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The Journal of Immunology, 1998, 161: 2968-2976.
Copyright © 1998 by The American Association of Immunologists

Limited Diversity of Human scFv Fragments Isolated by Panning a Synthetic Phage-Display scFv Library with Cultured Human Melanoma Cells1

Elvyra J. Noronha, Xinhui Wang, Smruti A. Desai, Toshiro Kageshita and Soldano Ferrone2

Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To broaden the specificity of the Abs recognizing human melanoma-associated Ags (MAAs), we have isolated human single-chain fragment of the V region (scFv) fragments by panning the synthetic phage Ab library (#1) with the human melanoma cell lines S5 and SK-MEL-28. All of the isolated scFv fragments reacted with the mouse mAb defined high molecular weight melanoma-associated Ag (HMW-MAA). scFv #70 immunoprecipitates the two characteristic subunits of HMW-MAA, while scFv #28 only immunoprecipitates its large subunit. These results challenge the current view regarding the structure of HMW-MAA and indicate that it consists of two independent subunits. The human scFv fragments share some similarities with the mouse anti-HMW-MAA mAb. Like mAb 149.53 and 225.28, scFv #28 reacts with rat B49 neural cells that express a homologue of HMW-MAA. scFv #70 reacts with a determinant that is spatially close to the one identified by mAbs 149.53, VT68.2, and VT86. Besides suggesting similarities in the recognition of human melanoma cells by the mouse and human Ab repertoire, these results indicate that the Abs isolated from synthetic Ab libraries resemble those that are found in natural Ab repertoires. The restricted diversity of the scFv fragments that were isolated by panning synthetic Ab libraries with different melanoma cell lines suggests that certain Ags, like HMW-MAA, are immunodominant in vitro. This phenomenon, which parallels the in vivo immunodominance of certain Ags, implies that the antigenic profile of the cells used for panning determines the specificity of the preponderant population of isolated Abs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Malignant transformation of human melanocytes is associated with qualitative and quantitative changes in their antigenic profile (1, 2, 3). These changes have been investigated extensively since they may play a role in the interaction of melanoma cells with the immune system of the host and may provide markers to develop and apply immunodiagnostic and immunotherapeutic approaches to malignant melanoma.

Several types of immunologic probes have been used to characterize the antigenic profile of human melanoma cells. Among them, mouse mAbs have been the most effective in identifying a number of human melanoma-associated Ags (MAAs)3 with distinct immunochemical and functional properties (1, 2, 3). A few of the mouse mAb-defined MAAs, including the high molecular weight (HMW)-MAA, p97 MAA, and GD3 ganglioside, have been used as markers for in vivo diagnosis and as targets for the immunotherapy of melanoma (4, 5, 6, 7, 8). Attempts have also been made to identify human MAAs using both sera from patients with malignant melanoma and human mAbs. The application of sera from patients with malignant melanoma to analyze melanoma cells by serologic and/or immunochemical techniques has been successful only in a limited number of cases, because it has been hampered by the low titer and polyclonality of Abs (9). Human mAbs have been successful only to a limited extent primarily because of the methodologic difficulties in constructing stable human mAb-secreting hybridomas and cell lines (10, 11, 12). More recently, sera from patients with malignant melanoma have been used to screen cDNA libraries from human melanoma cell lines (13, 14). These investigations have identified not only previously described MAAs but also a tumor Ag named HOM-MEL-40; this Ag is expressed in a significant proportion of human melanomas, colon cancers, hepatocarcinomas, and breast carcinomas but is not expressed in normal tissues except the testis (14). Although this approach has proved useful in identifying novel MAAs, many of the Ags identified, including HOM-MEL-40, are intracellular. Therefore, the MAAs identified by this approach are not likely to be useful targets for Ab-based immunotherapy.

Recently, libraries of human single-chain fragment of the V region (scFv) fragments or Fab fragments of Abs that are expressed on the surface of filamentous phages have been used by Cai and Garen (15, 16) and by Pereira et al. (17) to identify human MAAs with human Abs. The phage Ab libraries used in these studies were constructed from the PBLs of patients with malignant melanoma that had been immunized with autologous melanoma cells transduced with the IFN-{gamma} gene (15) or with a vaccinia virus melanoma oncolysate (17). Of the several scFv fragments isolated by panning with autologous melanoma cells, Cai and Garen (15) reported only one scFv fragment that showed a specific reactivity with melanoma cells. Among the phage-displayed Fab fragments analyzed by Pereira et al. (17), only one showed relatively strong binding to cultured melanoma cells and surgically removed melanoma lesions as well as weak binding to a limited number of normal tissues. No information is available with regard to the molecular profile of the MAAs that were identified with the human Abs in the two studies. Therefore, the relationship of these MAAs to those described in the literature cannot be assessed.

