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Immunology |
Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel [C. J. C., N. H., Y. R.]; Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel [M. F., L. E.]; Department of Pathology, Maastricht University, 6200 MD Maastricht, the Netherlands [H. R. H.]; and Dyax sa, Sart-Tilman, B-4000 Liege, Belgium [H. R. H.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In recent years, the advent of the application of tetrameric arrays of class I MHC-peptide complexes now enables us to detect and study rare populations of tumor antigen-specific T cells (4) . However, the study of antigen (MHC-peptide) presentation by tumor cells or professional APCs is lagging behind and very limited because of the lack of appropriate reagents that will enable direct detection of distinct tumor-associated MHC-peptide complexes.
The presence of tumor-specific MHC-peptide complexes on the surface of tumor cells may also represent a unique, specific target for an antibody-based therapeutic approach. However, new targeting moieties must be isolated such as recombinant antibodies that will recognize specific peptide-MHC complexes and will mimic the specific recognition by the TCR3 . These antibodies were therefore termed TCR-like antibodies.
In addition to being used as targeting agents, such antibodies would serve as a valuable tool for obtaining precise information about the presence, expression pattern, and distribution of the target tumor antigen, i.e., the MHC-peptide complex on the tumor’s cell surface, on tumor metastases, in lymphoid organs, and on professional APCs. Such molecules will, for the first time, enable measuring the antigen presentation ability of tumor cells by direct visualization of the specific MHC-peptide complex on the tumor cell surface. Attempts to use soluble TCRs for this purpose have proven difficult because of the inherent low affinity for their target and their instability as recombinant-engineered molecules.
Antibodies that specifically recognize class I peptide-MHC complexes have already been used in murine systems to study antigen presentation, to localize and quantify APCs displaying a T-cell epitope, or as a targeting tool in a mouse model (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19) . Antibodies with such a unique specificity directed toward human MHC-peptide complexes have proven difficult to make using conventional immunization strategies even in combination with in vitro selection from phage display libraries (19) .
In this study, we attempted to isolate human recombinant antibodies directed toward a T-cell epitope derived from the MUC1 antigen. MUC1 is an epithelial cell-associated mucin that is developmentally regulated and aberrantly expressed by carcinomas, which makes it an important marker in malignancy (20) . Structurally, this molecule exists as a large extended rod protruding from the apical cell membrane into the lumen of the ducts. MUC1 has an unusual structure, consisting mainly of a 20-aa sequence repeated in tandem on an average of 30–90 times. The tandem repeats serve as the scaffold for O-linked oligosaccharides that cover the polypeptide core (21 , 22) . In cancer, there are differences in expression that distinguish this protein as tumor specific. More specifically, there is a large increase in the amount of mucin expressed on the cells and in the circulation. Its distribution is no longer restricted to the apical surface of ducts and glands but rather is found throughout the tumor mass and on the surface of tumor cells. Importantly, the glycosylation is altered; oligosaccharide structures are shorter and fewer in number, revealing immunodominant peptide sequences in every tandem repeat that on normal surfaces would be concealed by glycosylation. Underglycosylation of MUC1 revealed peptide epitopes presented in the context of MHC molecules that are recognized by CTLs that can kill tumor cells expressing this form of MUC1 (23) . The recent description of MUC1 as a target for CTLs has raised interest in using this protein as a target for immunotherapy. It is expressed by most adenocarcinomas of the breast, lung, stomach, pancreas, colon, prostate, ovary, endometrium, and cervix, which makes MUC1 an attractive therapeutic target. Recently, Carmon et al. (24) characterized three new HLA-A2.1-restricted, MUC1-derived CTL epitopes. These peptides, which are not deduced from the extracellular Tandem Repeat Array, were shown to be processed and presented by a breast tumor cell line. Moreover, CTLs induced against these peptides lysed target cells pulsed with breast carcinoma-derived peptide extracts more efficiently than target cells pulsed with normal breast-derived peptides. One of these MUC1 epitopes, the D6 peptide (LLLTVLTVV), which exhibited high MHC-binding affinity, was positively correlated with preferential immunogenic properties in CTL assays.
In the present work, we have isolated a panel of high-affinity human recombinant antibodies possessing antigen-specific, MHC-restricted specificity of T cells directed toward HLA-A2 complexes that display the specific Mucin-1 D6 peptide. These molecules were used to directly visualize and quantitate the specific HLA-A2/MUC1-D6 epitope on APCs, as well as on the surface of tumor cells.
