricted, peptide-specific Ag binding properties, whereas the effector portions of the molecules, human IL-2 and IgG1, retain biological activity. Although these molecules are being developed as potential cancer therapeutics, we are concurrently developing immunohistochemical staining methods with another 264scTCR fusion protein to facilitate patient screening for clinical use.

In this report we describe the construction, characterization and utility of a 264scTCR fusion protein that is multimerized on streptavidin via an engineered biotinylation site. This fusion protein has been designated 264scTCR/multimer. We describe the use of this molecule for the quantitation of peptide/MHC complexes on the cell surface and the detection of naturally presented peptide/MHC complexes on the tumor cell surface and formalin-fixed tumor tissues. Successful development of methods using scTCR molecules as tumor-staining reagents will lead to improved tumor Ag target identification and validation. In addition, these methods should facilitate identification of patients who may benefit from soluble TCR-based therapeutics, T cell-based ex vivo therapy, and peptide/MHC-based vaccination treatment strategies.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Constructs

The TCR genes were cloned from the T cell clone A2.1 264#5 as described (13). We designated the TCR derived from this clone 264scTCR. To generate the 264scTCR/birA construct, the coding region of the three-domain 264scTCR was amplified using the 264scTCR/IL-2 fusion construct as template (7). The scTCR coding sequence was ligated into pUC shuttle vector containing the birA biotinylation tag sequence. The entire construct was then transferred into an expression vector and transfected into Chinese hamster ovary (CHO)-K1 cells followed by selection in medium containing 1 mg/ml G418 and limiting dilution cloning. The resulting transfected CHO cells secrete soluble 264scTCR/birA protein into the culture medium. The control fusion proteins, 264scTCR/IgG and CMVscTCR, were described previously (6). Briefly, 264scTCR/IgG is identical to 264scTCR/birA, with the exception that birA was replaced with an IgG1 H chain constant region and that CMVscTCR was cloned from CTLs stimulated with an HLA-A2-restricted CMVpp65495–503 peptide. The birA fragment was amplified from the 264scTCR/birA DNA to create the CMVscTCR/birA construct.

A soluble 264scTCR/birA fusion protein was purified from culture medium with an anti-mouse TCR Cbeta region mAb H57-597 coupled to a Sepharose 4 Fast Flow column (Amersham Biosciences). CMVscTCR/birA was purified from supernatant by immunoaffinity chromatography using the anti-human TCR Cbeta mAb (BF1) 8A3.31 mAb coupled to a Sepharose 4 Fast Flow column (Amersham Biosciences). The fusion proteins were biotinylated with biotin-protein ligase (Avidity) in the presence of excess biotin according to the manufacturer’s instructions. The biotinylated 264scTCR/birA fusion protein was then multimerized with R-PE-conjugated streptavidin, HRP-conjugated streptavidin (Jackson ImmunoResearch), or nonconjugated streptavidin (Pierce) at a TCR/streptavidin molar ratio of 4:1 or as indicated for at least 60 min at 4°C.

SDS-PAGE

The 264scTCR/birA, nonconjugated streptavidin, biotinylated 264scTCR/birA monomer, and 264scTCR/multimers were fractionated on a precast 4–20% gradient Tris-glycine polyacrylamide gel (Cambrex) under nonreducing condition. The gel was stained with SimplyBlue SafeStain (Invitrogen Life Technologies) according to the manufacturer’s instructions.

Surface plasmon resonance

Biotinylated p53264–272/HLA-A2.1 complexes or irrelevant control CMVpp65495–503/HLA-A2.1 complexes were bound to the streptavidin sensor chip (BIAcore) surface, allowing dilute solutions (1.6 µg/ml) of the fusion proteins to flow over the streptavidin-coated cells. For equilibrium affinity measurements, 915–964 resonance units of peptide-MHC complexes were bound to the relevant flow cell, and for kinetics measurements 1181–1514 resonance units were bound. Soluble 264scTCR/monomer or 264scTCR/multimer fusion proteins were diluted in PBS at various concentrations (0.25–20 µM) and were allowed to flow over the relevant cells at a rate of 10 µl/min at room temperature. Responses were recorded in real time using the BIAcore 1000 and analyzed using the BIAevaluation software 3.1 (BIAcore). Equilibrium dissociation constant (KD) were determined assuming a 1:1 interaction.

Cells

All cell lines were purchased from American Type Culture Collection. A375 cells were cultured in DMEM supplemented with 10% FBS. MDA-MB231, CaSKi, SW-480, AsPC-1, T2 lymphoblast hybrid cells, and CHO-K1 cells were cultured in IMDM supplemented with 10% FBS. HT-29 and SaOS-2 cells were cultured in McCoy’s medium supplemented with 15% FBS. The H57-597, W6/32, MA2.1, and BB7.2 mAb-producing hybridoma cells were cultured in RPMI 1640 medium supplemented with 20% FBS.

Abs and peptides

The anti-mouse TCR Cbeta mAb H57-597, class-I MHC-specific mAb W6/32, and HLA-A2-specific mAbs MA2.1 and BB7.2 were produced and purified from the supernatants of their relevant corresponding hybridoma cells by affinity chromatography using protein A. Anti-HLA-A2 mAbs (0397HA and 0791HA) were purchased from One Lambda. PE-conjugated H57-597 was purchased from BD Pharmingen. Anti-p53 Abs (PAb1801 and DO-12) recognizing aa 32–79 and 256–270 of human p53, respectively, were purchased from BD Pharmingen or Lab Vision. Rabbit anti-human GRB2 Ab was purchased from Cell Signaling. IRDye 800-conjugated anti-mouse IgG and IRDye 700DX-conjugated anti-rabbit IgG were purchased from Rockland.

All HLA-A2-restricted peptides (PeptidoGenic Research) were dissolved in DMSO and diluted in IMDM to the desired concentrations. The binding affinity of peptides to HLA-A2 was estimated using the algorithm developed by Reche et al. (14, 15) as provided at www.mif.dfci.harvard.edu/Tools/rankpep.html.

Flow cytometry

For studies using HLA-A2.1-positive T2 cells, 1 x 106 cells/ml were pulsed with peptides at the indicated concentration for 3–18 h at 26°C. To prepare tumor cells for staining, the cells were trypsinized, washed with IMDM supplemented with 10% FBS, resuspended in serum-free IMDM at 106 cells/ml, and incubated at 26°C for 2 h.

To compare binding abilities among the 264scTCR/multimer, the 264scTCR/birA monomer and the 264scTCR/IgG1 dimer, 2 x 105 T2 cells pulsed or not pulsed with 50 µM p53264–272 peptide were stained with the PE-conjugated 264scTCR/multimer (0.5 µg) or molar equivalent amounts of the 264scTCR/birA monomer or the 264scTCR/IgG1 dimer for 1 h. For the 264scTCR/birA monomer-stained or 264scTCR/IgG1 dimer-stained cells, cells were washed once in FACS buffer (PBS with 0.5% BSA and 0.1% sodium azide) and stained with PE-conjugated H57 for an additional hour. In these studies the flow cytometer was adjusted to detect specific staining by the 264scTCR/birA monomer. For other staining studies, 2 x 105 cells were stained with PE-conjugated 264scTCR/multimer (0.5 µg) in the presence or absence of the blocking reagents W6/32, MA2.1, or BB7.2 for 1 h at room temperature and washed once in FACS buffer before analysis. Cells stained with the PE-coupled streptavidin or the molar equivalent amounts of PE-conjugated CMVscTCR/multimer were used as controls and exhibited background fluorescence intensity similar to that of the nonstaining cells (data not shown).