In the present study, we have evaluated the usefulness of a synthetic phage scFv library in the identification of human MAAs. We have selected this library because a synthetic repertoire is not biased by immune selection and therefore has a broader range of Ab specificities. The melanoma cell lines S5 and SK-MEL-28 were used for panning, because they differ in the expression of mouse mAb-defined MAA (18) and consequently provide the opportunity to assess the influence of the antigenic profile of cell lines on the diversity of the scFv fragments isolated by panning a phage display Ab library.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Synthetic phage scFv library (#1)

The synthetic phage scFv library (#1) (a gift from Dr. Greg Winter, Medical Research Council Center for Protein Engineering, Cambridge, U.K.) was constructed as described previously (19).

mAbs and conventional antisera

The anti-HMW-MAA mAbs 149.53, 225.28, 763.74, TP41.2, TP61.5, VT68.2, and VT86 (20, 21, 22, 23); the anti-100-kDa MAA mAb 376.96 (24); the anti-HLA class I mAb TP25.99 (25); the anti-human ß2-microglobulin (ß2m) mAb NAMB-1 (26); the antiidiotypic mAbs MF9–10, MF9–36, MK1–94, MK1–148, MK1–181, MK1–193, MK1–264, and MK1–340 that were elicited with mAb 149.53 (27); the antiidiotypic mAb MK2–23 that was elicited with mAb 763.74 (27); the anti-rat NG2 mAb D4.11 (28), and the anti-c-myc oncoprotein mAb 9E10 (29) have all been described elsewhere. All mAbs were generated from BALB/c mice. The anti-CD8 mAb was purchased from Dako A/S (Glostrup, Denmark). The human anti-HMW-MAA scFv #61 and the human anti-antiidiotypic scFv #119 were obtained by panning the synthetic phage scFv library (#1) with purified HMW-MAA (18) and with the mAb MK2–23 (30), respectively.

mAbs were purified from ascites by sequential caprylic acid and ammonium sulfate precipitation (31). The purity and activity of the mAb preparations were monitored by SDS-PAGE and ELISA, respectively. Purified mAbs were biotinylated using sulfosuccinimidyl-6-(biotinamido)hexanoate biotin (Pierce, Rockford, IL) according to the manufacturer’s instructions.

Horseradish peroxidase (HRP)-conjugated sheep anti-M13 IgG Abs and purified rabbit anti-mouse IgG Abs were purchased from Pharmacia Biotech (Piscataway, NJ) and Jackson Immunoresearch Laboratories (West Grove, PA), respectively.

Cell lines, Ag preparations, and tissues

The human melanoma cell lines Colo 38, FO-1, Melur, S5, and SK-MEL-28; the human bladder carcinoma cell line T24; the human breast carcinoma cell line T47D; the human B lymphoid cell lines LG-2 and L14; the human T lymphoid cell line MOLT-4; and human fibroblasts were grown in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% FBS (BioWhittaker, Walkersville, MD). The guinea pig melanoma cell line GP5, the mouse melanoma cell line B16, and the rat neural cell line B49 were maintained in RPMI 1640 medium (Life Technologies) supplemented with 10% FBS and 2 mM L-glutamine (Life Technologies). All cell lines were maintained at 37°C in a 5% CO2 atmosphere.

Cell lysates were prepared by solubilizing the cells (1 x 107) that had been washed with HBSS (Life Technologies) in 1.2 ml of lysis buffer (10 mM Tris, 1 mM EDTA, 150 mM NaCl, and 1% Nonidet P-40 containing 1 mM PMSF). Ag-coated microtiter plates were prepared as described previously (18).

Lesions of melanocytic origin were obtained from patients who had undergone surgery in the Department of Dermatology at Kumamoto University School of Medicine (Kumamoto, Japan). The diagnosis of melanoma lesions was based on histopathologic characteristics. The frozen and formalin-fixed tissue sections were prepared as described previously (18).

Synthetic oligonucleotide primers and probes

The synthetic oligonucleotide primers complementarity determining region (CDR)FOR (5'-CAGGGTACCTTGGCCCCA) and CDRBACK (5'-GTGTATTACTGTGCAAGA) that were used to amplify the CDR3 (19); LMB3 (5'-CAGGAAACAGCTATGAC) (32) and LINKSEQ (5'-CGATCCGCCACCGCCAGAG) (33) that were used for PCR amplification and nucleotide sequencing; and VH family-specific oligonucleotide probes (32) were synthesized using a Cruachem PS250 Automated DNA Synthesizer (Cruachem, Sterling, VA). Oligonucleotide primers were radiolabeled with [{gamma}-32P]ATP (Amersham, Arlington Heights, IL) using the enzyme T4 polynucleotide kinase (Amersham).

Panning of synthetic phage scFv library (#1) with melanoma cells

The synthetic phage scFv library (~1 x 1013 transducing units/2 ml of PBS) was added to 2 ml of a melanoma cell suspension (5 x 106 cells/ml of 4% dry milk-PBS). The cell suspension was incubated for 90 min at room temperature on a turntable and then stood at room temperature for 30 min. Following six washings with PBS, bound phages were eluted by incubating the cells with 200 µl of 76 mM citric acid (pH 2.8) in PBS (34). After neutralization with 200 µl of 1 M Tris-HCl (pH 7.4), eluted phages were used to infect exponentially growing Escherichia coli TG1 cells. Bacteria were plated on trypticase-yeast extract (TYE) medium (35) containing ampicillin (100 µg/ml) (Boehringer Mannheim, Indianapolis, IN) and 1% w/v glucose (TYE-amp-glu). Phages were rescued using the helper phage VCS-M13 (Stratagene, La Jolla, CA) and were used for the next round of panning. A total of 2 µl of a phage suspension (~1 x 107 transducing units) that eluted after the fourth round of panning was diluted in 48 µl of HBSS and absorbed with LG-2 cells (1 x 106) for 2 h at room temperature in a 96-well tissue culture plate (Nunc A/S, Roskilde, Denmark) on a shaker. After seven absorptions, the phages were used to infect E. coli TG1 cells that were subsequently grown on TYE-amp-glu plates.

