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The Journal of Immunology, 2003, 171: 2197-2207.
Copyright © 2003 by The American Association of Immunologists

Selective Targeting of Melanoma and APCs Using a Recombinant Antibody with TCR-Like Specificity Directed Toward a Melanoma Differentiation Antigen 1

Galit Denkberg*, Avital Lev*, Lea Eisenbach{ddagger}, Itai Benhar{dagger} and Yoram Reiter2,*

* Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel; {dagger} Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel; and {ddagger} Department of Immunology, Weizmann Institute of Science, Rehovot, Israel


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tumor-associated, MHC-restricted peptides, recognized by tumor-specific CD8+ lymphocytes, are desirable targets for novel approaches in immunotherapy because of their highly restricted fine specificity. Abs that recognize these tumor-associated MHC-peptide complexes, with the same specificity as TCR, would therefore be valuable reagents for studying Ag presentation by tumor cells, for visualizing MHC-peptide complexes on cells, and eventually for developing new targeting agents for cancer immunotherapy. To generate molecules with such a unique, fine specificity, we immunized HLA-A2 transgenic mice with a single-chain HLA-A2, complexed with a common antigenic T cell HLA-A2-restricted epitope derived from the melanoma differentiation Ag gp100. Using a phage display approach, we isolated a recombinant scFv Ab that exhibits a characteristic TCR-like binding specificity, yet, unlike TCRs, it did so with a high affinity in the nanomolar range. The TCR-like Ab can recognize the native MHC-peptide complex expressed on the surface of APCs, and on peptide-pulsed or native melanoma cells. Moreover, when fused to a very potent cytotoxic effector molecule in the form of a truncated bacterial toxin, it was able to specifically kill APCs in a peptide-dependent manner. These results demonstrate the utility of high affinity TRC-like scFv recombinant Abs directed toward human cancer T cell epitopes. Such TCR-like Abs may prove to be very useful for monitoring and visualizing the expression of specific MHC-peptide complexes on the surface of tumor cells, APCs, and lymphoid tissues, as well as for developing a new family of targeting agents for immunotherapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The expression of specific peptides in a complex with MHC class I molecules on cells was shown to be associated with cancer and autoimmune disorders (1, 2, 3). In cancer, the discovery of these peptides emerged from the now well-established observation that human tumor cells often express Ags that are recognized by CTLs derived from patients (1, 2, 3, 4, 5).

Moreover, it has been demonstrated that the immune response against the tumor is insufficient to cause tumor regression and that tumor cells can develop effective mechanisms to escape such an immune attack (6, 7, 8, 9). Therefore, numerous approaches are being developed in the field of tumor vaccination in an attempt to augment the antitumor immune responses, including cancer peptide vaccines, autologous cancer vaccines, and the cancer-dendritic cell hybrid vaccine (7, 10, 11). Because the specificity of the immune response is regulated and dictated by these class I MHC-peptide complexes, it should be possible to use these very specific and unique molecular cell surface markers as targets to eliminate the cancer cells, while sparing the normal cells. Thus, it would be very desirable to devise new molecules in a soluble form that will mimic the fine, unique specificity of the TCR to the cancer-associated MHC-peptide complexes. One promising approach is to generate recombinant Abs that will bind the MHC-peptide complex expressed on the cancer cell surface with the same specificity as the TCR. These unique Abs can subsequently be armed with an effector cytotoxic moiety such as a radioisotope, a cytotoxic drug, or a toxin. For example, Abs that target cancer cells are genetically fused to powerful toxins originating from both plants and bacteria, thus generating molecules termed recombinant immunotoxins (12).

Abs with the MHC-restricted specificity of T cells are rare and have been difficult to generate by conventional hybridoma techniques because B cells are not educated to be self-MHC restricted (13, 14, 15, 16). The advantages of Ab phage-displayed technology make it possible to also select large Ab repertoires for unique and rare Abs against very defined epitopes. This has been demonstrated by the ability to isolate by phage display a TCR-like restricted Ab to a murine class I MHC H-2Kk complexed with a viral epitope (17), and more recently against the melanoma Ag MAGE-A1 in a complex with HLA-A1; however, this Ab exhibited a low affinity and could be used to detect the specific complexes on the surface of APCs only when expressed in a multimeric form on a phage and not as a soluble Ab (18).

In this work, we have immunized HLA-A2 transgenic mice with a single-chain HLA-A2 (scHLA-A2) 3 molecule complexed with a peptide derived from the melanoma differentiation Ag gp100 (19, 20, 21, 22). Using phage display, we isolated from the immune murine Ab library repertoire a soluble high affinity recombinant single-chain Ab with the Ag-specific MHC-restricted specificity of T cells directed toward a gp100-derived cancer epitope in complex with HLA-A2.

The isolated Ab binds with high affinity, but TCR-like specificity to the recombinant gp100-derived MHC-peptide complex and can detect the specific HLA-A2-peptide complex on the surface of APCs as well as melanoma cells. The Ab was used to directly visualize in situ the specific gp100-derived epitope on intact melanoma cells by immunohistochemistry. Moreover, we have fused the Ab gene to a truncated form of Pseudomonas exotoxin A (12) to form a recombinant immunotoxin and tested its ability to specifically target APCs. In this study, we show, for the first time, the ability of such a soluble fusion protein to kill, in a TCR-like restricted manner, those target cells that express a particular human cancer-associated MHC-peptide complex.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Production of biotinylated scMHC/peptide complexes

scMHC/peptide complexes were produced by in vitro refolding of inclusion bodies produced in Escherichia coli, as described previously (23, 24). Biotinylation was performed using the BirA enzyme (Avidity, Denver, CO), as previously described (25).

Mice immunization

We used the Db-/- x {beta}2-microglobulin ({beta}2m) null mice transgenic for a rHLA-A2.1/Db-{beta}2m single chain (HHD mice) (26) to immunize with an emulsion containing purified protein-derived peptide of tuberculin (PPD) covalently coupled with HLA-A2/G9-209 complexes, as described previously (17). Briefly, mice were initially immunized subdermally and subsequently s.c. for 2-wk intervals for a period of 3–5 mo with 20–30 µg/mice of the antigenic mixture in IFA. Spleens were collected 2 wk after the last immunization.

