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Cancer Research 68, 6360, August 1, 2008. doi: 10.1158/0008-5472.CAN-08-0928
© 2008 American Association for Cancer Research
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Immunology

Antitumor Activity of Immunotoxins with T-Cell Receptor–like Specificity against Human Melanoma Xenografts

Eynav Klechevsky1,2, Michael Gallegos2, Galit Denkberg1, Karolina Palucka2, Jacques Banchereau2, Cyril Cohen1 and Yoram Reiter1

1 Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel and 2 Baylor Institute for Immunology Research, Dallas, Texas

Requests for reprints: Yoram Reiter, Faculty of Biology, Technion-Israel Institute of Technology, Technion City, Room 333, Haifa 32000, Israel. Phone: 972-4-8292785; Fax: 972-4-8225153; E-mail: reiter{at}tx.technion.ac.il.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
In this study, we have explored the use of Fab-toxin proteins (immunotoxin) to target antigen-specific MHC-peptide complexes of in vitro and in vivo cancer cells. A human phage display library was used to screen for T-cell receptor (TCR)–like antibodies that are highly specific for the peptide melanoma-associated antigen MART-126-35 presented by HLA-A201. We also used previously selected TCR-like antibodies specific for the peptide melanoma-associated antigen gp100280-288 presented by HLA-A201. The recombinant immunotoxin constructs were generated by fusing the targeting Fab fragment to a truncated form of Pseudomonas exotoxin, PE38KDEL. These immunotoxins bound with high affinity to the EBV-transformed JY cell line pulsed with the aforementioned peptides and internalized within 30 min. A significant inhibition of protein synthesis, which resulted in cell death, was detected at 24 h. MART-1–specific and gp100-specific immunotoxins bound and killed HLA-A201 melanoma MART-1+ and gp100+ cell lines that were presented at natural levels but do not bind to HLA-A201– or to HLA-A201+ MART-1– and gp100– cell lines. In severe combined immunodeficient mice, MART-1 and gp100 immunotoxins significantly and discriminately inhibited human melanoma growth. These results show that MHC class I/peptide complexes can serve as a specific target for passive immunotherapy of cancer. [Cancer Res 2008;68(15):6360–7]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Observation of spontaneous antitumoral T-cell response in melanoma patients led to the identification of tumor-associated antigens (1, 2). Drugs that target these antigens are becoming a first-line treatment for some cancer types (3). Rituximab, a monoclonal antibody (mAb) that binds CD20 and promotes destruction of non–Hodgkin's lymphoma B cells via antibody-dependent cellular cytotoxicity (ADCC), represents a success in passive immunotherapy (4). Similarly, Herceptin, which targets HER-2+ cancer cells, is now the standard in breast cancer therapy (5). The efficacy of such drugs has prompted efforts to develop additional antibody agents that elicit ADCC or deliver toxin moieties to the cancerous cells. Although numerous melanoma-associated antigens have been identified (6), many are intracellular rather than surface proteins and therefore not accessible to antibodies. However, peptides derived from these intracellular antigens are presented as epitopes on MHC class I molecules of human cancer cells (7). These melanoma exclusive complexes represent prime targets for immunotoxins. HLA-A201–restricted CTLs derived from melanoma tumor-infiltrating lymphocytes (TIL) of patients were found to recognize epitopes from the melanocytic differentiation proteins gp100 and MART-1 (1, 2). However, progression of tumors leading to patient death suggests that these T cells are ineffective in eradicating the tumor. Several mechanisms are considered in this matter to control tumor growth, including the quality of the T cells (i.e., low versus high avidity; ref. 8) and the presence of local immunosuppressive processes. The specificity of the TILs remains attractive for therapeutic purposes. Recent studies show that these cells, expanded in vitro and adaptively transferred back to the patient, can elicit remarkable responses particularly in the lymphoablated patients (9, 10).