Preparation of phage-displayed and soluble scFv fragments

Phage-displayed scFv fragments were produced from single, ampicillin-resistant colonies by rescue with helper phage VCS-M13 as described previously (32). Soluble scFv fragments were produced from individual ampicillin-resistant colonies by induction with isopropyl-ß-D-thiogalactopyranoside (36). Alternatively, scFv fragments were harvested from the periplasmic space of individual colonies (37).

Binding assays

The ELISA to test the reactivity of the phage-displayed and soluble scFv fragments with viable cells in suspension (2 x 105/well), with cells grown as a monolayer and fixed with 0.24% glutaraldehyde and with mAb-captured Ags, was performed as described previously (18).

Cross-blocking assays were performed by incubating melanoma cells with various concentrations of unconjugated mAb at 4°C overnight. After washing with PBS-0.5% BSA, the cells were incubated for 2 h at 4°C with soluble scFv fragments that had been preincubated with the biotinylated mAb 9E10 or with biotinylated mAbs. The binding of scFv fragments and mAbs was detected by HRP-conjugated streptavidin (SA-HRP, Pierce) using orthophenylenediamine (Sigma, St. Louis, MO)-H2O2 as a substrate.

To test the reactivity of the mAbs and scFv fragments with antiidiotypic mAbs, flexible, 96-well microtiter plates were coated with antiidiotypic mAbs (100 µg/ml of 0.05 M NaHCO3, pH 9.6). The wells were blocked with PBS-2% BSA following an 18-h incubation at room temperature. Next, scFv fragments that had been preincubated with biotinylated mAb 9E10, or biotinylated mAbs were added and incubation was continued at room temperature for an additional 1 h. The binding of scFv fragments and mAbs was detected by a sequential incubation with SA-HRP and orthophenylenediamine-H2O2.

The indirect immunoperoxidase staining of frozen and formalin-fixed tissue sections was performed as described previously (18).

Determination of VH gene family and CDR3 length of scFv fragments

Colony hybridization to assign the VH gene family used by scFv fragments was performed as described previously (38) using {gamma}-32P-labeled VH family-specific oligonucleotide probes (32). The CDR3 length of the scFv fragments was also determined as described previously (19).

Determination of the nucleotide sequence of VH segments of scFv fragments

The nucleotide sequence of the VH segments was determined by the dideoxy chain-termination method (39). DNA templates for sequencing were prepared by PCR-amplifying scFv inserts (18) from selected clones using primers LMB3 and LINKSEQ (33). The PCR products were purified using a Geneclean II kit (Bio 101, Vista, CA) and were cycle sequenced (Cyclist Exo-Pfu DNA-sequencing kit, Stratagene) using the phosphorylated primer LINKSEQ. VH genes were assigned to a germline segment using the Ab database, V BASE (40).

Indirect immunoprecipitation and SDS-PAGE

The labeling of cells with iodine-125 (Na125I, Amersham) or with [35S]methionine (Trans-35S-label, ICN Biochemicals, Costa Mesa, CA) in the presence of tunicamycin (Sigma) was performed as described previously (41, 42). The solubilization of labeled cells, immunoprecipitation, SDS-PAGE, autoradiography, and fluorography were also performed as described previously (18).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Selection of melanoma cell-binding scFv fragments

Four rounds of panning of the synthetic phage scFv library (#1) with SK-MEL-28 cells resulted in a 3 x 103-fold enrichment of melanoma cell-binding scFv fragments. Isolated phages were absorbed with cultured human LG-2 B lymphoid cells to remove the phages that were binding to the Ags shared by human cells. Individual phage-displayed scFv fragments were then tested for reactivity with SK-MEL-28 cells and fibroblasts in a cell ELISA. Of the 232 scFv fragments screened, 30 reacted only with SK-MEL-28 cells, 110 reacted with both SK-MEL-28 cells and fibroblasts, and 92 did not react with either target. No scFv fragment reacted only with fibroblasts. Soluble scFv fragments were prepared from the 30 clones that reacted only with the SK-MEL-28 cells, and their reactivity was tested with SK-MEL-28 cells and fibroblasts. Only scFv #65 reacted with SK-MEL-28 cells in the soluble form; the reactivity was low. This finding may reflect the lower avidity of monomeric scFv fragments as compared to that of the multivalent scFv fragments that are displayed on phages (19). Soluble scFv fragments were prepared from an additional 114 clones and tested with SK-MEL-28 cells and fibroblasts by ELISA. A total of 30 scFv fragments reacted only with the SK-MEL-28 cells, while 25 fragments reacted with both targets and 59 reacted with neither target.

Four rounds of panning of the synthetic phage scFv library (#1) with S5 cells resulted in a 1.5 x 105-fold enrichment of melanoma cell-binding scFv fragments. Because of the low reactivity of the soluble scFv fragments despite the high binding to SK-MEL-28 cells of the corresponding phage-displayed scFv fragments, 80 clones that had been isolated after four rounds of panning with S5 cells followed by five absorptions with LG-2 B lymphoid cells were screened with S5 and LG-2 cells in an ELISA using soluble scFv fragments. Of the scFv fragments tested, 20 reacted only with S5 cells, 1 reacted with both targets, and 59 did not react with either target.