Library construction and selection of phage Abs on biotinylated complexes

Total RNA was isolated from immunized mice, and an Ab scFv library was constructed by RT-PCR from the mRNA, as described (27). The scFv repertoire was cloned as an SfiI-NotI fragment into the pCANTAB5E or pCC-CBD phagemid vectors (28). The complexity of both libraries was 1 x 108 independent clones. For panning, biotinylated scHLA-A2/G9-209 M complexes (20 µg) were incubated with streptavidin-coated magnetic beads (2 x 108), washed, and incubated with 1011 CFU of the libraries (1 h, room temperature (RT)). Starting with the second round, panning was performed in the presence of an excess (5 µg) of scHLA-A2/G9-280V complexes. Beads were washed extensively 10–12 times with 2% skimmed milk in PBS + 0.1% Tween. Bound phages were eluted by using 1 ml of triethylamine (100 mM, pH 12) for 5 min at RT, followed by neutralization with 0.1 ml of 1 M Tris-HCl, pH 7.4. Eluted phages were expanded in exponentially growing E. coli TG1 cells that were later superinfected with M13KO7 helper phage, as described (28).

Expression and purification of soluble recombinant scFv and scFv-P38 fusion protein

The G1 scFv gene was rescued from the phage clone by PCR and was subcloned into the phagemid vector pCANTAB6 by using the SfiI-NotI cloning sites. A Myc and hexahistidine tags were fused to the C terminus of the scFv gene. The scFv Ab was expressed in BL21 {lambda}DE3 cells, as previously described (29), and purified from the periplasmic fraction by metal-ion affinity chromatography. For the expression of the G1scFv-PE38 fusion protein, the scFv gene was subcloned as an NcoI-NotI fragment into the plasmid pIB-NN, which encodes the translocation and ADP-ribosylation domains of PE (PE38). Expression in BL21 {lambda}DE3 cells, refolding from inclusion bodies, and purification of G1scFv-PE38 were performed, as previously described (30).

ELISA with phage clones and purified scFv or scFv-PE38

Binding specificity studies were performed by ELISA using biotinylated scMHC-peptide complexes. Briefly, ELISA plates (Falcon) were coated overnight with BSA biotin (1 µg/well), washed, incubated (1 h, RT) with streptavidin (1 µg/well), again washed extensively, and further incubated (1 h, RT) with 0.5 µg of MHC/peptide complexes. Plates were blocked with PBS/2% milk (30 min, RT), incubated with phage clones (~109 phages/well, 1 h, RT) or 0.5–1 µg of soluble scFv or scFv-PE38, and afterward washed with 1:1000 HRP-conjugated/anti-M13, anti-myc Ab, or anti-PE Ab, respectively. The HLA-A2-restricted peptides used for specificity studies are gp100 (154), KTWGQYWQV; gp100 (209), IMDQVPFSV; gp100 (280), YLEPGPVTV; MUC1, LLLTVLTVL; HTLV-1 (TAX), LLFGYPVYV; hTERT (540), ILAKFLHWL; hTERT (865), RLVDDFLLV.

Flow cytometry

The B cell line RMAS-HHD transfected with a single-chain {beta}2m-HLA-A2 gene (26) or the EBV-transformed B-lymphoblast JY cells (106 cells) were washed twice with serum-free RPMI and incubated overnight at 26°C or 37°C, respectively, in medium containing 100 µM of the peptide. The APCs were subsequently incubated at 37°C for 2–3 h to stabilize cell surface expression of MHC-peptide complexes, followed by incubation with recombinant scFv (10–50 µg/ml, 60–90 min, 4°C) in 100 µl. The cells were then washed, incubated with FITC-labeled anti-Myc Ab (30–45 min, 4°C), and finally washed and analyzed by a FACStar flow cytometer (BD Biosciences, San Jose, CA).

Melanoma cells were pulsed at 37°C with 1–10 µM of peptide and then stained with the scFv, as described above.

Competition-binding assays

Flexible ELISA plates were coated with BSA biotin, and scMHC peptide complexes (10 µg in 100 µl) were immobilized, as previously described. The recombinant G1scFv-PE38 was labeled with 125I using the Bolton-Hunter reagent. Labeled protein was added to wells as a tracer (3–5 x 105 cpm/well) in the presence of increasing concentrations of the cold G1scFv-PE38 as a competitor and incubated at RT for 1 h in PBS. The plates were washed thoroughly with PBS, and the bound radioactivity was determined by a gamma counter. The apparent affinity of the G1scFv-PE38 was determined by extrapolating the concentration of a competitor necessary to achieve 50% inhibition of 125I-labeled G1scFv-PE38 binding to the immobilized scMHC-peptide complex. Nonspecific binding was determined by adding a 20- to 40-fold excess of unlabeled Fab.

Immunohistochemistry

Melanoma and control cells were incubated with 20–30 µg G1 scFv for 1 h on ice in RMPI containing 10% FCS, followed by incubation with HRP anti-Myc Ab (45 min, on ice). The cell suspension was applied onto glass slides precoated with 0.1% poly(L-lysine) (Sigma-Aldrich, St. Louis, MO). Cells were then incubated for 1 h at room temperature. Slides were washed three times with PBS, and incubated with a diaminobenzidine+ solution (DAKO, Glostrup, Denmark) for 1 min, followed by washing with PBS to remove excess of staining reagent. Cell nuclei were stained with hematoxylin (Sigma-Aldrich).