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MATERIALS AND METHODS |
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Selection of Phage-Antibodies on Biotinylated Complexes.
Selection of phage antibodies on biotinylated complexes was preformed as described previously (26
, 27)
. Briefly, a large human Fab library containing 3.7 x 1010 different Fab clones was used for the selection (28)
. Phages (1013) were first preincubated with streptavidin-coated paramagnetic beads (200 µl; Dynal, Oslo, Norway) to deplete the streptavidin binders. The remaining phages were subsequently used for panning with decreasing amounts of biotinylated scMHC-peptide complexes (500 nM for the first round and 100 nM for the following rounds). Bound phages were eluted with Triethylamine (100 mM), followed by neutralization with Tris-HCl [1 M (pH 7.4)] and used to infect E. coli TG1 cells (A600 nm = 0.5) for 30 min at 37°C.
The diversity of the selected antibodies was determined by DNA fingerprinting. The Fab DNA of different clones was PCR amplified using the primers pUC-reverse (5'-AGCGGATAACAATTTCACACAGG-3') and fd-tet-seq24 (5' TTTGTCGTCTTTCCAGACGTTAGT-3') followed by digestion with BstNI (New England Biolabs) (2 h, 37°C) and gel electrophoresis.
GenBank Accession Numbers.
The GenBank accession numbers for the Fab nucleotide sequences described in this manuscript are: AF527046 (M3A1 variable heavy chain); AF527047 (M3A1 variable light chain); AF527048 (M3B8 variable heavy chain); and AF527049 (M3B8 variable light chain).
Expression and Purification of Soluble Recombinant Fab Antibodies.
Fab antibodies were expressed and purified as described previously (19
, 26
, 27)
. TG1 or BL21 cells were grown to A600 nm = 0.8–1.0 and induced to express the recombinant Fab antibody by the addition of 1 mM IPTG for 3–4 h at 30°C. Periplasmic content was released using the B-PER solution (Pierce), which was applied onto a prewashed Talon column (Clontech). Bound Fabs were eluted using 0.5 ml of 100 mM imidazole in PBS. The eluted Fabs were dialyzed twice against PBS (overnight, 4°C) to remove residual imidazole.
ELISA with Phage Clones and Purified Fab Antibodies.
The binding specificities of individual phage clones and soluble Fab fragments were determined by ELISA using biotinylated scMHC-peptide complexes. ELISA plates (Falcon) were coated overnight with BSA-biotin (1 µg/well). After having been washed, the plates were incubated (1 h, room temperature) with streptavidin (1 µg/well), washed extensively, and additionally incubated (1 h, room temperature) with 0.5 µg of MHC-peptide complexes. The plates were blocked for 30 min at room temperature with PBS/2% skim milk and subsequently were incubated for 1 h at room temperature with phage clones (
109 phages/well) or various concentrations of soluble purified Fab. After having been washed, the plates were incubated with horseradish peroxidase-conjugated/antihuman Fab antibody (for soluble Fabs) or horseradish peroxidase-conjugated anti-M13 phage (for phage-displayed Fabs). Detection was performed using tetramelhylbenzidine reagent (Sigma). The HLA-A2-restricted peptides used for specificity studies of the Fab phage clones or purified Fab antibodies are as follows in Table 1
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Flow Cytometry.
The B cell line RMAS-HHD, which is transfected with a single-chain ß2M-HLA-A2 gene (29)
, the EBV-transformed HLA-A2+ JY cells, and the human breast tumor cells lines MDA-MB-231 (30)
and MCF-7 were used to determine the reactivity of the recombinant Fab antibodies with cell surface-expressed HLA-A2/peptide complexes. Peptide pulsing was performed as indicated: 106 cells were washed twice with serum-free RPMI and incubated overnight at 26°C or 37°C, respectively, in medium containing 1–30 µM of the peptide. The RMAS-HHD cells were subsequently incubated at 37°C for 2–3 h to stabilize cell surface expression of MHC-peptide complexes. Next, the cells were incubated for 60–90 min at 4°C with recombinant Fab antibodies (20 µg/ml) in 100 µl. After three washes, the cells were incubated with 1 µg of antihuman Fab (Jackson). After another three washes, the cells were resuspended in ice-cold PBS. Nonpulsed cells were harvested by trypsinization and resuspended in cold RPMI. All subsequent washes and incubations were performed under ice-cold conditions as already described for peptide-loaded cells. The cells were analyzed by a FACStar flow cytometer (Becton Dickinson), and the results were analyzed with the WinMDI program.4
Competition Binding Assays.