VH family and CDR3 length of scFv fragments isolated by panning with SK-MEL-28 and S5 melanoma cells

Of the 30 scFv fragments that were isolated by panning with SK-MEL-28 cells, 26 belong to the VH3 gene family and have a 4-aa-long CDR3; 4 belong to the VH1 gene family and have a 7-aa-long CDR3. All 20 scFv fragments that were isolated by panning with S5 cells belong to the VH3 gene family and have a 6-aa-long CDR3. scFvs #65 and #70, which belong to the VH1 and VH3 gene families, respectively, and scFv #28, which belongs to the VH3 gene family, were chosen for further studies among the clones that had been isolated by panning with SK-MEL-28 cells and S5 cells, respectively.

Nucleotide sequence of VH segments of scFvs #28, #65, and #70

An analysis of the deduced aa sequence of the VH segments of scFvs #28, #65, and #70 indicated that the VH segment of scFvs #28 and #70 is derived from the germline gene DP-38, while the segment of scFv #65 is derived from the germline gene DP-10 (Table IGo). The aa sequences of the CDR3 of the three scFv fragments display no homology with each other.


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  		Table I. VH gene family, germline origin, and VH CDR3 aa sequence of scFv #28, #65, and #70

 
Reactivity patterns of scFvs #28, #65, and #70 with human cell lines

When tested by ELISA with a panel of human cell lines, the phage-displayed and soluble scFvs #28, #65, and #70 reacted with Colo 38, FO-1, Melur, and SK-MEL-28 melanoma cells. scFvs #65 and #70 showed a lower reactivity with FO-1 cells than with Colo 38, Melur, and SK-MEL-28 cells. scFv #28 reacted with S5 melanoma cells, while scFvs #65 and #70 did not. The three scFv fragments did not react with the LG-2 and L14 B lymphoid cells, the MOLT-4 T lymphoid cells, the T24 bladder carcinoma cells, the T47D breast carcinoma cells, and the cultured human fibroblasts (Fig. 1Go).



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  		FIGURE 1. Reactivity in an ELISA of phage-displayed and soluble scFvs #28, #65, and #70 with human cell lines. Cultured human SK-MEL-28, Colo 38, Melur, FO-1, and S5 melanoma cells; human T24 bladder carcinoma cells; human T47D breast carcinoma cells; human LG-2 and L14 B lymphoid cells; human MOLT-4 T lymphoid cells; and human fibroblasts were incubated at 4°C for 2 h with phage-displayed scFvs #28, #65, and #70 (A) and also with soluble scFvs #28, #65, and #70 that had been preincubated with the biotinylated mAb 9E10 (B). The binding of the phage-displayed and soluble scFv fragments was detected using HRP-conjugated anti-M13 Abs and SA-HRP, respectively. Results are expressed as absorbance at 490 nm. Human anti-antiidiotypic scFv #119 was used as a control.

 
Immunochemical analysis of the specificity of scFvs #28 and #70

scFv #70 immunoprecipitated two components from 125I-labeled SK-MEL-28 cells; the electrophoretic profile of these components is very similar to those of the two subunits of HMW-MAA (250 kDa and >450 kDa) that were immunoprecipitated by the anti-HMW-MAA mAb 763.74. The latter had been used to monitor the expression of Ags by melanoma cells. scFv #28 immunoprecipitated only a >450-kDa component from 125I-labeled SK-MEL-28 cells (Fig. 2Go). scFv #65 was not used in these experiments because of its low reactivity with melanoma cells in the soluble form. Immunodepletion experiments were then performed to determine the structural relationship between the components that had been immunoprecipitated by the scFv fragments and by mAb 763.74. Immunodepletion of a 125I-labeled SK-MEL-28 cell lysate with mAb 763.74 resulted in a complete loss of the molecules identified by scFv #70 and a partial loss of the component recognized by scFv #28 (Fig. 3GoA). This immunodepletion is specific, because the anti-100-kDa MAA mAb 376.96 did not remove the molecules that were identified by mAb 763.74, scFv #28, and scFv #70 from a SK-MEL-28 cell extract. Immunodepletion of a 125I-labeled SK-MEL-28 cell lysate with scFv #28 resulted in a partial loss of the >450-kDa component recognized by scFv #70 and mAb 763.74 but did not cause a detectable reduction of the 250-kDa component (Fig. 3GoB). Immunodepletion of a 125I-labeled SK-MEL-28 cell lysate with scFv #70 resulted in a complete loss of the 250-kDa component recognized by mAb 763.74 and a partial loss of the >450-kDa component recognized by mAb 763.74 and scFv #28 (Fig. 3GoC). This immunodepletion is specific because scFv #119 did not remove the molecules identified by mAb 763.74, scFv #28, and scFv #70 from a SK-MEL-28 cell lysate. Also, scFvs #28 and #70 did not remove the 100-kDa molecule recognized by mAb 376.96 from a melanoma cell lysate.