Cytotoxicity assays

RMAS-HHD and JY APCs were loaded with the G9-209 peptide and control peptides, as previously described. Peptide-loaded cells were subsequently incubated with increasing concentrations of G1scFv-PE38, and the inhibition of protein synthesis was determined by measuring the uptake of [3H]leucine into cellular proteins, as previously described (30). IC50 was determined as the concentration of G1scFv-PE38 required to inhibit protein synthesis by 50%. In competition assays, excesses of specific and nonspecific HLA-A2-peptide complexes (35–50 µg/well) were added to wells 15 min before the addition of G1scFv-PE38.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of recombinant scMHC-peptide complexes with the melanoma gp100-derived peptide G9-209 M

Gp100 is a melanocyte lineage-specific membrane glycoprotein consisting of 661 aa that is expressed in most melanoma cells (19, 20, 21, 22). This protein is recognized by many HLA-A2-restricted, melanoma-reactive, tumor-infiltrating lymphocytes that have been isolated from melanoma patients (19, 20, 21, 22). Several T cell HLA-A2-restricted epitopes have been identified in gp100; they have been improved in MHC anchor positions for enhanced immunogenicity without altering T cell specificity (31). The G9-209 M (IMDQVPFSV) peptide is one of three major immunogenic epitopes (19, 20, 21, 22). The native anchor position 2 was changed from threonine to methionine to improve the peptide-binding affinity to the MHC (31). Recombinant MHC-peptide complexes that present the G9-209 M peptide were generated by using a scMHC construct expressed in E. coli that has been described previously (23, 24). The scMHC-peptide complexes are produced by in vitro refolding of inclusion bodies in the presence of the G9-209 M or other HLA-A2-restricted peptides, followed by a purification protocol using ion-exchange chromatography. The refolded gp100-derived and control scHLA-A2-peptide complexes were >95% pure, homogenous, and monomeric, as shown by analysis on SDS-PAGE and gel filtration chromatography (data not shown). The G9-209 M-containing scHLA-A2 complexes have been previously shown to be functional, by their ability to stimulate specific CTL lines and clones and stain G9-209 M-specific T cells in the form of tetramers (23, 24).

Construction of an Ab scFv phage library and selection of a phage that binds HLA-A2/G9-209 M complexes with TCR-like specificity

For immunization purposes, we coupled PPD to the purified complex and immunized the Db-/- x {beta}2m null mice transgenic for a rHLA-A2.1/Db-{beta}2m single chain (HHD mice) (26). These mice combine classical HLA transgenesis with selective destruction of murine class I H-2. Hence, unlike the classical HLA transgenics, these mice showed only HLA-A2.1-restricted responses with multiepitope proteins such as intact viruses. Moreover, we presume that these mice are a useful tool for immunization with HLA-A2-peptide complexes because they should be largely tolerant to HLA-A2 as a B cell immunogen, and thus may favor the generation of an Ab response directed against the MHC-restricted epitope when in complex with HLA-A2 (the specific tumor-associated peptide). PPD was used for conjugation because it is a highly reactive T cell immunogen (17).

Total spleen mRNA was isolated from immunized mice and reverse transcribed to cDNA. Specific sets of degenerated primers were used to PCR amplify the cDNA segments corresponding to the Ig H and L chain V domains. The VH and VL PCR pools were assembled into a scFv repertoire by a PCR overlap extension reaction and subsequently cloned into the pCANTAB5E phagemid vector or to the phagemid vector pCC-Gal6(Fv) in which the scFv is expressed as an in-frame fusion protein with a cellulose-binding domain (CBD) (28). The resulting libraries were transduced into E. coli TG1 cells by electroporation and expressed as fusion with the minor phage coat protein pIII after rescue with a helper phage. The library complexity consisted of 1 x 108 independent clones using both types of vectors.

The library was subjected to three to four panning cycles, followed by elution of bound phages and reamplification in E. coli. To enhance the efficiency of our selection, we used biotinylated scMHC-peptide complexes. A BirA sequence tag for site-specific biotinylation was engineered at the C terminus of the HLA-A2 gene, as previously described (25). Several selection strategies were used, the most successful of which resulted in the isolation of specific binders consisting of panning protocols with a negative depletion step starting from the second round of panning. The specific HLA-A2/G9-209 M-biotinylated complexes were immobilized onto streptavidin-coated magnetic beads, and the library was incubated with the immobilized complex in the presence of a large excess of HLA-2 complexes that displayed a different gp100-derived epitope, the G9-280V peptide. When this strategy is used, G9-209 M-specific phage will bind to streptavidin-biotin-immobilized complexes that are captured by a magnetic force, whereas pan-MHC binders that are not specific to the G9-209 M peptide in the complex will bind to the nonspecific complex in the solution, and thus can be separated and removed from the specific phage. As shown in Table I, a progressive, marked enrichment for phage that binds the immobilized complexes was observed after three to four rounds of panning, two of which were performed with the negative depletion strategy. Polyclonal phage ELISA was performed to determine phage specificity on biotinylated recombinant scMHCpeptide complexes immobilized to BSA-biotin-streptavidin-coated immunoplates. The BSA-biotin-streptavidin spacer enables the correct presentation and folding of the complexes, which can be distorted by direct binding to plastic. Phage analyzed already after the second and, more dramatically, after the third round of panning revealed a unique specificity pattern only directed toward the specific G9-209 M-containing HLA-A2 complexes (Fig. 1, A and B). No binding was observed with control HLA-A2 complexes that display the gp100-derived epitope, G9-280V, or the telomerase-derived epitope 865.


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  		Table I. Phage selection on scHLA-A2/G9-209 M complexesa

 


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  		FIGURE 1. Selection and characterization of phage clones directed toward scHLA-A2/gp100 complexes. A and B, Polyclonal phage ELISA on recombinant scHLA-A2-peptide complexes. Plates were coated with in vitro refolded scHLA-A2 complexes containing the melanoma differentiation Ag gp100-derived peptides G9-209 and G9-280 or with telomerase catalytic subunit-derived peptide 865. Shown is the binding of the polyclonal phage population from the initial library (L) or phages eluted after each round of panning (I-IV). The phage input was 109/well. Detection was with HRP-labeled anti-M13 Ab. A, Phages from the pCANTAB scFv library; B, phages from the scFv-CBD library. C and D, Differential binding of monoclonal phage clones to scHLA-A2/gp100 complexes. Monoclonal phage clones isolated after the third round of selection were tested for binding specificity on immobilized scHLA-A2 complexes containing the gp100-derived epitopes G9-209 (C) or G9-280 (D). The phage input was 109/well.

 
Individual monoclonal phage clones were isolated from the population of phages from the last round of panning (no further enrichment observed after a fourth round) and rescreened for specificity by phage ELISA (Fig. 1, C and D). Of the 93 clones tested, 85 (91%) reacted with the HLA-A2/G9-209 M complex (Fig. 1C). A total of 77 of the 85 reactive clones (90%) reacted specifically with the specific HLA-A2/G9-209 M complex, but not with the control G9-280V-containing complex (Fig. 1D). Only a small percentage of the clones (5 of 93; 5%) did not exhibit peptide specificity (Fig. 1C). Thus, the panning procedure yielded a successful enrichment of phage Abs with TCR-like specificity toward the HLA-A2/G9-209 M complex. Fingerprint analysis by means of multicutter restriction enzyme digestion revealed that 50 positive, HLA-A2/G9-209 M-specific clones had a similar digestion pattern, indicating that all are similar (data not shown). Similar results were obtained with the two libraries. Because they were constructed from the same genetic material (the same pool of mRNA), we further characterized phage clones derived from the pCANTAB5E scFv library.