All samples were analyzed on a FACScan flow cytometry using CellQuest software (BD Biosciences); a minimum of 10,000 live cells was analyzed per sample. Geometric mean was chosen as mean fluorescence intensity (MFI), which was displayed as a difference between the MFI of cells stained with PE-conjugated 264scTCR/multimer and the MFI of cells stained with PE-conjugated streptavidin.

In some studies, the MFI of cells was compared with the MFI from a standard curve of PE-coupled calibration beads (QuantiBRITE PE beads; BD Biosciences) carrying known quantities of PE per bead and an estimated amount of PE molecules per stained cell was determined. To determine the minimum number of peptide/MHC complexes per stained cells, each 264scTCR/multimer was assumed to also contain two PE molecules. The peptide/HLA tetramers are thought to cross-link at least three different TCRs on the T cell surface (16). Assuming the same kind of interaction for 264scTCR/multimers binding to cell surface peptide/HLA complexes, the following formula was used for calculation of peptide/MHC complexes on the cell surface: PE per cell/2 x 3 = peptide/MHC complexes per cell.

To stain cells with anti-HLA-A2 Abs, 2 x 105 cells were stained with 1 µg of MA2.1 for 30 min at room temperature and washed once. Cells were stained with 2 µg of the secondary Ab PE-conjugated goat anti-mouse Ab for 30 min at room temperature and washed once. Cells stained with the equivalent amount of anti-Stx2 Ab (clone 11E10) were set as the isotype control. All samples were analyzed as described above.

Western blotting and immunofluorescence

Tumor cell lines cultured in flasks were collected by trypsinization when cell concentrations were at 50% confluence. Cells were washed once with IMDM supplemented with 10% FBS and once with PBS. Cells were extracted in cell lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 1 mM Na3OV4, 5 mM NaF, and 1x protease inhibitor mixture (Sigma-Aldrich)). Protein concentrations of cell extracts were determined using protein assay dye reagent (Bio-Rad) at 595 nm using known concentration BSA as standard. Protein (100 µg) from each cell line was fractionated under reducing conditions in precast 4–20% gradient Tris-glycine polyacrylamide gel (Cambrex) and transferred onto a 0.2-µm nitrocellulose membrane (Pierce). The membranes were blocked with Odyssey blocking buffer (LI-COR) for 1 h. Mouse anti-human p53 Ab (DO-12) and the loading control, Ab rabbit anti-human GRB2 Ab, were diluted 1000-fold in Odyssey blocking buffer and incubated with the membrane overnight at 4°C. The membrane was washed four times at 5-min intervals with buffer (PBS plus 0.1% Tween 20) and incubated with IRDye 800-conjugated anti-mouse IgG or IRDye 700DX-conjugated anti-rabbit IgG (diluted 10,000-fold in Odyssey blocking buffer) for 1 h at room temperature. The washing steps were repeated, and the results were visualized with the Odyssey scanner using Odyssey analysis software version 1.1 (LI-COR). The IRDye 800 intensity was scored from – to ++++.

For immunofluorescence, tumor cell lines were cultured on BD Falcon culture slides (BD Biosciences) overnight. The cells were fixed with 3.7% formaldehyde for 10 min and permeabilized with 0.2% Triton X-100 for 10 min. Cells were stained with mouse anti-human p53 Ab (PAb1801) at 10 µg/ml in 10% normal goat serum for 1 h. Cells were washed twice with PBS and stained with FITC-conjugated goat anti-mouse IgG (1/100 dilution) for 1 h. Cells were washed twice with PBS and once with neutralized buffer (Molecular Probes). Slides were mounted in Slow-Fade medium (Invitrogen Life Technologies) and sealed with nail oil for the fluorescence photomicrography. The fluorescence intensity was scored from – to ++++.

Immunohistochemistry

A375, MDA-MB-231, CaSKi, SW-480, or HT-29 cells were s.c. implanted into athymic nude mice (nu/nu; Harlan Breeders). When tumors reached a volume of 100–200 mm3 they were excised, fixed in buffered formalin for 24 h, dehydrated, and embedded in paraffin. Tumors were sectioned to 5 µm and mounted on charged glass slides (Fisher Superfrost Plus; Fisher Scientific).

The Formalin-fixed, paraffin-embedded human breast cancer tissue microarrays (TMAs), and the normal TMAs were purchased from Cytomyx. Human colorectal cancer TMAs were purchased from Imgenex. For each of the TMAs, the vendors included the specimen clinical pathology report as well as the corresponding patient data.

For all immunohistochemistry, paraffin sections were dewaxed in xylene and rehydrated in a descending series of ethanol. Sections were treated with 3% H2O2 for 10 min to abolish any endogenous peroxidase activity followed by Ag retrieval with BD Retrievagen A (BD Pharmingen) for 20 min in a 97°C water bath and cooled for 20 min. Nonspecific binding was blocked with 1% normal mouse serum for scTCR staining or 1% normal goat serum for anti-p53 or anti-HLA-A2 staining for 30 min. For scTCR staining, tumor sections were incubated with HRP-conjugated 264scTCR/multimer (1.25–2.5 µg of monomeric 264scTCR) or equivalent amounts of control HRP-conjugated CMVscTCR/multimer for 1 h in the presence or absence of anti-HLA-A2 Abs (2 µl of 0397HA and 2 µl of 0791HA). For p53 or HLA-A2 staining, tumor sections were first stained with 3 µg of anti-p53 (PAb1801) or anti-HLA-A2 Abs (1/500 dilution) for 30 min, followed with HRP-conjugated goat anti-mouse IgG (2.5 µg) for 30 min. Between each step, slides were washed three times for 5 min each with TBS and 0.1% Tween 20. Peroxidase activity was developed using a 3,3'-diaminobenzidine tetrahydrochloride-hydrogen peroxide solution (DakoCytomation) for 2 min. Sections were counterstained with hematoxylin, dehydrated in ethanol, and mounted with Permount (Fisher Scientific).

Animals

All animals in these studies were treated in accordance to the provisions of the Animal Welfare Act of 1996 (Public Law 89.544) plus subsequent amendments as well as standards set forth in National Institutes of Health (Bethesda, MD) publication 85-23 (17). All research was approved by the Institutional and Animal Care and Use Committee of Altor Bioscience Corporation as required by the Health Resource Extension Act of 1995.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Construction of the TCR fusion proteins

We have constructed a fusion protein comprising a three-domain scTCR that recognizes an unmutated peptide spanning aa 264–272 derived from human p53 presented in the context of HLA-A2.1 (6, 7). The scTCR portion of this fusion protein is composed of the TCR V{alpha} domain linked to the Vbeta/Cbeta domains via a flexible linker (18). The Cbeta domain, which is directly linked to the Vbeta domain, is truncated at the amino acid residue just before the final cysteine to generate a soluble scTCR molecule. This scTCR is linked to a birA peptide tag to allow for biotinylation and subsequent multimerization in the presence of streptavidin.

Characterization of the 264scTCR/birA fusion protein

Purified 264scTCR/birA fusion protein was subjected to SDS-PAGE and Coomassie G-250 staining (Fig. 1A). The SDS gel analysis and size exclusion chromatogram showed that 264scTCR/birA was a monomeric protein with an approximate molecular mass of 60 kDa. This value is larger than the calculated molecular mass of the protein of 46.5 kDa, suggesting that the 264scTCR/birA is glycosylated. To confirm that the 264scTCR/birA fusion protein could be biotinylated, the 264scTCR/birA monomer was incubated in the presence of excess streptavidin at various molar ratios. At a 264scTCR/birA monomer/streptavidin molar ratio of 4:1, most of the 264scTCR/birA monomer was converted to dimers, trimers, or tetramers (i.e., 264scTCR/multimer), as displayed in Fig. 1A (Fig. 1A, lane 4). At ratios of 8:1 and 16:1 the multimerization process appeared to be less efficient, leading to the production of fewer multimers and excess unbound 264scTCR/birA monomer (Fig. 1A, lanes 5 and 6). Thus, the 264scTCR/birA fusion protein can be biotinylated to create 264scTCR/birA monomers, which can subsequently be used to form 264scTCR/multimers in the presence of streptavidin (Fig. 1B).