Another approach is to develop Fab fragments that bind melanoma-specific peptide-MHC complexes with the specificity of the T-cell receptor (TCR). Such ligands, when conjugated with therapeutic moieties (i.e., drugs, radioisotopes, or tumor cell toxins), constitute potential anticancer exogenous agents that may also avoid tumor-regulating immunosuppressive mechanisms. By screening a large human phage display library, we previously isolated high-affinity recombinant Fab antibodies Fab 2F1 and G2D12 that recognize HLA-A201 in complex with peptide gp100280-288 and gp100154-162, respectively (11). Herein, we describe the isolation of Fab antibodies (Fab CAG10 and Fab CLA12), which recognize MART-126-37 peptide in the context of HLA-A201. Fusion proteins composed of these Fab antibody fragments and a truncated form of Pseudomonas exotoxin (PE38KDEL) specifically kill in vitro and in vivo melanoma cells that present the corresponding peptide complexes on their surface.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Peptides and cell lines. The HLA-A201–restricted peptides used for specificity studies are gp100154-162 (KTWGQYWQV), gp100209-217 [IMDQVPFSV (G9-209)], gp100280-288 [LLLTVLTVL (G9-280)], HTLV-1 TAX11-19 [LLFGYPVYV (TAX)], CMV p65495-503 (NLVPMVATV), TARP29-37 (FLRNFSLML), XAGE-1 (GVFPSAPSPV), MART-126-35 [EAAGIGILTV (MART-1 26-35)], MART-127L [ELAGIGILTV (MART-1 27L)], and hTERT865-873 (RLVDDFLLV).

Cell lines used in this study were the following: B-cell line RMAS-HHD, which is transfected with a single-chain β2m-HLA-A201 gene, the EBV-transformed HLA-A201+ JY cells, and HLA-A201+ TAP-deficient T2 cells. Melanoma cell lines used were the following: HLA-A201+/gp100+/MART-1+ (Mel624.38, Mel526, Mel501A, FM3D, and Stilling), HLA-A201+/gp100–/MART-1– (Mel1938), HLA-A201–/gp100+/MART-1+ (HA24 and G-43), and HLA-A201–/gp100–/MART-1– (PC3).

Selection and characterization of recombinant Fabs with specificity for MART-1/HLA-A201. The generation and characterization of a panel of Fabs specific for peptide-HLA-A201 were previously described in detail (12). Phage antibodies were selected for binding to single-chain MHC-peptide complexes (13) using a large human Fab library containing 3.7 x 1010 different Fab clones (12). The binding specificity of the phage clones selected was tested against soluble MART-1/HLA-A201 complexes in ELISA assays. MART-1/HLA-A201–specific Fab antibodies were expressed and purified as previously described (12). The eluted Fabs were dialyzed twice against PBS (overnight, 4°C) to remove residual imidazole.

Construction, expression, and production of melanoma-specific Fab-PE38KDEL. The light chains and the heavy chain containing the variable and constant region 1 (VLCL or VHCH) of Fabs 2F1, CLA12, and H9 were cloned separately by PCR into T7 promotor-based expression vector pULI9 (14). The VLCL chain was fused to a gene encoding the toxin PE38KDEL for the construction of VLCL-PE38KDEL. The VHCH of the Fabs was cloned into the same expression vector after the toxin gene was removed. These constructs were expressed separately in Escherichia coli BL21 {lambda}DE3 cells. On induction with isopropyl-L-thio-B-D-galactopyranoside, intracellular inclusion bodies that contain large amounts of the recombinant protein accumulated. Inclusion bodies of both chains were purified, solubilized, reduced, and subsequently refolded at a 1:1 ratio in a redox-shuffling buffer system containing 0.1 mol/L Tris, 0.5 mol/L arginine, and 0.09 mmol/L oxidized glutathione (pH 8.0). Correctly folded Fab-PE38KDEL fusions were purified by ion exchange chromatography on Q-Sepharose and Mono-Q (Pharmacia).

ELISA with purified Fab antibody or Fab-PE38KDEL immunotoxin. The binding specificities of individual soluble Fabs and recombinant Fab-PE38KDEL immunotoxin were determined by ELISA using biotinylated single-chain MHC-peptide complexes. Peptide-MHC complexes were refolded using each peptide and coated via streptavidin on an ELISA plate (Falcon). After extensive washing, plates were blocked with PBS/2% skim milk and incubated with various concentrations of soluble purified Fab or Fab-PE38KDEL for 1 h at room temperature. Bound clones were detected with an anti-human Fab mAb coupled to horseradish peroxidase (HRP) or HRP-conjugated anti–Pseudomonas exotoxin (for Fab immunotoxin). Detection was performed using tetramethylbenzidine reagent (Sigma-Aldrich).

Measurement of melanoma-specific peptide-HLA-A201–specific Fab or Fab-PE38KDEL immunotoxin binding to cell surface peptide-MHC complexes. RMAS-HHD or JY cells (106) were pulsed overnight with 50 µmol/L peptide at 26°C or 37°C, respectively. RMAS-HHD cells were subsequently incubated at 37°C for 2 to 3 h to stabilize cell surface expression of MHC-peptide complexes. The cells were then washed in fluorescence-activated cell sorting (FACS) assay medium [PBS, 2% bovine serum albumin (BSA), and 0.09% sodium azide] and incubated for 1 h at 4°C with 20 µg/mL Fabs or Fab-toxin and FITC-labeled goat anti-human IgG (Fab-specific; The Jackson Laboratory). Cells were washed thrice with PBS and analyzed by FACSCalibur (BD Biosciences). Melanoma cells were trypsinized and stained with the Fab or Fab-toxin as described above. The level of total HLA-A201 expression was detected using the mouse anti-human HLA-A201 (clone BB7.2).