DNA sequencing of VH and VL V domains from 10 clones revealed that all were identical (data not shown), suggesting that they were all derived from a single productive Ab VH/VL combinatorial event.

Characterization of the soluble recombinant scFv Ab with TCR-like specificity

DNA sequencing revealed that the Ab VH sequence belongs to the mouse H chains subgroup III (D), and the VL sequence to mouse {kappa} L chains group IV (according to Kabbat). The nucleotide sequence and deduced amino acid sequence were submitted to GenBank, accession number bankit533067. To further characterize the binding specificity and the biological properties of the selected scFv Ab, termed G1, we used two expression systems: for the first, the scFv was subcloned into the phagemid vector pCANTAB6 in which a Myc and a hexahistidine tag are fused to the C terminus of the scFv gene; the second was a T7 promotor-driven expression system in which the scFv gene is fused to a truncated form of Pseudomonas exotoxin A (PE38) to generate a scFv immunotoxin (12). This truncated form of PE contains the translocation and ADP-ribosylation domains of whole PE, but lacks the cell-binding domain, which is replaced by the scFv fragment fused at the N terminus of the truncated toxin. The G1 scFv was produced in E. coli BL21 ({lambda}DE3) cells by secretion and was purified from periplasmic fractions by metal affinity chromatography using the hexahistidine tag fused to the C terminus (Fig. 2B). The G1scFv-PE38 was expressed in BL21 cells, and, upon induction with isopropyl {beta}-D-thiogalactoside, large amounts of recombinant protein accumulated as intracellular inclusion bodies. SDS-PAGE showed that inclusion bodies from cultures expressing G1scFv-PE38 contained >90% recombinant protein (data not shown). Using established renaturation protocols, we succeeded in refolding G1scFv-PE38 from solubilized inclusion bodies in a redox-shuffling refolding buffer and purified it by ion-exchange chromatography on Q-Sepharose and MonoQ columns, and later by size-exclusion chromatography. A highly purified G1scFv-PE38 fusion protein with the expected size of 63 kDa was obtained, as analyzed by SDS-PAGE under nonreducing conditions (Fig. 2A). The molecular profile of the G1 scFv and G1 scFv immunotoxin was analyzed by size-exclusion chromatography and revealed a single protein peak in a monomeric form with an expected molecular mass of 26 and 63 kDa, respectively (data not shown). The yield of the refolded G1 scFv immunotoxin was ~2%; thus, 2 mg of highly pure protein could be routinely obtained from the refolding of 100 mg of protein derived from inclusion bodies containing 80–90% of recombinant protein. This yield is similar to previously reported scFv immunotoxins that expressed well and were produced using a similar expression and refolding system (30). The yield of the G1 scFv was 3 mg from a 1-L bacterial culture.



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  		FIGURE 2. Purity and binding specificity of G1 scFv and G1scFv-PE38. Purified G1 scFv (A) and G1scFv-PE38 (B) were analyzed by SDS-PAGE analysis on 10% gels. Binding specificity of purified G1scFv-PE38 was tested by ELISA on Immunoplates coated with scHLA-A2-peptide complexes that were refolded in vitro and contain: the gp100-derived peptide G9-209 (wild type), G9-209 M (mutated anchor position 2), G9-154, and G9-280. Telomerase catalytic subunit-derived peptide 865 and 540, human T lymphotropic virus type 1 (HTLV-1)-derived peptide TAX, and epithelial-derived mucine peptide MUC. The binding of G1scFv-PE38 to immobilized complexes was monitored with HRP-labeled anti-PE Abs. The ELISA reactivity data with the various scHLA-A2/peptide complexes are a representative of 10 independent experiments.

 
The binding specificity of the soluble purified G1 scFv Ab and G1scFv-PE38 fusion protein was determined by ELISA on biotinylated MHC-peptide complexes immobilized to wells through BSA-biotin-streptavidin to ensure correct folding of the complexes. The correct folding of the bound complexes and their stability during the binding assays were determined by their ability to react with the conformational, specific mAb w6/32, which binds HLA complexes only when folded correctly and when it contains peptide (data not shown). When we used the soluble purified G1 scFv or G1scFv-PE38 protein, the ELISA revealed a very specific recognition pattern corresponding to the hallmarks of MHC-restricted T cell specificity (Fig. 2C). The G1 scFv selected to bind the G9-209 M-containing HLA-A2 complex reacted only with the specific complex and not with complexes displaying the G9-280 and G9-154 gp100-derived MHC-peptide complexes nor to other control complexes containing HLA-A2-restricted telomerase-derived epitopes 540 and 865 (32), a MUC1-derived peptide (33), or the HTLV-1-derived TAX peptide (34) (Fig. 2C). The G1 scFv, which was selected on HLA-A2 complexes containing the G9-209 M-modified peptide, in which position 2 of the peptide was altered to methionine to improve its binding affinity to the MHC, also recognized the native peptide G9-209 in complex with HLA-A2 (Fig. 2C). In these assays, the binding was detected with an anti-PE38 Ab. Similar results were obtained when using the unfused G1 scFv Ab in which detection was performed with anti-Myc tag Ab (data not shown). Thus, this Ag-specific scFv fragment exhibits binding characteristics and the fine specificity of a TCR-like molecule. The G1 scFv or G1scFv-PE38 did not recognize the peptide alone nor empty HLA-A2 molecules (which are difficult to produce because they are unstable in the absence of a peptide), neither streptavidin nor other protein Ags (data not shown).