Figure 1
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  		FIGURE 1. Multimerization of 264scTCR. A, SDS-PAGE analysis of the 264scTCR/birA fusion protein. Lane 1, 264scTCR/birA fusion protein; lane 2, streptavidin; lane 3, biotinylated 264scTCR/monomer; lane 4, 4:1 ratio of 264scTCR/monomer to streptavidin; lane 5, 8:1 ratio of 264scTCR/monomer to streptavidin; lane 6, 16:1 ratio of 264scTCR/monomer to streptavidin. B, Schematic representation of the 264scTCR/multimer.

 
264scTCR binding to peptide/MHC complexes

The binding ability of the 264scTCR/multimer was compared with that of the molar equivalents of the 264scTCR/birA monomer and a dimeric form of 264scTCR (264scTCR/IgG1) by staining p53264–272 peptide-loaded T2 cells. As shown in Fig. 2A, staining with the 264scTCR/birA monomer was weak, whereas staining with 264scTCR/IgG1 dimer or 264scTCR/multimer was substantially more intense. However, the background staining with 264scTCR/IgG1 was higher than with the PE-conjugated 264scTCR/multimer, likely due to nonspecific binding of the IgG1 portion of the molecule. Repeated experiments confirmed these observations, indicating that staining with 264scTCR/multimer was more intense with less background than staining with the 264scTCR/IgG1 dimer or the 264scTCR/birA monomer. Thus, it appears to be possible to improve the avidity/affinity of the 264scTCR molecule for peptide-loaded APCs by increasing its valency. The results of this experiment are in accord with recent observations made by Laugel et al. (19) that tetrameric TCRs bind to peptide/MHC complexes better than monomeric TCRs.


Figure 2
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  		FIGURE 2. Peptide/MHC binding ability of 264scTCR fusion protein. A, T2 cells pulsed with p53264–272 peptide were stained with the 264scTCR/monomer, 264scTCR/IgG1, or 264scTCR/multimer. Unpulsed T2 cells stained with these reagents were the controls. B, Level of fluorescence intensity of T2 cells pulsed with increasing concentrations of p53264–272 peptide and stained with PE-conjugated 264scTCR/multimer.

 
To evaluate the sensitivity of the 264scTCR/multimer as a staining reagent, we performed flow cytometric analysis with T2 cells loaded with various concentrations of cognate peptides. The 264scTCR/multimer was able to stain T2 cells loaded with as little as 12 nM p53264–272 peptide (Fig. 2B). To estimate the numbers of PE molecules per cell, the fluorescent staining intensities obtained at the various concentrations of peptide were compared with a standard curve of PE-coupled calibration beads (QuantiBRITE PE beads; BD Biosciences) carrying known quantities of PE per bead (Fig. 2B). When T2 cells were loaded with 12 nM cognate peptide, ~203 PE molecules on each cell were detected. Based on the assumed ratio of PE per peptide/MHC binding of the 264scTCR/multimer, this result suggests that a minimum of 300 p53264–272/HLA-A2.1 complexes can be detected on the cell surface with a PE-conjugated 264scTCR/multimer.

Binding avidity/affinity of 264scTCR/multimers

To determine whether the improved avidity/affinity of the 264scTCR/multimer could be attributed to its stronger staining capability compared with that of the 264scTCR/birA monomeric fusion protein, surface plasmon resonance analysis was performed to compare the binding affinities of the 264scTCR/multimer and the 264scTCR/birA monomer for peptide/MHC molecules (Table I). In this analysis, the equilibrium binding response of the interaction between the 264scTCR/birA monomer and p53264–272/HLA-A2.1 complexes was plotted for each protein concentration using a nonlinear fit of the Langmuir isotherm assuming a one to one interaction. The mean equilibrium binding response (KD) of three independent measurements was 0.527 µM, which falls in the high affinity range of typical KD values for TCR to peptide/MHC interactions (range of 0.1–90 µM) (20). The time necessary to reach equilibrium binding with p53264–272/HLA.A2.1 was similar for both the 264scTCR/birA monomer and the 264scTCR/multimer (Ka of 2.4 x 104 M–1s–1 and 2.3 x 104 M–1 s–1, respectively). The peptide/MHC specificity of the 264scTCR/multimer was not affected, as no binding was detected when irrelevant CMVpp65495–503/HLA-A2.1 complexes were immobilized on the flow cell (data not shown). However, the dissociation rate constant Kd of the 264scTCR/multimer (4.07 x 10–3 s–1) was greatly reduced relative to the Kd of 264scTCR/birA monomer (9.77 x 10–2 s–1), which corresponds to a binding half-life of 1201 s for the 264scTCR/multimer and 7.2 s for the 264scTCR/birA monomer. Thus, the 264scTCR/multimer exhibits a 167-fold longer binding half-life compared with the 264scTCR/birA monomer, which likely accounts for the enhanced binding ability of the 264scTCR/multimer fusion protein.


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  		Table I. Equilibrium and kinetic binding constants of 264scTCR monomer and 264scTCR multimer

 
Multimerized 264scTCR maintains specificity for p53264–272/HLA.A2.1

A potential problem with increasing the avidity/affinity of the 264scTCR molecule via multimerization is that its peptide specificity could be compromised. However, the 264scTCR/multimer retained its highly specific interaction with p53264–272/HLA-A2.1 complexes and did not bind nonspecifically to CMVpp65495–503/HLA-A2.1 complexes in the surface plasmon resonance analysis. To further demonstrate the peptide specificity of the 264scTCR/multimer, flow cytometry was used to evaluate peptide-specific binding when the PE-conjugated 264scTCR/multimer was applied to T2 cells pulsed with 10 HLA-A2.1-restricted noncognate peptides in addition to its cognate p53264–272 peptide. PE-conjugated 264scTCR/multimer intensely stained p53264–272 peptide-loaded T2 cells with a MFI of >400, whereas staining of noncognate peptide-loaded T2 cells remained at background levels (Fig. 3A), although these peptides bind to HLA-A2.1 with affinities similar to those of p53264–272 (Fig. 3A). The MFI of hepatitis B virus (HBV) polymerase (Pol)575–583-loaded (50 µM) T2 cells stained with PE-coupled 264scTCR/multimer was twice background level (Fig. 3A), but staining was reduced to background levels when the HBV Pol575–583 peptide concentration was reduced to 6 µM (Fig. 3B). However, the peptide specificity of the 264scTCR/multimer was best demonstrated by comparing staining of T2 cells pulsed with human p53264–272 to T2 cells pulsed with the mouse equivalent p53261–269 peptide. Mouse p53261–269 and human p53264–272 are identical with the exception of a single amino acid residue, and both demonstrate similar binding affinities to HLA-A2.1 (Fig. 3A) (21, 22, 23). However, despite this minor amino acid difference, the 264scTCR/multimer can discriminate between the two peptides as demonstrated by its robust staining of human p53264–272-pulsed T2 cells compared with background staining of mouse p53261–269-pulsed T2 cells (Fig. 3A).


Figure 3
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  		FIGURE 3. Specificity of 264scTCR/multimer. A, T2 cells pulsed with 50 µM various peptides known to be associated with HLA-A2 and stained with PE-conjugated 264scTCR/multimer. B, T2 cells pulsed with 0, 6, or 50 µM p53264–272 or human B-type hepatitis virus HBV Pol575–583 and stained with the PE-conjugated 264scTCR/multimer.