Flexible ELISA plates were coated with BSA-biotin and scMHC-peptide complexes (10 µg in 100 µl) were immobilized as described previously. The binding of soluble purified Fabs was performed by competitive binding analysis, which examined the ability of purified Fab to inhibit the binding of 125I-Fab to the specific immobilized scMHC-peptide complex. The recombinant Fab antibodies were labeled with [125I] using the Bolton-Hunter reagent. The labeled Fab was added to the wells as a tracer (3–5 x 105 cpm/well) in the presence of increasing concentrations of the cold Fab fragments as a competitor. The binding assays were performed at room temperature for 1 h in PBS. The plates were washed (five times) with PBS, and the bound radioactivity was determined using a gamma counter. The apparent affinity of the Fabs was determined by extrapolating the concentration of competitor necessary to achieve 50% inhibition of 125I-labeled Fab binding to the immobilized scMHC-peptide complex. Nonspecific binding was determined by adding a 20–40-fold excess of unlabeled Fab.
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RESULTS |
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Selection of Recombinant Antibodies with TCR-like Specificity to HLA-A2-restricted T-cell Epitope of MUC1.
The large antibody phage library, consisting of a repertoire of 3.7 x 1010 human recombinant Fab fragments (28)
, was first depleted of streptavidin binders and used for the subsequent panning in solution on soluble recombinant MHC-peptide complexes containing the MUC1-derived T-cell epitope. A 560-fold enrichment in phage titer was observed after three rounds of panning using the MUC1-derived D6 peptide-MHC complexes (Table 2)
. The fine specificity of the selected phage antibodies was determined by a differential ELISA analysis on streptavidin-coated wells incubated with biotinylated scMHC HLA-A2 complexes containing either the specific MUC1-derived D6 peptide or control complexes containing other HLA-A2-restricted peptides. Phage clones analyzed after the third round of selection exhibited two types of binding patterns toward the MHC-peptide complex: one class of antibodies consisted of pan-MHC binders that cannot differentiate between the various MHC-peptide complexes; the second type consisted of antibodies that bound the MHC-peptide complex in a peptide-specific manner. The ELISA screen revealed that 76 of 90 clones randomly selected clones screened (84%) from the third round of panning appeared to be binding to the HLA-A2/peptide complex.
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, 80% of the clones exhibited antigen-specific, MHC-restricted binding characteristics of T cells. Thus, they bound only to the MHC peptide complex containing the specific D6 MUC1-derived peptide and did not bind to control complexes containing other HLA-A2-restricted peptides. These apparent MHC/peptide-specific positive clones remained specific in a secondary screening on more complexes (data not shown, see "Materials and Methods" for list of HLA-A2- restricted peptides tested). Fig. 1
shows a representative analysis of five TCR-like Fab clones. Clones M2B1 and M2F5 are from the second round of panning and clones M3A1, M3B8, and M3C8 are from the third round. The different MUC1-D6-specific clones tested reacted only with the scMHC-MUC1-D6 complexes and not with MHC-peptide complexes displaying the MUC1-derived A7 epitope, the melanoma gp100-derived epitope, G9–154, and the viral TAX11–19 epitope (Fig. 1)
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Characterization of Recombinant Soluble Fab Antibodies with TCR-like Specificity.
Using E. coli BL21 or TG1 cells, we produced soluble Fab fragments from the phage clones (analyzed above, Fig. 1
) that exhibited the specific binding pattern to the MUC1-derived HLA-A2-peptide complexes.
These Fab antibodies were purified by metal affinity chromatography from the periplasm by use of the hexahistidine tag fused to the CH1 domain of the Fabs. SDS-PAGE analysis of the affinity-purified material revealed homogenous, pure Fab antibodies with the expected molecular weight (Fig. 2A)
. Approximately 0.5–2 mg of pure material could be obtained from 1 liter of bacterial culture. For additional manipulations (to increase the avidity of the monomeric Fab), we also produced the Fabs by in vitro refolding; the Fd and light chains were subcloned into a T7-promotor pET-based expression vector and upon induction with IPTG, large amounts of recombinant protein accumulated in intracellular inclusion bodies (Fig. 2B)
. In vitro refolded purified monomeric Fab was obtained in high yields (4–6 mg of purified Fab were obtained from two 1-liter cultures, each expressing the Fab Fd or light chain domain; Fig. 2C
). The minor contaminating bands below the Fab band reflect Fd and light chains from Fab in which the interchain disulfide did not form.