Next, the binding properties of the TCR-like soluble purified G1scFv-PE38 were determined using a saturation ELISA in which biotinylated complexes were bound to BSA-biotin-streptavidin-coated plates to which increasing amounts of G1scFv-PE38 were added. The binding of G1scFv-PE38 to the specific gp100-derived HLA-A2/G9-209 M complex was dose dependent and saturable (Fig. 3A). Extrapolating the 50% binding signal revealed that this Ab possessed high affinity, with a binding affinity in the nanomolar range. To determine the apparent binding affinity of the TCR-like scFv fragments to its cognate MHC-peptide complex, we performed a competition-binding assay in which the binding of 125I-labeled G1scFv-PE38 was competed with increasing concentrations of unlabeled protein. These binding studies revealed an apparent binding affinity in the low nanomolar range of 5 nM (Fig. 3B). Importantly, these results underscore our success in isolating a high affinity scFv Ab with TCR-like specificity from the phage-displayed Ab repertoire of immunized HLA-A2 transgenic mice.



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  		FIGURE 3. Binding characteristics of the TCR-like G1 scFv. A, Titration ELISA of purified soluble G1 scFv. Wells were coated with the scHLA-A2/G9-209 complex, and the binding of increasing amounts of G1 scFv was tested. Detection was with HRP-labeled anti-Myc tag Ab. The ELISA-binding data are representative of four independent experiments. B, Competitive binding analysis of the ability of purified G1scFv-PE38 to inhibit the binding of 125I-labeled G1scFv-PE38 to the HLA-A2/G9-209 M complex. The apparent binding affinity of the recombinant scFv was determined as the concentration of competitor (soluble purified G1scFv-PE38) required for 50% inhibition of the binding of the 125I-labeled tracer. The competition-binding data are representative of three independent experiments.

 
Binding of G1 scFv to APCs displaying the gp100-derived epitope

To demonstrate that the isolated soluble G1 scFv can bind the specific MHC-peptide complex, not only in its recombinant soluble form, but also in the native form, as expressed on the cell surface, we used two APC systems. One consists of the murine TAP2-deficient RMA-S cells that were transfected with the human HLA-A2 gene in a single-chain format (26) (HLA-A2.1/Db-{beta}2m single chain) (RMA-S-HHD cells). The gp100-derived peptide and control peptides were loaded on the RMA-S-HHD cells, and the ability of the selected G1 scFv Ab to bind to peptide-loaded cells was monitored by FACS (Fig. 4). Peptide-induced MHC stabilization of the TAP2 mutant RMA-S-HHD cells was determined by analyzing the reactivity of the conformational anti-HLA Ab w6/32 and the anti-HLA-A2 mAb BB7.2 with peptide-loaded and unloaded cells (Fig. 4, A and B). The G1 scFv, which recognized the G9-209 M-containing HLA-A2 complex, reacted only with RMA-S-HHD cells loaded with the G9-209 M peptide, but not with cells loaded with the G9-280 peptide (Fig. 4C) or control cells not loaded with peptide. The G1 scFv did not bind to cells loaded with other HLA-A2-restricted control peptides such as TAX, MUC1, or telomerase-derived peptides used for the specificity analysis (see Fig. 2C).



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  		FIGURE 4. Binding of G1 scFv to peptide-pulsed APCs and melanoma cells. RMAS-HHD, JY, or melanoma FM3D cells were pulsed with the gp100-derived peptides G9-209, G9-209 M, or G9-280, as indicated. Peptide-loaded cells were then incubated with the soluble purified G1 scFv Ab. Monitoring of binding was by flow cytometry using FITC-labeled anti-Myc Ab for detection. In A and B, RMAS-HHD cells were loaded with the G9-209 and G9-280 peptides and stained with the anti-HLA Ab w6/32 (A) or anti-HLA-A2 Ab BB7.2 (B) to demonstrate the stabilization/expression of HLA-A2 complexes on the surface of peptide-loaded, but not on peptide-unloaded cells. Control RMAS-HHD cells that were not loaded with peptide are used as reference. In C, RMAS-HHD cells were loaded with the G9-209 or G9-280 peptides, as marked, and were subsequently stained with the G1 scFv. The binding to cells loaded with the G9-209 peptide, but not to control cells loaded with G9-280 peptide is shown. Control unloaded cells are used as reference. In D–F, melanoma FM3D cells were pulsed with the gp100-derived peptides G9-209 M (D, F), G9-209 (D, E), and control G9-280 when indicated, and subsequently stained with the anti-HLA-A2 Ab BB7.2 (D) or G1 scFv (E, F). The reactivity of FM3D-loaded cells with the G1 scFv is demonstrated only when the G9-209/G9-209 M peptide was used for loading, but not the G9-280 peptide. Cells not pulsed with peptide were used as reference. As negative controls, HLA-A2-negative KB3-1 cells were used (G, H) that were loaded with the G9-209 M and G9-280 gp100-derived peptides and stained with the anti-HLA-A2 Ab BB7.2 (G) or the G1 scFv Ab (H).

 
We also used a second type of APC, namely the EBV-transformed B lymphoblast JY cells, which express HLA-A2; these cells were incubated with the gp100-derived or control peptides. They are TAP+, and consequently, the displaying of the exogenously supplied peptide is facilitated by peptide exchange. Using this strategy, we observed a similar binding specificity with the G1 scFv Ab (data not shown). These results demonstrate that the scFv Ab can specifically recognize its corresponding native HLA-A2 complex on the surface of cells.

Detection of complexes formed by active intracellular processing

To examine the ability of the TCR-like G1 scFv to detect HLA-A2/G9-209 complexes produced by physiological Ag processing, we transfected the gp100 gene into the EBV-transformed B cell HLA-A2-positive Ag-presenting JY cells and into HLA-A2-negative APD cells as controls. Twenty-four hours after transfection, we tested the reactivity of the HLA-A2/G9-209-specific Ab G1 scFv by flow cytometry. As shown in Fig. 5A, significant staining above control could be clearly seen only with HLA-A2-positive JY cells transfected with the gp100 gene, but not with control nontransfected JY cells (Fig. 5B) or control HLA-A2-negative cells transfected or not transfected with the g100 gene (Fig. 5, C and D). The peptide-specific, MHC-restricted pattern of reactivity by G1 scFv is not due to differences in transfection efficiency or HLA expression of JY and APD cells because the percentage of transfected cells was similar, as determined in control experiments transfecting green fluorescence protein into these cells (Fig. 5E), and the staining intensity of these cells with w6/32, a pan-MHC mAb, was similar (data not shown). These results indicate that the TCR-like scFv G1 Ab is capable of detecting the specific MHC-peptide complex after active and naturally occurring endogenous intracellular processing.