Fab-toxin affinity measurement. Fab-toxin was labeled with 125I using Bolton-Hunter reagent. 125I-labeled Fab-toxin [3 x 105 to 5 x 105 counts per minute (cpm)/106 cells] was incubated with JY cells that were loaded with specific or irrelevant peptide and with increasing concentrations of unlabeled Fab-toxin for 1 h at room temperature. The cells were washed extensively with PBS, and the bound radioactivity was measured by a gamma counter. Nonspecific binding was determined by adding a 20-fold excess of unlabeled Fab-toxin and was 25% of the total bound radiolabel. Values of nonspecific binding were subtracted to calculate specific binding. Specifically bound 125I-labeled Fab 2F1-PE38KDEL was expressed as the percentage of the maximal bound 125I-labeled toxin and is plotted against the concentration of the competitor. The apparent binding affinity of the recombinant immunotoxin was determined using nonlinear regression as the concentration of competitor (soluble purified Fab-toxin) required for 50% inhibition of 125I-labeled Fab-toxin binding to the cells. Data are representative of two independent experiments.

Internalization assay. JY cells were loaded with specific or control peptide, washed, and incubated with 20 to 30 µg/mL FITC-labeled Fab-toxin for 1 h on ice. Cells were then washed and resuspended in RPMI 1640 containing 10% FCS. Half of the cells were kept on ice, whereas the other half was incubated at 37°C. At indicated time points, a sample was removed, washed, and fixed in Tris/glycerol/polyvinyl alcohol mounting solution. Specimens were examined with a Zeiss confocal laser fluorescence inverted microscope (LSM 410, Carl Zeiss) using simultaneous lasers with excitation wavelength 488 nm.

Cytotoxicity assays on JY antigen-presenting cells and melanoma cell lines. JY cells were incubated overnight with 0.1 mmol/L specific peptide or control peptides at 37°C. Peptide-loaded cells were then washed twice with medium and incubated for 24 h with increasing concentrations of recombinant Fab-PE38KDEL. For melanoma killing assay, 5 x 104 cells were plated in each well of a flat-bottomed 96-well plate for 36 h. Graduate amounts of Fab-toxin were then added for an additional 24 h. Protein synthesis inhibition is measured by incorporation of [3H]leucine into cell proteins. IC50 is the concentration of immunotoxin that causes 50% inhibition of protein synthesis.

Antitumor activity (in vivo antitumor assay). The antitumor activity of Fab-PE38KDEL fusions was determined in severe combined immunodeficient (SCID) mice bearing human cancer cells. Mel526 cells (10 x 106) were injected s.c. into irradiated nonobese diabetic (NOD)-SCID β2M-deficient mice on day 0. When tumors developed in the animals for ~0.05 cm3 in size (by days 6–10), treatment with immunotoxin was initiated. Animals were treated with four i.v. injections of CLA12 Fab-PE38KDEL, 2F1 Fab-PE38KDEL, or H9 Fab-PE38KDEL diluted in 0.2 mL of PBS once every other day. Fifth and sixth injections were given 4 and 8 d later. Treatment groups consisted of four animals. Tumors were measured with a caliper every other day, and the volume of the tumor was calculated by using the following formula: tumor volume (cm3) = length x (width)2 x 0.5.