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shows a representative analysis of four soluble Fab antibodies directed to the HLA-A2/MUC1-D6 complexes. All four antibodies reacted specifically with the D6-containing HLA-A2 complexes but not with 10 control HLA-A2/peptide complexes containing the A7 MUC1-derived MHC-peptide complex, nor with HLA-A2 complexes containing melanoma gp100-derived epitopes (154, 209, 280), melanoma MART1 epitope, telomerase epitopes (540, 865) or the viral epitopes (TAX, cytomegalovirus, EBV; see Table 3
for list of peptides). Thus, these peptide-specific and MHC-restricted Fab fragments exhibit the binding characteristics and fine specificity of a TCR-like molecule. The Fab antibodies did not recognize the peptide alone when immobilized on the plate, nor with streptavidin or other protein antigens such as BSA, IgG, RNase, and Chymotrypsin (data not shown).
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, the binding of two specific Fabs (M3A1 and M3B8) was dose dependent and saturable. Extrapolating the 50% binding signal of either antibody revealed that their affinity was in the nanomolar range.
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revealed an apparent binding affinity of 15 nM for the M3A1 antibody and 25 nM for the M3B8 antibody specific for the MUC1-D6 epitope.
Binding of Fab fragments to APCs Displaying the MUC1-derived Epitope.
To demonstrate that the isolated Fab fragments can bind the specific MHC-peptide complex not only in the recombinant soluble form but also in the native form, as expressed on the cell surface, we used murine TAP2-deficient RMA-S cells transfected with the human HLA-A2 gene in a single-chain format (Ref. 32
; HLA-A2.1/Db-ß2m single chain, RMA-S-HHD cells). The MUC1-derived D6 and control peptides were loaded on RMA-S-HHD cells, and the ability of the selected Fab antibodies to bind to peptide-loaded cells was monitored by fluorescence-activated cell sorter. Peptide-induced MHC stabilization of the TAP2 mutant RMA-S-HHD cells was demonstrated by the reactivity of monoclonal antibodies w6/32 (HLA conformation-dependent) and BB7.2 (HLA-A2-specific) with peptide-loaded but not unloaded cells (data not shown). Fabs M3A1 and M3B8 reacted only with D6-loaded RMA-S-HHD cells and not with cells loaded with the gp100-derived G9–154 peptide (Fig. 5, A and B)
. Similar results were observed in fluorescence-activated cell sorter analysis using 10 other HLA-A2-restricted peptides (data not shown).
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. As a control, we used peptide-loaded HLA-A2-/HLA-A1+ APD B cells. No binding of the Fab antibodies to these cells was observed (data not shown).
Increasing the Avidity of the TCR-like Fabs and Binding to MUC1-expressing Tumor Cells.
To confirm that the MUC 1-specific TCR-like Fab antibodies can bind endogenously derived MHC-peptide complexes on the surface of tumor cells, we performed flow cytometry analysis on various tumor cells that express MUC1 and HLA-A2.