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  		FIGURE 5. Detection of the specific HLA-A2/G9-209 complex by G1 scFv after naturally occurring active intracellular processing. JY HLA-A2+ APCs were transfected with the whole gp100 gene (pCDNA-gp100) (A) or control empty vector (pCDNA) (B). Twelve to 24 h after transfection, cells were stained by flow cytometry using the HLA-A2/G9-209-specific G1 scFv. As controls, HLA-A2-negative (HLA-A1+) APD cells were transfected (C) or not (D) with the gp100 gene and stained with the G1 scFv. Detection of staining by G1 scFv was with HRP-labeled anti-Myc Ab. The efficiency of gene transduction into JY and APD cells was similar, as monitored by transfection with a pCDNA vector carrying the green fluorescence protein gene (E).

 
Binding of G1 scFv to melanoma cells

To demonstrate that the G1 scFv can bind the specific MHC-peptide complex displayed on the surface of tumor cells, we used HLA-A2+ melanoma FM3D cells (Fig. 4D) that were pulsed with the specific gp100-derived G9-209 peptide or control peptide. Similarly to the JY APCs, pulsing the melanoma cells enables the display of the exogenously supplied peptide facilitated by peptide exchange. Using this strategy, we obtained a mixture of exogenously and endogenously derived peptides presented on HLA-A2 that are displayed on the cell surface. As shown in Fig. 4E, the G1 scFv Ab was able to stain and detect HLA-A2/G9-209 M complexes on G9-209 M-pulsed FM3D cells, but not on FM3d cells pulsed with the gp100-derived G9-280 peptide. The G1 scFv Ab was able also to stain FM3D cells pulsed with the native nonmodified G9-209 peptide (Fig. 4F); however, control HLA-A2-negative KB3-1 cells (Fig. 4G) pulsed with the G9-209 M or G9-209 peptides were not recognized by the G1 scFv Ab (Fig. 4H).

Next, we attempted to detect the gp100-derived G9-209 epitope on the surface of nonpulsed melanoma cells by flow cytometry. As shown in Fig. 6, A and D, G1 scFv was able to detect the specific HLA-A2/G9-209 epitope on the surface of HA12 HLA-A2+ and gp100-positive FM3D melanoma cells, but not on HLA-A2-negative gp100-positive G-43 melanoma cells nor on the surface of HLA-A2-positive, gp100-negative, MDA-MB-231 breast carcinoma cells. These results demonstrate that the G1 scFv Ab can recognize the specific MHC-peptide complex on the surface of peptide-pulsed as well as nonpulsed tumor melanoma cells.



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  		FIGURE 6. Binding of G1 scFv to melanoma cells. FM3D (HLA-A2+/gp100+) (A), G43 (HLA-A1+/gp100+) (B) melanoma cells, and MDA-MB-231 (HLA-A2+/gp100-) breast carcinoma cells were stained with G1 scFv (50 µg/ml) and analyzed by flow cytometry. Staining was detected with FITC-labeled anti-Myc Ab. Control cells are stained with secondary FITC-labeled anti-Myc Ab only. In D, the relative fluorescence intensity of eight independent measurements is shown.

 
In situ detection of HLA-A2/G9-209 peptide-MHC complexes on melanoma cells

Another major potential use for TCR-like Abs is the direct in situ visualization of ligand-bearing cells within undisturbed tissues using immunohistological methods. As a first step to assess this potential, we determined whether G1 scFv can be used for such studies by testing its ability to detect in situ HLA-A2/G9-209 complexes on melanoma cells. Melanoma gp100-expressing, HLA-A2-positive and HLA-A2-negative melanoma cells were subjected to immunohistochemistry staining with the G1 scFv Ab, followed by a secondary staining step with HRP-labeled anti-Myc Ab. As shown in Fig. 7, these experiments showed a strong positive staining of the gp100-expressing, HLA-A2-positive melanoma lines FM-3-29, FM3-D, M-ww, M-PAT, and M-141 (Fig. 7, A–D and F), but not of gp100-expressing, HLA-A2-negative (HLA-A1+) melanoma G-43 cells (Fig. 7E) nor HLA-A2-positive, gp100-negative MDA-MB-231 breast carcinoma cells (Fig. 7G). The G1 scFv did not stain additional six nonmelanoma cell lines tested (data not shown). These data show the ability of the G1 scFv TCR-like Ab to detect specific peptide-MHC complexes in situ on cells and potentially in tissue sections after naturally occurring active intracellular processing. To our knowledge, this is the first demonstration of in situ detection of a tumor-derived T cell epitope using TCR-like scFv recombinant Abs.



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  		FIGURE 7. Immunohistochemical staining of melanoma cells with G1 scFv. HLA-A2-positive or HLA-A2-negative gp100-expressing melanoma cells or control MDA-MB-231 breast carcinoma cells were stained in situ by G1 scFv, followed by detection with HRP-labeled anti-Myc Ab. FM-3-29 and FM3-D are HLA-A2+ established melanoma cells lines; G-43 are HLA-A2- melanoma cell line. M-ww, M-PAT, and M-141 are lines established from a fresh melanoma tissue. MDA-MB-231 are HLA-A2+ breast carcinoma cell line.