Statistical analysis. Tumor sizes in animal experiments are expressed as mean ± SD. For comparison between the two experimental groups, Mann-Whitney test was used. P < 0.05 is considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Generation of recombinant antibodies specific for HLA-A201 MART-1 peptide complexes. HLA-A201/MART-126-35 (EAAGIGILTV) complexes were exposed to a large naive repertoire of 3.7 x 1010 human recombinant Fab fragments displayed on the surface of phage. A 1,000- to 2,500-fold enrichment in phage titer was observed after three rounds of panning. Two Fab phage clones, CAG10 and CLA12 antibodies, were selected and produced in a soluble form in E. coli BL21 cells and then purified by IMAC as described (12). The binding specificity of these purified Fab fragments was determined by ELISA with biotinylated MHC-peptide complexes immobilized to wells through BSA-biotin-streptavidin. As shown in Fig. 1A , Fab CLA12 binds to MART-1/HLA-A201–restricted MHC peptides but not to other HLA-A201 peptide complexes (gp100 G9-209, TARP, tyrosinase, XAGE, and gp100 G9-280). As shown in Fig. 1B and C, Fabs CAG10 and CLA12, respectively, bound in a peptide-specific manner to HLA-A201+ RMAS-HHD cells that were loaded with the MART-1 26-35 peptide but not control HTLV-1–derived HLA-A201–restricted peptide (TAX11-19). The Fabs also bound HLA-A201+ RMAS-HHD cells that were loaded with the anchor-modified MART-1–derived peptide 27L, where the alanine in position 2 is replaced by a leucine residue, giving stronger binding to HLA-A201 (15, 16). The MART-1 26-35, 27L-35, and the TAX peptide were all presented on the surface of the pulsed RMAS-HHD as shown by the binding of mAb W6/32, a conformational antibody that recognizes peptide-loaded MHC class I (Fig. 1D).


Figure 1
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  		Figure 1. Binding of Fabs CAG10 and CLA12 to peptide-loaded APCs. A, fine specificity of the purified Fab clones by ELISA. TCR-like Fabs were tested by ELISA for binding refolded peptide-MHC complexes. B and C, detection of MART-1 peptide-HLA-A201 complexes on APC RMAS-HHD. RMAS cells were loaded with specific MART-1 (26-35 or 27L) or control TAX peptide. Complexes were probed with recombinant purified Fab CAG10 (B) and CLA12 (C) and analyzed by FACS using FITC-labeled goat anti-human Fab. D, mAb W6/32 binding shows the total peptide-HLA on the APC cell surface.

 
Thus, Fab CAG10 and CLA12 antibodies exhibit TCR-like fine specificity and recognize cell surface-HLA-A201 complexes bearing MART-1 26-35 peptide.

MART-1–specific and gp100-specific TCR-like Fab antibodies bind to HLA-A201+ melanoma cells. To test whether the melanoma-specific TCR-like Fab antibodies can bind naturally processed HLA-A201-peptide complexes on the surface of tumor cells, we performed flow cytometry studies on HLA-A201+ melanoma tumor cell lines. As shown in Fig. 2 , MART-1–specific Fab antibody CLA12 reacted with the HLA-A201+/MART-1+ melanoma lines 501A (Fig. 2A), 624.38 (Fig. 2C), FM3D (Supplementary Fig. S1A), and Stilling (Supplementary Fig. S1B) but not with HLA-A201+/MART-1– melanoma 1938 (Fig. 2B) or with melanoma G-43 cells, which are HLA-A201–/MART-1+ (Fig. 2D). As expected, the HLA-A201/MART-1 complexes represent a minor fraction out of the total surface HLA-A201 complexes as monitored by the binding of the HLA-A201–specific mAb BB7.2. These results indicate that the TCR-like antibody CLA12 can detect the native HLA-A201/MART-1 epitope on the surface of melanoma cells.


Figure 2
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  		Figure 2. Binding of Fab CLA12 to melanoma cell lines. A to D, detection of MART-1 peptide-HLA-A201 complexes on HLA-A201+/MART-1+ melanoma cell lines 501A (A) and 624.38 (C) and on control cell lines [the HLA-A201+/MART-1– melanoma 1938 (B) or the HLA-A201–/MART-1+ melanoma G-43 (D)] by Fab CLA12. Complexes were detected by FACS using recombinant purified Fab CLA12 and FITC-labeled goat anti-human Fab. mAb BB7.2 recognizes total HLA-A201 and was used to validate HLA-A201 expression by the indicated cell lines.

 
The TCR-like antibody Fab 2F1, directed against the HLA-A201/gp100 G9-280 epitope, also binds HLA-A201+/gp100+ 624.38 and 501A melanoma cells (Supplementary Fig. S2A and B) but does not bind to either HLA-A201+/gp100– 1938 melanoma (Supplementary Fig. S2C) or HLA-A201–/gp100– PC3 cells (Supplementary Fig. S2D).

These results show that these MART-1–specific and gp100-specific TCR-like antibodies bind in a peptide-dependent HLA-A201–restricted manner to target cells that express naturally processed endogenously derived peptide-HLA-A201 complexes.