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  		FIGURE 2. SDS-PAGE analysis of Ags immunoprecipitated from 125I-labeled human SK-MEL-28 melanoma cells by scFvs #28 and #70. A 1% Nonidet P-40 extract of 125I-labeled SK-MEL-28 cells was immunoprecipitated with scFvs #28 and #70. Ags were eluted and analyzed by SDS-PAGE in an 8% polyacrylamide gel. The gel was fixed, dried, and autoradiographed for <=3 days at -80°C using Kodak XAR-5 film. A 1% Nonidet P-40 extract of cultured human LG-2 B lymphoid cells, the anti-HMW-MAA mAb 763.74, the anti-HLA class I mAb TP25.99, the antiidiotypic mAb MK2–23, and anti-antiidiotypic scFv #119 were used as controls.

 


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  		FIGURE 3. Analysis of the structural relationship between the molecules recognized by scFvs #28 and #70 and the molecules recognized by the mouse anti-HMW-MAA mAb 763.74 in an SK-MEL-28 melanoma cell extract. A 1% Nonidet P-40 extract of 125I-labeled SK-MEL-28 cells was immunodepleted with the mAb 763.74 (A), scFv #28 (B), or scFv #70 (C). Each immunodepleted cell extract was immunoprecipitated with the insolubilized mAb 763.74, scFv #28, and scFv #70. Ags were eluted and analyzed by SDS-PAGE in an 8% polyacrylamide gel. Gels were fixed, dried, and autoradiographed for <=3 days at -80°C using Kodak XAR-5 film. A 1% Nonidet P-40 extract of 125I-labeled SK-MEL-28 cells that had been immunodepleted with the anti-100-kDa MAA mAb 376.96 and with anti-antiidiotypic scFv #119 was used as a control.

 
The >450-kDa component immunoprecipitated by scFv #28 is similar in electrophoretic mobility to the component immunoprecipitated by scFv #61 that had been isolated by panning the synthetic phage scFv library (#1) with purified HMW-MAA (18). The two scFv fragments recognize the same molecule, since the immunodepletion of a 125I-labeled Colo 38 melanoma cell lysate with scFv #28 resulted in a complete loss of the >450 kDa-component that was identified by scFv #61 (Fig. 4Go). This immunodepletion is specific because scFv #119 did not remove the component identified by scFv #61 in a Colo 38 cell lysate.



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  		FIGURE 4. Analysis of the structural relationship between molecules recognized by scFv #28 and anti-HMW-MAA scFv #61 in a Colo 38 melanoma cell extract. Following immunodepletion with scFv #28, a 1% Nonidet P-40 extract of 125I-labeled Colo 38 melanoma cells was immunoprecipitated with scFvs #28 and #61. Ags were eluted and analyzed by SDS-PAGE in an 8% polyacrylamide gel. The gel was fixed, dried, and autoradiographed for <=3 days at -80°C using Kodak XAR-5 film. A 1% Nonidet P-40 extract of 125I-labeled SK-MEL-28 cells that had been immunodepleted with anti-antiidiotypic scFv #119 was used as a control.

 
Additional proof of the specificity of scFvs #28 and #70 for HMW-MAA is provided by the reactivity of these scFv fragments in an ELISA with HMW-MAA that had been isolated from a melanoma cell lysate by binding to anti-HMW-MAA mAb TP41.2- coated microtiter plates. scFv #70, both in the phage and in the soluble form, reacted strongly with HMW-MAA, while both the phage and soluble forms of scFv #28 showed weak reactivity with isolated HMW-MAA (Fig. 5Go). The weak reactivity of scFv #28 with HMW-MAA is not caused by steric hindrance due to the spatially close relationship of the determinants recognized by scFv #28 and by mAb TP41.2, because scFv #28 showed a weak reactivity with HMW-MAA that had been isolated from a melanoma cell lysate using the anti-HMW-MAA mAbs 149.53, 763.74, and TP61.5 (Fig. 6Go). The latter three mAbs recognize determinants that are spatially distant from each other and from the determinant recognized by mAb TP41.2 (27). It is noteworthy that the phage-displayed but not the soluble scFv #65 reacted with isolated HMW-MAA.



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  		FIGURE 5. Reactivity in an ELISA of phage-displayed and soluble scFvs #28, #65, and #70 with HMW-MAA captured from a melanoma cell extract using an anti-HMW-MAA mAb TP41.2-coated microtiter plate. Phage-displayed scFvs #28, #65, and #70 (A) and soluble scFvs #28, #65, and #70 that were preincubated with the biotinylated mAb 9E10 (B) were incubated for 2 h at room temperature with HMW-MAA that had been isolated from a melanoma cell extract by binding to an anti-HMW-MAA mAb TP41.2-coated microtiter plate ({blacksquare}). The binding of the phage-displayed and soluble scFv fragments was detected using HRP-conjugated anti-M13 Abs and SA-HRP, respectively. Results are expressed as absorbance at 490 nm. HLA class I Ags that were isolated from a LG-2 cell lysate by binding to an anti-ß2m mAb NAMB-1-coated microtiter plate () and anti-antiidiotypic scFv #119 were used as controls.