 
Cytotoxic activity of G1scFv-PE38 toward APCs

To determine the ability of the G1 scFv Ab to serve as a targeting moiety for T cell-like specific elimination of APCs, we constructed, as described, the G1scFv-PE38 molecule in which the very potent truncated form of Pseudomonas exotoxin A is fused to the C terminus of the scFv gene and tested its ability to kill peptide-loaded APCs. RMAS-HHD or JY cells were loaded with the gp100-derived epitopes G9-209 M and G9-280V as well as with other control HLA-A2-restricted peptides. FACS analysis with anti-HLA-A2 Ab revealed a similar expression pattern of HLA-A2 molecules with G9-209 M, G9-280V, and other control peptide-loaded cells (Fig. 4B). As shown in Fig. 8A, cytotoxicity by G1scFv-PE38 was observed only on RMAS-HHD cells loaded with the G9-209 peptide with an IC50 of 10–20 ng/ml. No cytotoxic activity was observed on RMAS-HHD cells that were loaded with the gp100-derived G9-280V epitope or with other control HLA-A2-restricted peptides or cells that were not loaded with peptide. G9-209 M-loaded RMAS-HHD cells were not killed with an irrelevant immunotoxin in which an anti-human Lewis Y scFv Ab is fused to PE38 (B3(Fv)-PE38) (Fig. 8A). In the EBV-transformed JY cells, which express normal TAP, the display of the exogenously supplied peptide is facilitated by peptide exchange. Using this strategy, we observed similar sp. act. in which G1scFv-PE38 kills only cells loaded with the G9-209 M peptide (Fig. 8B). Additional proof for specificity is demonstrated in competition experiments in which excess specific and control soluble scHLA-A2-peptide complex was present in solution, to compete for binding and inhibit cytotoxicity by G1scFv-PE38. An example of this type of assay is shown in Fig. 8C, in which excess soluble G9-209 M-containing HLA-A2, but not the G9-280V/HLA-A2 complex competed and inhibited the cytotoxic activity of G1scFv-PE38 toward G9-209 M-loaded JY cells.



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  		FIGURE 8. Cytotoxic activity of G1scFv-PE38 on peptide-loaded APCs. RMAS-HHD (A) or JY (B) cells were loaded with the HLA-A2-restricted peptides G9-209 M and G9-280 derived from gp100 or peptides 865 and TAX derived from the telomerase catalytic subunit or HTLV-1 Tax protein, respectively. Peptide-loaded cells were incubated with increasing concentrations of G1scFv-PE38. Protein synthesis was determined by incorporation of [3H]leucine into cellular proteins. The control immunotoxin B3(Fv)-PE38 directed to the Lewis Y Ag was used as a control nonspecific immunotoxin. In C, excess (0.15–0.25 mg/ml) of the indicated scHLA-A2-peptide complex is added to wells before the addition of G1scFv-PE38 (25–50 ng/ml). In the control, no MHC-peptide complex was added. In D, melanoma cells FM3-29, FM3-D, HA141, ZA (HLA-A2/gp100+), and G43 (HLA-A1/gp100+) or breast carcinoma MDA-MB-231 (HLA-A2/gp100-) cells were incubated with G1scFv-PE38 at 500 ng/ml. Protein synthesis was determined by incorporation of [3H]leucine into cellular proteins. The cytotoxicity data are representative of five independent experiments.

 
Next, we attempted to test the activity of G1scFv-PE38 on melanoma cells that are HLA-A2 positive and express gp100. As shown in Fig. 8D, G1scFv-PE38 exhibited specific cytotoxicity on gp100-expressing FM3-29, FM3-D, and HA141 melanoma cells, but not on the HLA-A2-negative, gp100-positive G43 cells or on HLA-A2-positive breast carcinoma MDB-MB-231 cells. A low degree of activity was also observed on ZA (HLA-A2+/gp100+) melanoma cells (Fig. 8D). These results further demonstrate the fine and unique specificity of the G1 scFv Ab and its ability to serve as a targeting moiety to deliver a cytotoxic effector molecule with Ag (peptide)-specific, MHC-restricted specificity of T cells directed toward a human tumor T cell epitope.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In recent years, the advent of the application of recombinant class I MHC-peptide complexes and their tetrameric arrays now enables us to detect and study rare populations of Ag-specific T cells (25, 35, 36). However, fundamental questions in immunology in general, and in tumor immunology in particular, regarding Ag presentation are still open because of the lack of reagents that will enable phenotypic analysis of Ag (MHC-peptide) presentation, the other side of the coin to MHC-peptide-TCR interactions. One way to generate such reagents is by making TCR-like Abs; however, only a few publications have reported the generation of self MHC-restricted Abs by conventional means such as the hybridoma technology (13, 14, 15, 16). The major reason for these past difficulties may be found in the molecular nature and the resolved structures of MHC-peptide complexes. More specifically, the peptides are deeply buried inside the MHC-binding groove, and therefore they are presented as extended mosaics of peptide residues intermingled with the MHC residues. It has been shown that no more than 100–300 Å2 of class I MHC-bound peptide faces outward and thus is available for direct recognition, whereas Abs recognizing protein molecules engage ~800 Å2 of their ligand (17). Thus, when generating TCR-like Abs, these molecules will presumably recognize the peptide, but will also have to be dominated by the MHC.

Until now, Abs with TCR-like specificity have been generated against murine MHC-peptide complexes using various strategies of immunization (17). Recently, a large human Fab library was used to select for HLA-A1-MAGE-A1-specific binding Abs (18). One specific clone, G8, was selected that exhibited TCR-like specificity, but revealed a relatively low affinity of 250 nM.

In this study, we have demonstrated the ability to select from an immune repertoire of murine scFv fragments, a high affinity Ab directed toward a human T cell epitope derived from a cancer Ag, the melanoma-associated Ag gp100.

G1 scFv exhibits a very specific and special binding pattern; it can bind in a peptide-specific manner to HLA-A2 complexes. Hence, this is a recombinant Ab with TCR-like specificity. In contrast to the inherent low affinity of TCRs, this molecule displays the high affinity binding characteristics of Abs, while retaining TCR specificity. Our study strikingly demonstrates the power of the phage display approach and its ability to select especially fine specificities from a large repertoire of different Abs.