 
Detection of p53264–272/HLA-A2.1 complexes naturally displayed on tumor cells

To determine whether the discriminatory potential of the 264scTCR/multimer extended to endogenous p53264–272/HLA-A2.1 complexes presented on the surface of human tumor cells, flow cytometry was performed on a panel of tumor cell lines which varied with respect to p53 and HLA-A2.1 expression. The HLA-A2.1 status of these cell lines were confirmed by flow cytometry assays with anti-HLA-A2 Ab (MA2.1); the p53 status of these cell lines were confirmed by Western blotting and immunofluorescence with the anti-p53 Abs (DO-12 and PAb1801, respectively) as described in the Materials and Methods and summarized in Fig. 4C.


Figure 4
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  		FIGURE 4. HLA-A2.1 restricted detection of endogenous p53/HLA-A2.1 complexes on the surface of tumor cells with 264scTCR/multimer. A, A375, MDA-MB-231, CaSKi, SW-480, HT-29, SaOS-2, and AsPC-1 cell lines were stained with mouse anti-HLA-A2 Ab MA2.1 and analyzed with flow cytometry. The average MFI ± SD from 4 to 11 experiments are reported. The average MFIs of A375, MDA-MB-231, CaSKi, and SW-480 cells were significantly higher than the average MFIs of HT-29 cells (p values of <0.03; Student’s t test). The average MFIs among HT-29, SaOS-2, and AsPC-1 cells were not significantly different (p values of >0.1). B, A375, MDA-MB231, and CaSKi cells were stained with PE-coupled 264scTCR/multimer in the absence (black thick line) or presence of the various blocking Abs, MA2.1 (black thin line), BB7.2 (light gray thick line), or W6/32 (light gray thin line) or with PE-conjugated streptavidin as a negative control (light gray shade). C, The MFIs of staining with the anti-HLA-A2.1 Ab (MA2.1) were graded on a – to ++++ scale. The cell lines were stained with anti-p53 Abs (PAb1801 or DO-12) and analyzed with immunofluorescence photomicrography or Western blotting. The data from immunofluorescence and Western blotting were exhibited as – to ++++. The cell lines were stained with PE-conjugated 264scTCR/multimer and analyzed by flow cytometry. Numbers represent calculated p53264–272/HLA-A2.1 complexes per cell (p53A2.1).

 
PE-conjugated 264scTCR/multimer could detect p53264–272/HLA-A2.1 complexes on all four p53+/HLA-A2.1+ tumor cell lines (A375, MDA-MB-231, CaSKi, and SW-480) but not on p53+/HLA-A2.1– HT-29 cells, p53–/HLA-A2.1+ SaOS-2 cells, or p53–/HLA-A2.1– AsPC-1 cells (Fig. 4A). However, three p53+/HLA-2.1+ cell lines, BT-549, MCF-7, and SCC-9, were not stained with PE-conjugated 264scTCR/multimer (data not shown), although these tumor cell lines can be recognized by p53264–272/HLA-A2.1-specific CTLs (23, 24). This finding suggests that the peptide displayed by these tumor cells might be below the level of detection for the 264scTCR/multimer. Therefore, peptide displaying >500 copies could be readily and specifically detected with the 264scTCR/multimer (Fig. 4C).

The specificity of the PE-conjugated 264scTCR/multimer was further confirmed by blocking experiments using various HLA-specific Abs. When the HLA class I-specific (W6/32) or HLA-A2-specific (MA2.1) Abs were added to 264scTCR/multimer staining experiments, specific staining of the three p53+/HLA-A2.1+ human tumor cells was reduced. Staining intensity was not reduced in the presence of the BB7.2 Ab (Fig. 4B). Because the binding sites of W6/32, MA2.1, and BB7.2 on HLA class I and HLA-A2 are well defined (25, 26), these data suggest that the epitopes recognized by W6/32 and MA2.1 are critical for 264scTCR/multimer binding. This possibility is consistent with other reports suggesting that the BB7.2 Ab does not block target cell recognition by p53264–272 specific T cells (21, 24).

Detection of p53264–272/HLA-A2.1 complexes on tumor xenografts

To determine whether the 264scTCR/multimer could specifically recognize peptide/MHC complexes in solid tumors, various human tumor cells were grown in nu/nu mice. Established tumors were excised, fixed in formalin, and stained with a HRP-conjugated 264scTCR/multimer to assess the ability of the molecule to stain more clinically relevant tumor samples. Tumor sections made from the p53+/HLA-A2.1+ cell lines (A375, MDA-MB-231, SW-480, and CaSKi) stained strongly with the 264scTCR/multimer (Fig. 5A), whereas staining of the p53+/HLA-A2.1– HT-29 tumors was similar to that of background. To confirm the specificity of staining, A375 tumors were stained with molar equivalents of either the control CMVscTCR/multimer or the 264scTCR/multimer in the presence or absence of anti-HLA-A2 Abs. The A375 tumor stained intensely with 264scTCR/multimer but did not stain with CMVscTCR/multimer. Furthermore, staining with the 264scTCR/multimer was reduced in the presence of the anti-HLA-A2 Abs (Fig. 5B). To the best of our knowledge, this is the first evidence showing that a soluble TCR-based molecule has the ability to bind to peptide/MHC complexes on formalin-fixed tissue sections.


Figure 5
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  		FIGURE 5. Detection of p53/HLA-A2.1 complexes on xenograft tumor with the 264scTCR/multimer. A, Xenograft tumor sections derived from p53+/HLA-2.1+ cells lines, A375 cells, MDA-MB231 cells, CaSKi cells, SW-480 cells, or p53+/HLA-2.1– HT-29 cells were stained with HRP-conjugated 264scTCR/multimer or HRP-conjugated streptavidin as a control (magnification x400). B, Xenograft sections derived from p53+/HLA-2.1+ A375 cells stained with irrelevant HRP-conjugated CMVscTCR/multimer or HRP-conjugated 264scTCR/multimer in the presence or absence of the anti-HLA-A2 Ab blocking reagent (magnification x400).

 
p53264–272/HLA-A2.1 complexes are not detected on normal human tissues

To determine the feasibility of using the 264scTCR/multimer to stain human tissues, we obtained normal human TMAs. The formalin-fixed, paraffin-embedded normal TMAs, including sections from adrenal gland, brain, tonsil, breast, colon, heart, kidney, liver, lung, lymph node, muscle, placenta, prostate, spleen, testis, and thyroid, were stained with 264scTCR/multimer and the control CMVscTCR/multimer as well as HLA-A2 and p53 Abs (data not shown). Cytoplasmic staining and cell membrane staining were observed with anti-HLA-A2 Abs in 18 of 65 normal tissue specimens. Positive nuclear staining for p53 was observed in only 3 of 65 normal tissue specimens, which is consistent with p53 expression generally being below levels of detection in normal cells (27, 28). For the majority of normal tissues, little to no staining was observed using 264scTCR/multimer or control CMVscTCR/multimer. In some tissues such as liver, kidney, and muscle, some background staining was observed (data not shown). The observation that normal colon and breast tissues showed background staining with 264scTCR/multimer prompted us to explore the staining of actual tumor samples derived from these human tissues.