Because the density of a particular peptide-HLA complex on these tumor cells is expected to be low compared with peptide-pulsed APCs, we increased the avidity of Fab M3A1 by making Fab tetramers, which are directly tagged with a fluorescent probe. This approach was used previously to increase the binding avidity of peptide-MHC complexes to the TCR or to increase the sensitivity of recombinant antibody molecules (33)
. Another advantage of using fluorescently labeled tetramers is that only a single staining step is required, whereas monomeric unlabeled Fabs require a fluorescently labeled secondary antibody. We thus used our Fab tetramers, which were generated with fluorescently labeled streptavidin, to measure the expression of MUC1-derived D6 peptide-MHC complexes on the surface of MUC1-expressing tumor cells. As shown in Fig. 6A
, the intensity of the binding, as measured by flow cytometry with peptide-pulsed JY cells, was dramatically increased by two logs compared with the staining intensity with the M3A1 Fab monomer. Next, we tested the ability of the Fab M3A1 tetramer to stain breast cancer HLA-A2+ MDA-MB-231 tumor cells pulsed with the MUC1-derived D6 peptide. As shown in Fig. 6B
, substantial staining of peptide-pulsed MDA-MB-231 cells was observed with the tetramer, whereas a lower degree of staining was observed when cells were stained using the Fab monomer. Similar results were observed when we used HLA-A2+ mature dendritic cells pulsed with the MUC1-derived D6 peptide (Fig. 6C)
. As expected, the titration of peptide-pulsed JY and MDA-MB-231 cells with different concentrations of the MUC1-derived D6 peptide demonstrated that the staining intensity was dependent on the concentration of the peptide used for pulsing and that peptide concentration at the nM range was sufficient to detect binding when using the Fab M3A1 tetramer (data not shown). This titration was performed on cells that have normal antigen processing and consequently, displaying the exogenous peptide is facilitated by peptide exchange. Similar experiments were performed on MUC1-expressing MCF-7 breast carcinoma cells. The staining intensity of peptide-pulsed JY or MDA-MB-231 cells observed with M3A1 was compared with calibration beads that display known fix number of phycoerythrin sites. This comparison enabled us to determine the number of D6 peptide-HLA-A2 complexes displayed on the surface of cells that are pulsed with the D6 MUC1-derived peptide (Table 3)
. The binding of the M3A1 tetramer to the MDA-MB-231 and MCF-7 cells was observed only when the MUC1-D6 peptide was used for pulsing (Fig. 7, A and B)
; when a control HLA-A2-restricted peptide derived from HTLV-1 (TAX) was used, no binding was detected (Fig. 7, A and B)
. We also attempted to detect the natural occurrence of HLA-A2/Mucin1-D6 complexes on cells without peptide pulsing, using the Fab M3A1 tetramer. We used MCF-7 and MDA-MB 231 cells that are also HLA-A2+ and express MUC1. These cells represent the normal situation in which the specific MHC-peptide complex is expected to be present at a much lower density on the cell surface compared with peptide-loaded APCs or peptide-pulsed tumor cells. The specificity of binding was assessed first on peptide-pulsed MCF-7 and MDA-MB 231 (Fig. 7, A and B)
. MUC1 expression in these cells was visualized by staining with an anti-Mucin1 antibody (data not shown). As shown in Fig. 7C
, the M3A1 tetramer was able to detect MUC1-D6 complexes on the surface of MUC1-expressing, HLA-A2+, MCF-7, and MDA-MB 231 cells but not on MUC1-negative, HLA-A2+ melanoma FM3D cells nor on HLA-A2 and MUC1-negative G-43 cells. The M3A1 tetramer exhibited a low background staining with the MUC1-positive, HLA-A2-negative SK-BR3 cells (Fig. 7C)
. The staining intensity of the cells was statistically significant, representing measurements of 10 independent experiments. These results demonstrate the ability of these high-affinity TCR-like antibodies to detect MHC-peptide complexes on the surface of tumor cells. Thus, these TCR-like antibodies can bind to cells that express the specific MHC-peptide complex at a density most likely to be found on tumor cells, APCs such as dendritic cells, and other cells involved in tumor-antigen presentation to the immune system.
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, whereas pulsing of tumor MDA-MB-231 cells resulted in the display of 1 x 104 complexes/cell. Next, we attempted using the same strategy to determine the number of D6-MUC1-derived complexes on the surface of MUC1-expressing tumor cells (naturally, without peptide pulsing). As shown in Table 3
, we could estimate the number of D6-HLA-A2 complexes to be several hundreds/cell. To our knowledge, this is the first attempt to quantify, using a direct mean, the number of tumor-associated TCR ligands (peptide-MHC complexes) on the surface of tumor cells. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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These antibodies exhibit a very specific and special binding pattern: they can bind with a peptide-specific manner only to HLA-A2/MUC1-D6 complexes; hence, these are recombinant antibodies with T-cell antigen receptor-like specificity. In contrast to the inherent low affinity of TCRs, these molecules display the high-affinity binding characteristics of antibodies while retaining TCR specificity. Most importantly, these recombinant antibodies specifically recognize native MUC1-D6-derived MHC-peptide complexes on the surface of cells, including binding to MUC1-positive tumor cells. The recent description of MUC1 as a target for CTLs has raised interest in using this protein as a target for immunotherapy (21 , 24 , 26) . It is expressed by most adenocarcinomas of the breast, lung, stomach, pancreas, colon, prostate, ovary, endometrium, and cervix, which makes MUC1 an attractive therapeutic target (22) .