 


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  		FIGURE 6. Reactivity in an ELISA of soluble scFv #28 with HMW-MAA captured from a melanoma cell extract by anti-HMW-MAA mAb 149.53-coated, 763.74-coated, and TP61.5-coated microtiter plates. Following preincubation with the biotinylated mAb 9E10, soluble scFv #28 was incubated for 2 h at room temperature with HMW-MAA that had been isolated from a melanoma cell extract by binding to the anti-HMW-MAA mAbs 149.53- ({blacksquare}), 763.74- (), and TP61.5- () coated microtiter plates. The binding of scFv fragments was detected using SA-HRP. Results are expressed as absorbance at 490 nm. HLA class I Ags that had been isolated from a LG-2 cell lysate by binding to an anti-ß2m mAb NAMB-1-coated microtiter plate ({boxtimes}), anti-HMW-MAA scFv #61, and anti-antiidiotypic scFv #119 were used as controls.

 
Differential role of carbohydrates in the expression of the determinants recognized by scFvs #28 and #70

N-linked oligosaccharides are involved in the expression of the epitope recognized by scFv #28, since this scFv fragment did not immunoprecipitate any component from Colo 38 melanoma cells that had been intrinsically radiolabeled with [35S]methionine in the presence of tunicamycin, an inhibitor of N-glycosylation of glycoproteins (43). In contrast, N-linked oligosaccharides are not involved in the expression of the determinant recognized by scFv #70, since the latter immunoprecipitated the 220-kDa core protein of HMW-MAA from tunicamycin-treated Colo 38 cells (Fig. 7Go).



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  		FIGURE 7. Differential role of N-linked glycosylation in the expression of the antigenic determinants recognized by scFvs #28 and #70 on HMW-MAA isolated from a Colo 38 melanoma cell extract. A 1% Nonidet P-40 extract of Colo 38 cells that had been labeled with [35S]methionine in the presence of tunicamycin (2 and 3 µg/ml, respectively) was immunoprecipitated with scFvs #28 and #70. Ags were eluted and analyzed by SDS-PAGE in an 8% polyacrylamide gel. Gels were fixed, dried, and processed for fluorography for <=3 days at -80°C using Hyperfilm-ECL. The anti-HMW-MAA mAb 763.74 and anti-antiidiotypic scFv #119 were used as controls.

 
Mapping of the antigenic determinants recognized on melanoma cells by scFvs #28, #70, and mouse anti-HMW-MAA mAb

In cross-blocking experiments, the anti-HMW-MAA mAbs 225.28, 763.74, and TP41.2 that recognize distinct and spatially distant antigenic determinants did not inhibit the binding of scFvs #28 and #70 to Colo 38 melanoma cells. However, the anti-HMW-MAA mAbs 149.53, VT68.2, and VT86, which cross-block each other, significantly inhibited the binding of scFv #70 to Colo 38 cells (Fig. 8Go). This cross-blocking most likely reflects the recognition of distinct but spatially close antigenic determinants, because mAbs VT68.2 and VT86 and scFv #70 did not react with a panel of antiidiotypic mAbs that were elicited with mAb 149.53 (Fig. 9Go).



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  		FIGURE 8. Mapping on cultured human Colo 38 melanoma cells of the antigenic determinants recognized by scFv #70 and by the anti-HMW-MAA mAbs 149.53, VT68.2, and VT86. Colo 38 melanoma cells were incubated with the mAbs 149.53 (A), VT68.2 (B), VT86 (C), and TP41.2 (D) at 4°C overnight. Following washing with PBS-0.5% BSA, the cells were incubated for 2 h at 4°C with soluble scFvs #28 ({blacktriangledown}) and #70 ({diamondsuit}) that had been preincubated with the biotinylated mAb 9E10. The binding of the scFv fragments was detected using SA-HRP. The inhibitory activity of the mAb 149.53, VT68.2, and VT86 preparations was monitored by testing their ability to inhibit the binding to Colo 38 cells of the autologous biotinylated mAbs 149.53 (•), VT68.2 ({blacktriangleup}), and VT86 ({blacksquare}). The mAb TP41.2 ({circ}), which binds to an epitope that is distinct from the one identified by mAb 149.53, VT68.2, and VT86, was used as a specificity control.

 


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  		FIGURE 9. Lack of expression on scFv #70 and on the anti-HMW-MAA mAbs VT68.2 and VT86 of the Ids recognized by antiidiotypic mAb elicited with the anti-HMW-MAA mAb 149.53. Soluble scFvs #28 () and #70 () that had been preincubated with the biotinylated mAb 9E10 and biotinylated mAbs 149.53 ({blacksquare}), VT68.2 (), and VT86 () were added to antiidiotypic mAb MF9–10-, MF9–36-, MK1–94-, MK1–148-, MK1–181-, MK1–193-, MK1–264-, and MK1–340-coated microtiter plates. After a 2 h incubation at room temperature, the binding of scFv fragments and mAbs was detected using SA-HRP. The results are expressed as absorbance at 490 nm. The anti-HMW-MAA mAb TP41.2 ({boxtimes}) and anti-antiidiotypic scFv #119 () were used as controls.

 
Comparison of the reactivity of scFv fragments and mouse anti-HMW-MAA mAb with surgically removed melanoma lesions

Both scFvs #28 and #70 stained frozen melanoma tissue sections, but only scFv #28 stained formalin-fixed melanoma tissue sections. The staining pattern that was obtained with scFvs #28 and #70 was similar to but not identical with that obtained with mouse anti-HMW-MAA mAb 149.53. mAb 149.53 stained the cell membrane and sometimes stained the cytoplasm of the melanoma cells and blood vessels (Fig. 10GoA). scFvs #28 and #70 stained the membrane and cytoplasm of melanoma cells in a few lesions. A representative result is shown in Figure 10GoB. In addition to melanoma cells, the two scFv fragments stained the connective tissue and/or the basement membrane-like structure surrounding the melanoma nest in the majority of lesions tested. A representative staining pattern is shown in Figure 10GoC. Furthermore, scFv #28 also stained normal keratinocytes and blood vessels.