Detection of p53264–272/HLA-A2.1 complexes on tumors from cancer patients

To determine whether the 264scTCR/multimer could stain human tumor sections, formalin-fixed, paraffin-embedded human colorectal cancer TMAs (59 specimens) and human breast cancer TMAs (138 specimens), along with their normal tissue counterparts, were stained with either 264scTCR/multimer, p53-, or HLA-A2.1-specific Abs. As summarized in Table II, 49% of the colorectal cancer specimens and 34% of the breast cancer specimens were stained positively with 264scTCR/multimer as compared with its control CMVscTCR/multimer (data not shown). Collectively, 41% of the colorectal cancer specimens and 75% of the breast cancer specimens stained positively with anti-HLA-A2 Ab, whereas 37% of the colorectal cancer specimens and 26% of the breast cancer specimens showed tumor cell nuclei staining with the anti-p53 Ab. One of six of the normal colorectal tissue specimens and two of fifteen of the normal breast tissue specimens stained positively with anti-HLA-A2 Abs, whereas none of the normal colorectal tissue specimens and only three of sixteen normal breast tissue specimens stained positively with anti-p53 Abs (data not shown). A highly significant correlation (Tables III and IV) was observed between HLA-A2-positive status of the 264scTCR/multimer-positive specimens in both the colorectal cancer (p < 0.019) and breast cancer (p < 0.003) TMAs. Approximately 94% of 264scTCR/multimer-positive breast cancer specimens and 88% of the 264scTCR/multimer-positive colorectal cancer specimens were stained with the HLA-A2 Ab, as anticipated based on the HLA-restricted binding of the 264scTCR reagent. These results also indicate that the level of false positive 264scTCR/multimer staining (i.e., 264scTCR+/HLA-A2–) of the whole sample set was <5%. Interestingly, 264scTCR+ status did not correlate with positive nuclear staining by the anti-p53 Ab (Tables III and IV). Of the HLA-A2+ 264scTCR/multimer+ specimens, ~55% of the colorectal cancer samples and 74% of the breast cancer samples were p53– (Table II). These cases may exemplify differences in the nature of the p53 Ags recognized by the 264scTCR/multimer and anti-p53 Abs and suggest that a large population of p53264–272/HLA-A2.1 peptide-presenting tumors cannot be identified solely using Ab-based reagents. Similar results were observed in tumor cell staining studies where the enhanced ability of the 264scTCR/multimer to detect p53 peptide presentation on cell surfaces appeared in some cases to be independent of the level of nuclear staining with p53 Abs (Fig. 4, A and C).


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  		Table II. Colorectal and breast cancer TMAs stained with antibodies against p53 or HLA-A2 or with the 264scTCR/multimera

 

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  		Table III. Correlation between 264scTCR/multimer positive staining with other patient variables from the colorectal carcinomas TMAs

 

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  		Table IV. Correlation between 264scTCR/multimer positive staining with other reported patient variables from the breast cancer TMAs

 
For 15% of the colorectal cancer specimens and 9% of the breast cancer specimens, identical positive staining patterns were observed with all three reagents (Table II). A sample representative staining result with human normal or cancerous breast and colorectal tissues using the 264scTCR/multimer, the control CMVscTCR/multimer, and the HLA-A2.1 and p53 Abs is shown in Fig. 6. The 264scTCR/multimer exhibits Ag-specific staining capability on formalin-fixed cancer tissues (Fig. 6B). The background staining with this reagent on normal colorectal and breast tissues is low (Fig. 6A), which exemplifies the utility of 264scTCR/multimer as a reagent to detect presentation of tumor-associated peptide/MHC complexes and aid in the identification of patients that might respond to TCR-based immunotherapeutics.


Figure 6
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  		FIGURE 6. Detection of p53/HLA-A2.1 complexes on TMAs from normal or cancerous human tissues with 264scTCR/multimer. A, TMAs from normal colorectal or breast tissues were stained with HRP-conjugated 264scTCR/multimer. B, TMAs from cancerous colorectal and breast tissues stained with HRP-conjugated 264scTCR/multimer, negative control HRP-conjugated 264scTCR/multimer, anti-HLA-A2 Ab, or anti-p53 Ab (magnification x200).

 
To determine whether there was a correlation between 264scTCR/multimer staining and other reported patient variables (as listed in Tables III and IV), we performed statistical analyses ({chi}2 test) for both the 59 colorectal carcinomas (stages II–IV) and 138 breast carcinomas (stages 0–IV or unknown). Among colorectal cancer patients, positive specimen staining with the 264scTCR/multimer was significantly associated with positive staining by the HLA-A2 Abs (p < 0.019). In contrast, positive staining with the 264scTCR/multimer was not correlated with other variables such as age, gender, tumor location, tumor differentiation, tumor type, tumor stage, or TNM classification. Likewise among breast cancer patients, positive specimen staining with the 264scTCR/multimer was significantly associated with positive staining by the HLA-A2 Abs (p < 0.003), but not with other patient characteristics. Collectively, these results demonstrate that scTCR multimers could be used as tissue staining reagents in a traditional pathology laboratory setting for Ag verification.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In these studies we have described the construction and characterization of a multimeric form of novel 264scTCR protein (264scTCR/multimer), which recognizes a distinct, naturally processed peptide fragment of human p53 presented in the context of HLA-A2.1. The multimer is capable of specifically staining unmanipulated human tumor cells in flow cytometric analyses. In immunohistochemical staining studies, the multimer can be used to differentiate normal tissues from human tumor tissues. The multimeric 264scTCR can also be used to quantify the p53264–272/HLA-2.1 complexes on the surface of tumor cells. Thus, we have demonstrated that multimeric scTCRs can be used as valuable new tools to visualize and quantify peptide/MHC complexes presented on the surface of tumors or APCs in a simple laboratory setting.

TCRs are known to have low affinity for cognate peptide/MHC complexes due to negative selection in the thymus during the T cell maturation process (29, 30). In addition, the density of a particular epitope displayed in the context of MHC on a particular APC or tumor surface is predictably low (31, 32). These two factors have raised significant doubts about whether it is feasible to use TCRs as staining reagents. However, we have evidence that these difficulties can be overcome by simply increasing the avidity of the scTCR molecules. In our previous study (6) we demonstrated that dimeric 264scTCR fused to the Ig H chain (264scTCR/IgG1) has sufficient affinity to function as an Ag recognition molecule in vitro and can be used as a staining reagent for unmanipulated tumor cells. In the more comprehensive study reported here, we observed that the 264scTCR/IgG1 fusion molecule exhibits some degree of nonspecific background staining. The nonspecific staining of tissue attributed to the 264scTCR/IgG1 fusion molecule is likely due to nonspecific binding of the IgG1 domain of the fusion molecule by the Fc{gamma}R displayed on cell surfaces. The use of 264scTCR/multimer overcomes this technical shortcoming, because the IgG1 H chain portion of the molecule has been removed, leaving only the portion expected to bind to peptide/MHC complexes.

The avidity/affinity of the 264scTCR/birA monomer for its cognate peptide/MHC complex falls in the high affinity range of the typical KD values for TCR/MHC-peptide interactions (33). The association rate constants (Ka values) for the 264scTCR/multimer and 264scTCR/birA monomer were similar; however, the dissociation rate constant (Kd) of 264scTCR/multimer was greatly reduced relative to the Kd of the 264scTCR/birA monomer. Thus, multimerization of the scTCR results in a reagent that binds to peptide/MHC complexes with a half-life of 20 min compared with ~7 s for the monomer. Similar observations related to changes in peptide/MHC binding kinetics with this type of molecule by multimerization have been reported by other laboratories (19, 34). In addition, we have previously shown that dimerization of 264scTCR using an Ig H chain tail did increase both the Ka and Kd of the molecule while binding to the p53264–272/HLA-A2.1 complexes. Such a difference in the binding kinetics of these molecules for identical cognate peptide/MHC complexes may be the result of the spatial arrangement differences between the binding domains on the Ig frame or the streptavidin molecule surface. It is unclear at this time whether such a difference in spatial display would affect the performance of these molecules for various applications.