Recombinant antibodies with TCR-like specificity, represent a valuable new tool in molecular immunology for several major fields of research: (1) for studying antigen presentation in cancer, (2) for developing new targeting molecules; for immunotheraphy, and (3) for studying structure-function relationships in TCR-peptide-MHC interactions. We have shown that these antibodies can be used to detect and visualize the presence of specific T-cell epitopes (MHC-peptide complexes) by standard methods of flow cytometry. With appropriate conservation of the MHC-peptide complexes during fixation, it may even be possible to detect such complexes by immunohistochemistry, opening the door for their widespread use in pathology. As such, they should be very useful for the study and analysis of antigen presentation on tumor cells by determining the expression of specific tumor-related MHC-peptide complexes on the surface of tumor cells, metastases, APCs, and lymphoid cells. Moreover, such antibodies are expected to be particularly useful for determining the alterations in MHC-peptide complex expression on APCs before, during, and after vaccination protocols with peptides, APCs loaded with tumor cell extracts, or dendritic tumor cell hybrid vaccinations (6, 7, 8) . Direct detection of particular MHC-peptide complexes using flow cytometry or immunohistochemistry would allow quantitation of TCR ligands on individual cells, as demonstrated in this study, phenotyping of such APCs or tumor cells, and localization of these APCs within normal or pathological tissues.
Confocal immunofluorescence microscopy would permit analysis of the intracellular site(s) of peptide-MHC molecule interaction and trafficking. In situ localization of APCs bearing particular peptide-MHC TCR ligands would be especially valuable in characterizing the cell-cell interactions involved in initiation, propagation, and maintenance of T-cell immune responses. Multicolor histochemistry could be used to reveal not only the type and location of APCs but also the phenotype of interacting T cells, including the set of cytokines elicited.
Indeed, we attempted in this study to quantify the number of distinct TCR ligands (peptide-MHC complexes) on the surface of cells. An important feature of the TCR-like Fab, M3A1, used for these studies is its capacity to detect peptide-MHC at cell surface densities near the threshold limit for T-cell signaling. The numbers obtained herein of several hundreds sites/cell on unpulsed tumor cells and several 103-104 sites/cell on peptide-pulsed APCs are in agreement with several estimates of the threshold number of peptide-MHC ligands needed to elicit nuclear responses from T cells such as cytokine secretion or sensitize target cells for lysis by CTL (32 , 34) . Using peptide-pulsed APCs, we could show that the M3A1 antibody was able to detect complexes on cells pulsed with peptide concentrations similar to those needed to activate a T-cell hybridoma or CTL line to cytokine secretion and within a few fold of the minimal concentration able to sensitize target cells for lysis in a short-term cytotoxicity assay.
Interestingly, we were able to isolate a repertoire of several antibodies against the MUC1-derived epitope. Until now, antibodies with TCR-like specificity had been generated against murine MHC-peptide complexes using various strategies of immunizations (10, 11, 12, 13, 14, 15, 16, 17, 18) . Most strikingly, here we were able to select several different TCR-like antibodies, whereas all previous experiments were able to isolate only a single antibody clone (10 , 11 , 20) . The fact that 80% of the MHC-peptide binding antibodies had the fine specificity of a TCR-like molecule, i.e., presumably bind to a small portion of the total area of the MHC-peptide complex, is nevertheless surprising, especially because they were selected from a nonimmune repertoire considered not to be biased toward such specificity. More recently, we have been able to isolate from the same phage library recombinant Fabs against a large variety of MHC-peptide complexes containing other cancer-associated or viral HLA-A2-restricted peptides (26 , 27) , indicating that this behavior is not MUC1 related or peptide related. The unexpected high frequency of these antibodies and our ability to isolate several different antibodies is even more surprising in view of previous reports in which the use of immunized or naïve phage libraries resulted in only a single antibody clone. It is possible that one particular antibody family or antibody V-gene segment could have an intrinsic propensity to bind HLA-A2 molecules and that the high frequency could be explained by a high abundance of such antibodies in the nonimmune library. However, the sequences of the selected clones are derived from many different antibody families and germ-line segments, without any biases visible in the complementary determining regions either (data not shown). The high frequency and high affinities for some of the antibodies isolated here suggest that these molecules may be present at a high frequency in the antibody repertoires from the B-cell donors of the phage library, but an in vivo role for such antibodies remains unclear.