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  		FIGURE 10. Immunoperoxidase staining of surgically removed melanoma lesions with scFvs #28 and #70 and with the anti-HMW-MAA mAb 149.53. Frozen (A and C) and paraffin-embedded (B) sections of a primary (a and b) and a metastatic melanoma lesion (c and d) were stained with mAb 149.53 (A, a and c), scFv #28 (B, a and c), and scFv #70 (C, a and c) in an avidin-biotin-complexed immunoperoxidase reaction using 3,3'-diaminobenzidine as a chromogen. Anti-CD8 mAb (A, b and d) and anti-antiidiotypic scFv #119 (B and C, b and d) were used as controls (x100 magnification).

 
A comparison of the staining patterns of lesions of melanocytic origin showed a lower reactivity of scFv #70 than of mAb 149.53. scFv #70 stained melanoma cells in two of six primary lesions and in two of five metastatic lesions, while mAb 149.53 stained all of the primary lesions tested and four of the five metastatic lesions tested. In addition, scFv #70 stained a lower percentage of melanoma cells than did mAb 149.53 both in primary and metastatic lesions, although the intensity of staining by the two probes was similar. Both scFv #70 and mAb 149.53 stained all three of the nevus pigmentosus lesions tested.

Reactivity of scFvs #28, #65, and #70 with xenogeneic melanoma cell lines and with rat neural cell line B49

The anti-HMW-MAA mAbs 149.53 and 225.28 have been shown to cross-react with cultured guinea pig GP5 melanoma cells (44) and with cultured rat B49 neural cells (data not shown). The latter cells express NG2, which is a homologue of human HMW-MAA (45, 46). Therefore, the cross-reactivity of human scFvs #28, #65, and #70 was tested with the xenogeneic melanoma cell lines B16 and GP5 and with rat B49 neural cells. The anti-rat NG2 mAb D4.11 was used to monitor the expression of NG2 by B49 cells. Soluble scFv #28 reacted strongly with the rat neural cell line B49 and showed a very weak reactivity with the mouse melanoma cell line B16 and the guinea pig melanoma cell line GP5. In contrast, soluble scFvs #65 and #70 did not react with B16, B49, and GP5 cells (Fig. 11Go).



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  		FIGURE 11. Reactivity in an ELISA of soluble scFvs #28, #65, and #70 with the xenogeneic melanoma cell lines B16 and GP5 and the neural cell line B49. Mouse B16 melanoma cells, guinea pig GP5 melanoma cells, and rat B49 neural cells were incubated for 2 h at 4°C with soluble scFvs #28, #65, and #70 that had been preincubated with the biotinylated mAb 9E10. The binding of scFv fragments was detected using SA-HRP. Results are expressed as absorbance at 490 nm. Human SK-MEL-28 melanoma cells and human LG-2 B lymphoid cells were used as controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Panning of the synthetic phage scFv library (#1) with two cultured human melanoma cells has resulted in the isolation of scFv fragments with limited diversity. Several lines of evidence indicate that the scFvs #28, #65, and #70 that we have characterized recognize distinct antigenic determinants of HMW-MAA. First, the three scFv fragments react selectively with melanoma cells when tested with a panel of human cell lines and with purified HMW-MAA in binding assays. Second, the human scFv fragments stain melanoma cells when tested with surgically removed melanoma lesions using the immunoperoxidase reaction. Third, scFv #70 immunoprecipitates molecules with the characteristic electrophoretic profile of the two subunits of HMW-MAA from a melanoma cell lysate. These two subunits are not detected when the melanoma cell lysate that is used as an Ag source has been immunodepleted with anti-HMW-MAA mAb 763.74. It is noteworthy that immunodepleting a melanoma cell lysate with scFv #70 completely removes the 250-kDa component that is immunoprecipitated by mAb 763.74 but causes only a partial depletion of the >450-kDa chondroitin sulfate proteoglycan (CSP) component.This noncoordinate depletion of the two subunits of HMW-MAA by scFv #70 argues against the current view that HMW-MAA consists of two noncovalently associated subunits (20, 47, 48, 49), but suggests that the two subunits are independently expressed by melanoma cells. This possibility is corroborated by the immunoprecipitation of the >450-kDa CSP component alone from melanoma cells by scFv #28. Furthermore, immunodepletion of a melanoma cell extract with scFv #28 removed part of the >450-kDa CSP component of the HMW-MAA that had been immunoprecipitated by scFv #70 and the mouse mAb 763.74 without causing detectable changes in the level of the 250-kDa component that had been immunoprecipitated by the two Abs.The reactivity of scFv #28 alone with the >450-kDa CSP component of HMW-MAA is not unique to this scFv fragment, because a similar specificity is displayed by scFv #61 that has been isolated by panning the synthetic phage scFv library (#1) with purified HMW-MAA (18).