One potential problem with increasing the avidity/affinity of a soluble TCR molecule by multimerization is that its peptide specificity may be compromised. Fortunately, this does not appear to be the case for 264scTCR. The 264scTCR/multimer recognized HLA-A2.1-presented cognate peptides (p53264–272-pulsed T2 cells) but not HLA-A2.1-presented noncognate peptides (noncognate peptide-pulsed T2 cells). Even a single amino acid difference between mouse p53261–269 and human p53264–272 could be discriminated by the 264scTCR/multimer fusion protein.

There have been several other attempts to develop TCR-based flow cytometry staining methods for detecting peptide/MHC presentation (19, 34, 35, 36). Although detection of artificially manipulated peptide/MHC complexes (e.g., loaded with exogenous peptide or elevated by beta2-microglobulin stabilization or IFN-{gamma} treatment) was observed, the reported methods were not sensitive enough to detect endogenous Ag presentation by unmanipulated cells (19, 34, 35, 36). Comparisons between the minimum amounts of exogenous peptide required to achieve TCR staining suggest that the methods described here are significantly more sensitive than those reported by others. For example, Laugel et al. (19) reported that staining peptide-loaded APCs with either JM22 (Flu matrix-specific) or A6 TCR (human T cell lymphotropic virus type 1 Tax-specific) multimers required at least 10 µM cognate peptide. This detection threshold could be lowered to 1 µM peptide following MHC stabilization by exogenous beta2-microglobulin. However, we found that the 264scTCR/multimers could stain cells pulsed with as little as 12 nM peptide and that the addition of beta2-microglobulin was not required. Because the A6 TCR has a KD of ~1 µM for Tax11–19 peptide/HLA-A2.1 complex (19), which is similar to that of the 264scTCR for its peptide/MHC complex, the difference in sensitivity between the 264scTCR and A6 TCR in flow cytometry analyses is not due to large differences in affinities for their cognate peptide/MHC complexes. Whether this reflects a difference in the relative stability of the peptide/MHC complexes per se or structural differences resulting from using a single chain vs a heterodimer format for each TCR multimer is unknown.

TCR-based staining methods could have a number of applications in analyzing Ag presentation in different cells or tissues. In this study, we have shown that we can use the multimeric 264scTCR in flow cytometry analyses to quantify the number of p53264–272/HLA-A2.1 complexes on the surface of a panel of unmanipulated tumor cell lines that vary with respect to p53 and HLA-A2 expression. HLA-A2-restricted T cell epitopes on human tumor cell surfaces have been reported to number ~20–4000 molecules per cell (37), and T cells have been reported to be able to recognize as few as one antigenic molecule per cell (38, 39). Our studies with peptide-loaded T2 cells demonstrate that 264scTCR/multimer can detect as few as 300 p53264–272/HLA-A2 complexes on the cell surface by flow cytometry with a detection limit for PE at >150 molecules per cell. The four p53+/HLA-A2+ cell lines that display the p53264–272 peptide (A375, MDA-MB-231, CaSKi, and SW480) stained intensely with 264scTCR/multimer. Based on this level of staining, we estimate that these cell lines display between 500 and 2000 p53264–272/HLA-A2.1 complexes on their surfaces. However, three other p53+/HLA-A2.1+ cell lines (SCC-9, MCF-7, and BT549), which are documented to be targeted by HLA-A2.1-restricted and p53 peptide-specific CTLs (23, 40, 41), stained very weakly with 264scTCR/multimer (data not shown), suggesting that these cell lines do not display high levels of p53264–272 peptide on their surfaces. The p53264–272 peptide levels displayed on the cell surface required the presence of both HLA-A2 and p53 but did not necessarily correlate with their expression levels as measured by the intensity of staining with their respective Abs. For example, A375 and CaSKi cells expressed moderate levels of HLA-A2 and low levels of p53 but were intensely stained with 264scTCR/multimer. MDA-MB-231 and SW-480 cells expressed high levels of both HLA-A2 and p53 but were only moderately stained with 264scTCR/multimer when compared with A375 and CaSKi cells. This finding suggests that the 264scTCR/multimer measures different parameters than either of the anti-HLA-A2 or anti-p53 Abs alone. This may be the result of differences in Ag processing and presentation among various cell lines. If that is indeed the case, the 264scTCR/multimer represents a new generation reagent that may be used to directly estimate epitope presentation on APCs.

One potential application is the use of scTCR/multimers in the development of dendritic cell-based vaccine approaches. Vaccination with peptide-pulsed dendritic cells has been shown to induce tumor Ag-specific immunity and to provide clinical benefit in cancer patients. However, characterization of the peptide-pulsed cells generally relies on indirect and lengthy methods, such as CTL-specific cytotoxicity assays, to assess Ag presentation. We have found that amount of peptide (i.e., ~10 nM) required to stain T2 cells with the 264scTCR/multimer is the same as the amount reported to be required for CTL-mediated T2 cell lysis with the parental T cell line expressing the p53264–272 TCR (21). Thus, scTCR/multimer staining methods appear to be as sensitive as current methods and could provide a more direct approach to optimizing and validating the production of APC therapies.

Another application is the development of a simple immunochemistry approach for Ag identification and validation and for patient eligibility screening for TCR-based therapeutics. In this study, we have demonstrated that the 264scTCR/multimer could be used as an immunohistochemical staining reagent to detect and visualize the presence of p53264–272/HLA-A2.1 complexes on human tumor tissues either from xenografts or TMA collections after chemical fixation. These results indicate that the conformation of the peptide/MHC complexes recognized by scTCRs is well preserved during standard tissue fixation. However, the converse situation with HLA tetramers has not held true, as fixation results in decreased staining (42). We believe that this might simply reflect effects of the fixing reagent on certain domains of TCRs necessary for HLA tetramer binding. It is well known that fixatives can affect the ability of cell surface Ags to be stained by Abs (independent of the level of Ag present). Thus, TCR/multimer reagents could be a valuable tool in pathology laboratories to screen patients for targeted immunotherapeutics and to monitor peptide/MHC complex expression/alterations on tumor cells vs distant metastases. We are currently expanding our initial study to examine a broader range of tissue samples and to develop a tiered scoring system to assess staining with the 264scTCR/multimer, anti-HLA-A2 Ab, and anti-p53 Ab reagents.

TCR-like Abs, which share many characteristics with scTCR multimers, have recently been isolated and characterized (43, 44, 45, 46, 47, 48). These Abs could prove useful as staining reagents to visualize or quantify peptide/MHC complexes on the surface of APCs. However, detailed studies comparing the peptide/MHC recognition of a panel of such Abs and TCRs reveal a number of important differences, suggesting that the TCRs and Abs bind to overlapping but nonidentical components on the peptide/MHC complex (37, 49, 50). For example, the MHC component appears to play a more significant role in Ab recognition than what is seen in TCR to peptide/MHC interactions. In addition, variants of the peptide-MHC complex can significantly inhibit or enhance recognition by the Abs without having any effect on TCR binding (37). Because of these differences in fine specificity, it is likely that the "TCR-like" Abs will react to a different spectrum of MHC/peptide complexes than those recognized by TCR reagents or by T cells themselves. Such an outcome may limit the usefulness of these Abs in validating targets of T cell-based therapies.

We envision that our scTCR/multimer methods could also be applied to the characterization of viral Ags. In addition, further improvement in Ag recognition affinity should yield an even more sensitive and specific screening reagent. Previous studies have shown that it is possible to increase the affinity of scTCRs 100-1000-fold by mutating the CDR3{alpha} loop region without significantly compromising their Ag recognition specificity (51). We are currently using a mutagenesis strategy and phage display of functional scTCR/selection methodologies to improve the affinity scTCR staining reagents (52).