Our ability to select high affinity TCR-like Abs, despite the fact that such peptide-specific binders are thought to be rare and difficult to isolate, may result from the following considerations. One is the mode of immunization and selection: the fact that we use a transgenic animal for immunization combined with the power of various selection strategies used by phage display. We think that using HLA-A2 transgenic mice is an advantage because they are usually tolerant to HLA-A2 complexes, unless a new foreign peptide is presented on the complex. The ability to isolate TCR-like Ab molecules may represent a situation in which lymphocytes that were tolerant to HLA-A2 are now exposed to new epitopes contributed by the melanoma gp100-derived peptide presented on HLA-A2. The panning procedure that combined an excess of nonspecific complex in solution significantly contributed to the selection process and allowed us to isolate a rare Ab clone (1 of 108 clones). Another important issue relates to the state of the Ag used in the selection process. The conformation of the Ag has to be as natural as possible, especially when produced in a recombinant form. We have found that in vitro refolding from inclusion bodies produced in E. coli of a scMHC molecule complexed with various peptides yields large amounts of correctly folded and functional protein. The fact that G1 scFv was isolated from a relatively small library of ~108 clones, yet is highly specific with an affinity in the nanomolar range, strongly indicates that the HLA-A2 transgenic mice we used for immunization indeed developed high affinity Abs to the HLA-A2/G9-209 complexes. The observation that only a single anti-HLA-A2/G9-209 Ab was isolated may reflect that only one such specificity exists or that other specificities were not generated during the immune response because such a response could not be easily generated and tolerated by the HLA-A2 transgenic mice. Quite astonishing is the fact that similar results were reported in the past for a murine MHC-peptide system, in which, using phage display, a recombinant TCR-like Ab directed toward a class I murine H-2Kk molecule in complex with the influenza hemagglutinin peptide Ha255–262 was isolated (17). Similarly to the results presented in this work, of the 50 clones tested, 7 reacted specifically with the H-2Kk/Ha255–262 complexes only, and not with other H-2Kk/peptide complexes. Interestingly, the DNA sequences of these specific clones were determined and found to be identical (17). Similar observations in two independent studies using different strains of mice and different class I MHC-peptide complexes indicate that these Abs are quite rare.

Despite the fact that these Abs are rare, this and our study demonstrate the power of the phage display approach, which can be applied in a generic form to isolate recombinant Abs with TCR-like specificity to a variety of MHC-peptide complexes related to various pathological conditions such as cancer, viral infections, and autoimmune diseases.

Recombinant Abs with TCR-like specificity represent a new, valuable tool for future research in two major areas of tumor immunology. First, these Abs may now be used to detect and directly visualize the presence of specific T cell epitopes or MHC-peptide complexes by standard methods of flow cytometry and immunohistochemistry. They should be very useful for the study and analysis of Ag presentation in cancer by determining the expression of specific tumor-related MHC-peptide complexes on the surface of tumor cells, metastasis, APCs, and lymphoid cells. Moreover, such Abs can be used to analyze immunotherapy-based approaches by determining the alterations in MHC-peptide complex expression on APCs before, during, and after vaccination protocols with peptides or with APCs loaded with tumor cell extracts or dendritic-tumor cell hybrid vaccinations (7, 8, 9, 10, 11). Thus, questions relating to how and where certain events occur during Ag presentation may be directly addressed, for the first time, and the expression of T cell epitopes on the APC may be visualized and quantitated. Second, Abs with such exquisitely fine specificity directed toward a very specific and unique human tumor Ag present new opportunities for use as targeting moieties for various Ab-based immunotherapeutic approaches. This includes using such Abs to construct recombinant immunotoxins (12), for fusion with cytokine molecules (37), or for bispecific Ab therapy (38). The open question with respect to these applications relates to the low density of the specific epitope on the surface of the target cell. It has been previously demonstrated, using the murine H-2Kk/influenza hemagglutinin peptide complex and a similar Ag-presenting system, as was used in this study, that to achieve efficient killing with a TCR-like immunotoxin molecule, a density of several thousand particular MHC-peptide complexes is required for the selective elimination of APCs (39). The results of this study support these findings, achieving a similar cytotoxic potential of a T cell-like immunotoxin.

Thus, a major application of TCR-like Abs is for the development of a new class of Ab-based tumor-targeting agents. In this approach, small recombinant fragments of Abs are being used to specifically target a cytotoxic drug or a toxin to cancer cells. The Fv targeting moiety of the Ab is composed of the H and L chain V regions (VH and VL), which are held together by a peptide linker, thereby forming an scFv molecule. scFv immunotoxins are being developed against a wide variety of cancer-specific targets and are being tested in various clinical trials, some of which already present promising anticancer activity (12). scFv immunotoxins have many advantages over conventional immunoconjugates in which the whole Ab is chemically conjugated to the toxin. 1) The former is produced directly in a single-chain form in bacteria, thereby avoiding complex chemical modification steps. 2) They can be produced in large quantities and are chemically homogenous as opposed to conventional immunoconjugates. 3) The most important advantage of scFvs and scFv immunotoxins is their small size, which allows them to penetrate into tumors more efficiently. ScFv fragments are also being developed as tumor-imaging agents. Further preclinical characterization is required to determine whether the G1scFv-PE38 molecule can be used to specifically eliminate melanoma cells that express the gp100-derived MHC-peptide complex. Our results indicate that recombinant Abs can be used to selectively target tumor cells that have been shown to express a particular MHC-peptide complex. Thus, the number of specific MHC-peptide complexes on the surface of human cancer cells could be sufficient to serve as a target for such a recombinant cytotoxic molecule. An obvious advantage arising from the use of these targets, such as gp100-derived epitopes, as an Ag for the specific targeting of drugs and toxins to tumor cells is the fact that gp100 is a differentiation Ag that is selectively expressed only on melanoma cells (or on normal melanocytes), but not on other essential tissues of a different histological origin. As such, these are considered very specific and unique targets for immunotherapy. To improve the targeting and detection capabilities of these TCR-like Ab molecules, two Ab engineering approaches can be used: 1) to increase the affinity of the parental Ab by affinity maturation strategies without altering its TCR-like fine specificity (40), and 2) to increase the avidity of these recombinant monovalent molecules by making them bivalent (38). Combining these strategies will result in second generation, improved molecules that will be valuable tools for immunotherapeutic approaches as well as serve as innovative research tools for studying the interaction of tumor cells and the human immune system.


   Footnotes
 
1 This work was supported by a research grant from the Israel Science Foundation administered by the Israel National Academy for Sciences and Humanities (Jerusalem, Israel) and by a Cancer Research Foundation Research Career Development Award, New York. Back

2 Address correspondence and reprint requests to Dr. Yoram Reiter, Faculty of Biology, Technion-Israel Institute of Technology, Technion City, Room 333, Haifa 32000, Israel. E-mail address: reiter{at}tx.technion.ac.il Back

3 Abbreviations used in this paper: sc, single-chain; {beta}2m, {beta}2-microglobulin; CBD, cellulose-binding domain; PPD, purified protein derivative; RT, room temperature. Back

Received for publication January 29, 2003. Accepted for publication June 18, 2003.


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 Introduction
 Materials and Methods
 Results
 Discussion
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