Whatever the reason for this high frequency of antibodies to MHC-peptides may be, it appears that this phage-based approach can be successfully applied to isolate recombinant antibodies with TCR-like specificity to a large variety of MHC-peptide complexes. Thus, it may now be possible to elucidate the role of antigens under various pathological conditions such as cancer, viral infections, and autoimmune disease, not only at the level of the T cell, using MHC tetramers, but also at the level of the APC and diseased cell, using antibodies of the type described here.
There are now opportunities to use these particular molecules, which recognize a very specific human tumor antigen, as candidates for targeting moieties using various antibody-based immunotherapeutic approaches. This includes the use of these antibodies to construct recombinant immunotoxins (35) , for fusions with cytokine molecules (36) , for bispecific antibody therapy (37) or immunogene therapy.
Another interesting application for using these TCR-like Fab antibodies is for structure-function studies of MHC-peptide TCR interactions. By mutating particular residues in the specific peptide and testing, the influence of these mutations on the binding of the Fab antibodies and peptide-specific T-cell clones, it may be possible to obtain important data on the structure-function relationship and the different nature of the recognition process between the TCR-like Fabs and the native TCR (38) .
To improve the sensitivity and targeting capabilities of these TCR-like antibody molecules, two antibody engineering approaches can be used: one increases the affinity of the parental antibodies by affinity maturation strategies without altering their TCR-like fine specificity (39) , and the second, already used in this study, increases the avidity of these recombinant monovalent molecules by making them multivalent. Combining these strategies may result in improved second-generation antibody molecules that will be sensitive enough and specific for immunotherapeutic approaches as well as for studying the interaction of tumor cells and the human immune system.
In recent years the advent of the application of tetrameric arrays of class I MHC-peptide complexes now enables us to detect and study rare populations of antigen-specific T cells (4) . Our approach produces antibody molecules that enable phenotypic analysis of antigen (MHC-peptide) presentation, the other side of the coin to MHC-peptide-TCR interactions. Combining these two new approaches will significantly enhance our ability to understand immune responses in health, as well as under various pathological conditions such as cancer, viral infections, and also when applied to class II MHC molecules, autoimmune diseases.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported, in part, by research grants (to Y. R.) from the Israel Science Foundation administered by the Israel National Academy for Sciences and Humanities, Jerusalem, Israel, a Research Career Development Award by the Israel Cancer Research Foundation, New York, and by The Mallat Family Fund for Research in Life Sciences. C. J. C. was supported by a short-term European Molecular Biology Organization fellowship. 
2 To whom requests for reprints should be addressed, at Faculty of Biology, Technion-Israel Institute of Technology, Technion City, Room 333, Haifa 32000, Israel. Phone: 972-4-8292785; Fax: 972-4-8225153; E-mail: reiter{at}tx.technion.ac.il
3 The abbreviations used are: TCR, T-cell receptor; APC, antigen-presenting cell; MUC1, Mucin 1 tumor-associated antigen; TR, tandem repeat; scMHC, single-chain MHC; IPTG, isopropyl-1-thio-ß-D-galactopyranoside; PE, phycoerithrin.
4 J. Trotter, http://facs.scripps.edu/.
Received 5/ 6/02. Accepted 8/14/02.
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X. Zhu, H. J. Belmont, S. Price-Schiavi, B. Liu, H.-i. Lee, M. Fernandez, R. L. Wong, J. Builes, P. R. Rhode, and H. C. Wong Visualization of p53264-272/HLA-A*0201 Complexes Naturally Presented on Tumor Cell Surface by a Multimeric Soluble Single-Chain T Cell Receptor. J. Immunol., March 1, 2006; 176(5): 3223 - 3232. [Abstract] [Full Text] [PDF]
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S. Oh, M. Terabe, C. D. Pendleton, A. Bhattacharyya, T. K. Bera, M. Epel, Y. Reiter, J. Phillips, W. M. Linehan, C. Kasten-Sportes, et al. Human CTLs to Wild-Type and Enhanced Epitopes of a Novel Prostate and Breast Tumor-Associated Protein, TARP, Lyse Human Breast Cancer Cells Cancer Res., April 1, 2004; 64(7): 2610 - 2618. [Abstract] [Full Text] [PDF]
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 A. Admon, E. Barnea, and T. Ziv Tumor Antigens and Proteomics from the Point of View of the Major Histocompatibility Complex Peptides Mol. Cell. Proteomics, June 1, 2003; 2(6): 388 - 398. [Abstract] [Full Text] [PDF]
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