   Acknowledgments
 
We thank Kimberlyn F. Card, Altor BioScience Corp., Miramar, FL, for thoughtful review of this manuscript, and Drs. Joseph T. Neary and Yuan Kang of University of Miami, Miami, FL, for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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 study was supported in part by National Institutes of Health Grants 1R43 CA105816 and 1R43 CA097550. Back

2 Address correspondence and reprint requests to Dr. Hing C. Wong, Altor BioScience Corp., 2810 North Commerce Parkway, Miramar, FL 33025. E-mail address: hingwong{at}altorbioscience.com Back

3 Abbreviations used in this paper: scTCR, single-chain T-cell receptor; 264scTCR, scTCR-recognized wild type human p53 peptide (aa 264–272) presented in the context of HLA-A2.1; CMVscTCR, scTCR-recognized CMV pp65 peptide (aa 495–503) presented in the context of HLA-A2.1; CHO, Chinese hamster ovary; HBV, hepatitis B virus; MFI, mean fluorescence intensity; Pol, polymerase; TMA, tissue microarray. Back

Received for publication September 30, 2005. Accepted for publication December 30, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Mosolits, S., B. Nilsson, H. Mellstedt. 2005. Towards therapeutic vaccines for colorectal carcinoma: a review of clinical trials. Expert Rev. Vaccines 4: 329-350. [Medline]
  2. Restifo, N. P., S. A. Rosenberg. 2005. Use of standard criteria for assessment of cancer vaccines. Lancet Oncol. 6: 3-4. [Medline]
  3. Hughes, M. S., Y. Y. Yu, M. E. Dudley, Z. Zheng, P. F. Robbins, Y. Li, J. Wunderlich, R. G. Hawley, M. Moayeri, S. A. Rosenberg, R. A. Morgan. 2005. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum. Gene Ther. 16: 457-472. [Medline]
  4. Zhou, J., M. E. Dudley, S. A. Rosenberg, P. F. Robbins. 2005. Persistence of multiple tumor-specific T-cell clones is associated with complete tumor regression in a melanoma patient receiving adoptive cell transfer therapy. J. Immunother. 28: 53-62.
  5. Mocellin, S.. 2005. Cancer vaccines: the challenge of developing an ideal tumor killing system. Front. Biosci. 10: 2285-2305. [Medline]
  6. Mosquera, L. A., K. F. Card, S. A. Price-Schiavi, H. J. Belmont, B. Liu, J. Builes, X. Zhu, P. A. Chavaillaz, H. I. Lee, J. A. Jiao, et al 2005. In vitro and in vivo characterization of a novel antibody-like single-chain TCR human IgG1 fusion protein. J. Immunol. 174: 4381-4388. [Abstract/Free Full Text]
  7. Card, K. F., S. A. Price-Schiavi, B. Liu, E. Thomson, E. Nieves, H. Belmont, J. Builes, J. A. Jiao, J. Hernandez, J. Weidanz, et al 2004. A soluble single-chain T-cell receptor IL-2 fusion protein retains MHC-restricted peptide specificity and IL-2 bioactivity. Cancer Immunol. Immunother. 53: 345-357. [Medline]
  8. Yu, Z., N. P. Restifo. 2002. Cancer vaccines: progress reveals new complexities. J. Clin. Invest. 110: 289-294. [Medline]
  9. Goldenberg, M. M.. 1999. Trastuzumab, a recombinant DNA-derived humanized monoclonal antibody, a novel agent for the treatment of metastatic breast cancer. Clin. Ther. 21: 309-318. [Medline]
  10. Birner, P., G. Oberhuber, J. Stani, C. Reithofer, H. Samonigg, H. Hausmaninger, E. Kubista, W. Kwasny, D. Kandioler-Eckersberger, M. Gnant, R. Jakesz. 2001. Evaluation of the United States Food and Drug Administration-approved scoring and test system of HER-2 protein expression in breast cancer. Clin. Cancer Res. 7: 1669-1675. [Abstract/Free Full Text]
  11. Ang, K. K., B. A. Berkey, X. Tu, H. Z. Zhang, R. Katz, E. H. Hammond, K. K. Fu, L. Milas. 2002. Impact of epidermal growth factor receptor expression on survival and pattern of relapse in patients with advanced head and neck carcinoma. Cancer Res. 62: 7350-7356. [Abstract/Free Full Text]
  12. Nakagawa, K., Y. Matsuno, H. Kunitoh, A. Maeshima, H. Asamura, R. Tsuchiya. 2005. Immunohistochemical KIT (CD117) expression in thymic epithelial tumors. Chest 128: 140-144. [Abstract/Free Full Text]
  13. Theobald, M., T. Ruppert, U. Kuckelkorn, J. Hernandez, A. Haussler, E. A. Ferreira, U. Liewer, J. Biggs, A. J. Levine, C. Huber, et al 1998. The sequence alteration associated with a mutational hotspot in p53 protects cells from lysis by cytotoxic T lymphocytes specific for a flanking peptide epitope. J. Exp. Med. 188: 1017-1028. [Abstract/Free Full Text]
  14. Reche, P. A., J. P. Glutting, E. L. Reinherz. 2002. Prediction of MHC class I binding peptides using profile motifs. Hum. Immunol. 63: 701-709. [Medline]
  15. Reche, P. A., J. P. Glutting, H. Zhang, E. L. Reinherz. 2004. Enhancement to the RANKPEP resource for the prediction of peptide binding to MHC molecules using profiles. Immunogenetics 56: 405-419. [Medline]
  16. McMichael, A. J., C. A. O’Callaghan. 1998. A new look at T cells. J. Exp. Med. 187: 1367-1371. [Free Full Text]
  17. Committee on Care and Use of Laboratory Animals. 1985. Guide for the Care and Use of Laboratory Animals, National Institutes of Health Publication 85-2 Department of Health and Human Services, Washington, DC.
  18. Huston, J. S., D. Levinson, M. Mudgett-Hunter, M. S. Tai, J. Novotny, M. N. Margolies, R. J. Ridge, R. E. Bruccoleri, E. Haber, R. Crea, et al 1988. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc. Natl. Acad. Sci. USA 85: 5879-5883. [Abstract/Free Full Text]
  19. Laugel, B., J. M. Boulter, N. Lissin, A. Vuidepot, Y. Li, E. Gostick, L. E. Crotty, D. C. Douek, J. Hemelaar, D. A. Price, et al 2005. Design of soluble recombinant T cell receptors for antigen targeting and T cell inhibition. J. Biol. Chem. 280: 1882-1892. [Abstract/Free Full Text]
  20. Davis, M. M., J. J. Boniface, Z. Reich, D. Lyons, J. Hampl, B. Arden, Y. Chien. 1998. Ligand recognition by {alpha}beta T cell receptors. Annu. Rev. Immunol. 16: 523-544. [Medline]
  21. Theobald, M., J. Biggs, D. Dittmer, A. J. Levine, L. A. Sherman. 1995. Targeting p53 as a general tumor antigen. Proc. Natl. Acad. Sci. USA 92: 11993-11997. [Abstract/Free Full Text]
  22. Theobald, M., J. Biggs, J. Hernandez, J. Lustgarten, C. Labadie, L. A. Sherman. 1997. Tolerance to p53 by A2.1-restricted cytotoxic T lymphocytes. J. Exp. Med. 185: 833-841. [Abstract/Free Full Text]