In addition to their different distribution on the components of HMW-MAA, the antigenic determinants that are recognized by scFvs #28 and #70 display several distinct features. N-linked carbohydrates play an important role in the expression of the antigenic determinant that is recognized by scFv #28, since this Ab did not immunoprecipitate any component from melanoma cells that had been treated with the N-glycosylation inhibitor tunicamycin. In contrast, the antigenic determinant that is recognized by scFv #70 is expressed on the HMW-MAA core protein synthesized by melanoma cells in the presence of tunicamycin. Furthermore, the antigenic determinants recognized by scFvs #28 and #70 display a differential expression on xenogeneic cells. scFv #70 did not cross-react with mouse B16 melanoma cells, guinea pig GP5 melanoma cells, and rat B49 neural cells, while scFv #28 did cross-react with B49 cells. The latter cells express the NG2 Ag, which shares an 81% homology in its aa sequence with human HMW-MAA (45, 46). Most of the homology lies in the first two domains of the Ag. Hence the antigenic determinant recognized by scFv #28 is most likely located in the first or second domain of HMW-MAA, provided that a peptide moiety plays a role in its expression. Lastly, the antigenic determinant recognized by scFv #28 is not spatially close to any of the antigenic determinants defined by mouse anti-HMW-MAA mAbs that we have tested. In contrast, the binding of scFv #70 to melanoma cells is inhibited by the mouse mAbs 149.53, VT68.2, and VT86. The latter finding reflects the recognition by mouse mAb and human scFv #70 of spatially close but distinct antigenic determinants, since the mouse mAb 149.53 cross-reacts with guinea pig GP5 melanoma cells and rat B49 neural cells, while; scFv #70 does not cross-react with these cells. Furthermore, like the mAbs VT68.2 and VT86, scFv #70 does not react with a panel of antiidiotypic mAbs that are elicited by mAb 149.53. Lastly, like the mAbs VT68.2 and VT86, scFv #70 does not react with a peptide that was isolated by panning a recombinant peptide library with mAb 149.53 (data not shown). The similarity between the human anti-HMW-MAA scFv fragments and the mouse anti-HMW-MAA mAb is not unique, since it has been shown that the human Abs reacting with melanoma-associated glycoproteins gp75 and gp95/p97 resemble the mouse mAb reacting with these Ags (50, 51). These results also show that it is possible to successfully use synthetic Ab libraries to isolate the Ab specificities that are present in the natural Ab repertoire.

One unexpected result of our investigations has been the restricted diversity of the scFv fragments that we have isolated by panning the synthetic phage scFv library (#1) with two human melanoma cell lines. The isolated scFv fragments are all specific for HMW-MAA, although these fragments recognize distinct antigenic determinants of this Ag. These findings suggest that in vitro like in vivo, Ags expressed by cells may be classified according to their immunodominance. According to our data, the HMW-MAA expressed by melanoma cells appears to be immunodominant in vitro. This Ag is also immunodominant in vivo, as suggested by the large panel of anti-HMW-MAA mAb-secreting hybridomas that were isolated from BALB/c mice that had been immunized with melanoma cells. Whether this is a general phenomenon cannot be assessed at present, since, to the best of our knowledge, scFv fragments have only been isolated by Cai and Garen (15, 16) and by Pereira et al. (17) by panning phage display Ab libraries with cultured human melanoma cells. In both studies, the isolated Ab fragments were characterized by their reactivity with a panel of human cell lines in binding assays and with a limited number of surgically removed malignant lesions and normal tissues in immunohistochemical reactions. No information is currently available with regard to the molecular profile of the Ags recognized by the isolated Ab fragments. Therefore, it is not possible to assess the extent of their diversity.

If the phenomenon of the immunodominance of certain Ags in vitro that we have observed in the present study is a general occurrence, it is likely that the diversity of the Abs that can be isolated by panning a phage Ab library with a particular type of cell will be restricted. The specificity of the preponderant population of isolated Abs will be determined by the antigenic profile of the cells used for panning.


   Acknowledgments
 
We thank Dr. M. Herlyn (The Wistar Institute, Philadelphia, PA) for providing the normal human fibroblasts, Dr. W. B. Stallcup (La Jolla Cancer Research Foundation, La Jolla, CA) for providing the B49 cell line and mouse anti-rat NG2 mAb D4.11, and Dr. G. Winter (Medical Research Council Center for Protein Engineering, Cambridge, U.K.) for providing the synthetic scFv library (#1).


   Footnotes
 
1 This work was supported by U.S. Public Health Service Grants CA37959 and CA51814 awarded by the National Cancer Institute, Department of Health and Human Services. Back

2 Address correspondence and reprint requests to Dr. Soldano Ferrone, Department of Microbiology and Immunology Basic Sciences Building, Rm. 308, New York Medical College, Valhalla, NY 10595. E-mail address: Back

3 Abbreviations used in this paper: MAA, melanoma-associated Ag; CDR, complementarity determining region; CSP, chondroitin sulfate proteoglycan; TYE, trypticase-yeast extract; HMW-MAA, high molecular weight melanoma-associated Ag; scFv, single-chain fragment of the V region; HRP, horseradish peroxidase; SA, streptavidin; ß2m, ß2-microglobulin. Back

Received for publication April 20, 1998. Accepted for publication May 19, 1998.


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 Results
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