  23. Sherman, L. A., M. Theobald, D. Morgan, J. Hernandez, I. Bacik, J. Yewdell, J. Bennink, J. Biggs. 1998. Strategies for tumor elimination by cytotoxic T lymphocytes. Crit. Rev. Immunol. 18: 47-54. [Medline]
  24. Ropke, M., J. Hald, P. Guldberg, J. Zeuthen, L. Norgaard, L. Fugger, A. Svejgaard, S. Van der Burg, H. W. Nijman, C. J. Melief, M. H. Claesson. 1996. Spontaneous human squamous cell carcinomas are killed by a human cytotoxic T lymphocyte clone recognizing a wild-type p53-derived peptide. Proc. Natl. Acad. Sci. USA 93: 14704-14707. [Abstract/Free Full Text]
  25. Hogan, K. T., S. L. Brown. 1992. Localization and characterization of serologic epitopes on HLA-A2. Hum. Immunol. 33: 185-192. [Medline]
  26. Maziarz, R. T., J. Fraser, J. L. Strominger, S. J. Burakoff. 1986. The human HLA-specific monoclonal antibody W6/32 recognizes a discontinuous epitope within the {alpha}2 domain of murine H-2Db. Immunogenetics 24: 206-208. [Medline]
  27. Hollstein, M., D. Sidransky, B. Vogelstein, C. C. Harris. 1991. p53 mutations in human cancers. Science 253: 49-53. [Abstract/Free Full Text]
  28. Hussain, S. P., M. H. Hollstein, C. C. Harris. 2000. p53 tumor suppressor gene: at the cross-roads of molecular carcinogenesis, molecular epidemiology, and human risk assessment. Ann. NY Acad. Sci. 919: 79-85. [Abstract/Free Full Text]
  29. Goldrath, A. W., M. J. Bevan. 1999. Selecting and maintaining a diverse T-cell repertoire. Nature 402: 255-262. [Medline]
  30. Sherman, L. A., D. J. Morgan, C. T. Nugent, F. J. Hernandez, H. T. Kreuwel, A. Murtaza, A. Ko, J. Biggs. 2000. Self-tolerance and the composition of T cell repertoire. Immunol. Res. 21: 305-313. [Medline]
  31. Rammensee, H. G., K. Falk, O. Rotzschke. 1993. Peptides naturally presented by MHC class I molecules. Annu. Rev. Immunol. 11: 213-244. [Medline]
  32. Christinck, E. R., M. A. Luscher, B. H. Barber, D. B. Williams. 1991. Peptide binding to class I MHC on living cells and quantitation of complexes required for CTL lysis. Nature 352: 67-70. [Medline]
  33. van der Merwe, P. A., S. J. Davis. 2003. Molecular interactions mediating T cell antigen recognition. Annu. Rev. Immunol. 21: 659-684. [Medline]
  34. Subbramanian, R. A., C. Moriya, K. L. Martin, F. W. Peyerl, A. Hasegawa, A. Naoi, H. Chhay, P. Autissier, D. A. Gorgone, M. A. Lifton, et al 2004. Engineered T-cell receptor tetramers bind MHC-peptide complexes with high affinity. Nat. Biotechnol. 22: 1429-1434. [Medline]
  35. Holler, P. D., L. K. Chlewicki, D. M. Kranz. 2003. TCRs with high affinity for foreign pMHC show self-reactivity. Nat. Immunol. 4: 55-62. [Medline]
  36. O’Herrin, S. M., M. S. Lebowitz, J. G. Bieler, B. K. al-Ramadi, U. Utz, A. L. Bothwell, J. P. Schneck. 1997. Analysis of the expression of peptide-major histocompatibility complexes using high affinity soluble divalent T cell receptors. J. Exp. Med. 186: 1333-1345. [Abstract/Free Full Text]
  37. Biddison, W. E., R. V. Turner, S. J. Gagnon, A. Lev, C. J. Cohen, Y. Reiter. 2003. Tax and M1 peptide/HLA-A2-specific Fabs and T cell receptors recognize nonidentical structural features on peptide/HLA-A2 complexes. J. Immunol. 171: 3064-3074. [Abstract/Free Full Text]
  38. Sykulev, Y., M. Joo, I. Vturina, T. J. Tsomides, H. N. Eisen. 1996. Evidence that a single peptide-MHC complex on a target cell can elicit a cytolytic T cell response. Immunity 4: 565-571. [Medline]
  39. Irvine, D. J., M. A. Purbhoo, M. Krogsgaard, M. M. Davis. 2002. Direct observation of ligand recognition by T cells. Nature 419: 845-849. [Medline]
  40. Gnjatic, S., Z. Cai, M. Viguier, S. Chouaib, J. G. Guillet, J. Choppin. 1998. Accumulation of the p53 protein allows recognition by human CTL of a wild-type p53 epitope presented by breast carcinomas and melanomas. J. Immunol. 160: 328-333. [Abstract/Free Full Text]
  41. Chikamatsu, K., K. Nakano, W. J. Storkus, E. Appella, M. T. Lotze, T. L. Whiteside, A. B. DeLeo. 1999. Generation of anti-p53 cytotoxic T lymphocytes from human peripheral blood using autologous dendritic cells. Clin. Cancer Res. 5: 1281-1288. [Abstract/Free Full Text]
  42. Skinner, P. J., M. A. Daniels, C. S. Schmidt, S. C. Jameson, A. T. Haase. 2000. Cutting edge: in situ tetramer staining of antigen-specific T cells in tissues. J. Immunol. 165: 613-617. [Abstract/Free Full Text]
  43. Cohen, C. J., O. Sarig, Y. Yamano, U. Tomaru, S. Jacobson, Y. Reiter. 2003. Direct phenotypic analysis of human MHC class I antigen presentation: visualization, quantitation, and in situ detection of human viral epitopes using peptide-specific, MHC-restricted human recombinant antibodies. J. Immunol. 170: 4349-4361. [Abstract/Free Full Text]
  44. Held, G., M. Matsuo, M. Epel, S. Gnjatic, G. Ritter, S. Y. Lee, T. Y. Tai, C. J. Cohen, L. J. Old, M. Pfreundschuh, et al 2004. Dissecting cytotoxic T cell responses towards the NY-ESO-1 protein by peptide/MHC-specific antibody fragments. Eur. J. Immunol. 34: 2919-2929. [Medline]
  45. Cohen, C. J., N. Hoffmann, M. Farago, H. R. Hoogenboom, L. Eisenbach, Y. Reiter. 2002. Direct detection and quantitation of a distinct T-cell epitope derived from tumor-specific epithelial cell-associated mucin using human recombinant antibodies endowed with the antigen-specific, major histocompatibility complex-restricted specificity of T cells. Cancer Res. 62: 5835-5844. [Abstract/Free Full Text]
  46. Denkberg, G., C. J. Cohen, A. Lev, P. Chames, H. R. Hoogenboom, Y. Reiter. 2002. Direct visualization of distinct T cell epitopes derived from a melanoma tumor-associated antigen by using human recombinant antibodies with MHC- restricted T cell receptor-like specificity. Proc. Natl. Acad. Sci. USA 99: 9421-9426. [Abstract/Free Full Text]
  47. Lev, A., G. Denkberg, C. J. Cohen, M. Tzukerman, K. L. Skorecki, P. Chames, H. R. Hoogenboom, Y. Reiter. 2002. Isolation and characterization of human recombinant antibodies endowed with the antigen-specific, major histocompatibility complex-restricted specificity of T cells directed toward the widely expressed tumor T-cell epitopes of the telomerase catalytic subunit. Cancer Res. 62: 3184-3194. [Abstract/Free Full Text]
  48. Denkberg, G., E. Klechevsky, Y. Reiter. 2002. Modification of a tumor-derived peptide at an HLA-A2 anchor residue can