Construction and purification of TCR-like antibody-toxin fusion proteins. We generated fusion molecules in which the different TCR-like antibody Fabs are fused to truncated form of Pseudomonas exotoxin A (PE38KDEL). This truncated form of Pseudomonas exotoxin contains the translocation and ADP-ribosylation domains of Pseudomonas exotoxin but lacks the cell-binding domain, which is replaced by the Fab fragment. In addition, the five COOH-terminal amino acids REDLK of the native Pseudomonas exotoxin were replaced with KDEL, which increases the toxin cytotoxicity (17) due to increased binding to the endoplasmic reticulum (ER) retention receptor (18). The truncated PE38KDEL gene was fused at its NH2 terminus to the COOH terminus of each Fab light chain as shown schematically in Fig. 3A and produced and purified as described in Materials and Methods.


Figure 3
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  		Figure 3. Analysis of recombinant purified Fab 2F1-PE38KDEL. A, schematics of the Fab 2F1-PE38 fusion protein. The gene encoding PE38 that contains the translocation and ADP-ribosylation domains of Pseudomonas exotoxin was fused to the COOH terminus of Fab 2F1 (L{kappa}) {kappa} chain. B, binding of 2F1 Fab-PE38KDEL to peptide-loaded APCs. JY cells were loaded with 50 µmol/L G9-280 or G9-209 peptides, incubated at 4°C in the presence of 2F1 Fab-toxin, and analyzed by flow cytometry. C, competitive binding analysis of the ability of purified recombinant Fab 2F1-PE38KDEL to inhibit the binding of 125I-labeled Fab 2F1-PE38KDEL to JY cells loaded with 0.05 mmol/L G9-280. Specifically bound 125I-labeled Fab 2F1-PE38KDEL is expressed as the percentage of the maximal bound 125I-labeled toxin and is plotted versus the concentration of the competitor. Values of nonspecific binding, as measured by binding of 125I-labeled toxin in the presence of excess amount of the competitor, were subtracted to calculate specific binding. Value of maximum binding: 2,011 cpm; value of nonspecific binding: 511 cpm. Data are representative of two independent experiments. D, binding of 2F1 Fab-PE38KDEL to HLA-A201+gp100+ melanoma cell line Mel526. Complexes were detected by flow cytometry using FITC-labeled goat anti-human Fab.

 
The binding specificity of the soluble purified Fab-PE38KDEL fusion proteins was first determined by ELISA on biotinylated MHC-peptide complexes immobilized to wells through BSA-biotin-streptavidin. The binding was assessed with either anti-human Fab or anti-PE38 antibodies. Fab 2F1-PE38KDEL reacts specifically with the immobilized HLA-A201/gp100 G9-280 complexes and not with control HLA-A201-peptide complexes (gp100 G9-209 and CMV p65; Supplementary Fig. S3).

The binding affinity of the soluble 2F1-PE38KDEL fusion protein was determined using a competition binding assay in which the binding of 125I-labeled Fab 2F1-PE38KDEL to the specific HLA-A201/G9-280 complexes on antigen-presenting cells (APC; Fig. 3B) is competed with increasing concentrations of unlabeled fusion protein. Nonspecific binding of the soluble 125I-labeled 2F1-PE38KDEL fusion protein was determined on APCs loaded with the specific peptide gp100 G9-280 in the presence of 20-fold excess of the unlabeled 2F1-PE38KDEL fusion protein. The specific binding was calculated by subtracting the background value from the bound cpm. The percentage of the specific maximal binding was plotted against the concentration of the competitor. Relative binding affinities were inferred from the IC50 values of four-variable curve fits (Fig. 3C) and determined as 249 nmol/L. The binding of the labeled Fab 2F1 and the 2F1-toxin fusion to cells was indistinguishable (data not shown). Thus, we have generated immunotoxin with comparable binding properties to the unconjugated Fab.

The Fab-PE38KDEL fusion proteins bind to APCs and melanoma cells displaying the relevant gp100-derived and MART-1–derived epitopes. As found with the original MART-1–specific TCR-like Fabs CAG10 and CLA12 (Fig. 1), the derived immunotoxins CAG10-PE38KDEL and CLA12-PE38KDEL bound to T2 cells loaded with MART-1 (Supplementary Fig. S4A) but did not bind T2 cells loaded with irrelevant peptides. As shown in Fig. 3B, Fabs 2F1-PE38KDEL, like the original gp100 G9-280–specific TCR-like Fab 2F1 (11), bound to JY cells loaded with gp100 G9-280 peptide. Thus, the fusion protein could still bind to peptide-MHC complexes expressed at high density on peptide-loaded APCs, leaving the question open as to whether the fusion protein would retain sufficient affinity for peptide-MHC complexes expressed at low levels as on melanoma cells. Indeed, as the original Fab (Fig. 2; Supplementary Fig. S2), the Fab-PE38KDEL fusion proteins bound to HLA-A201+ and gp100/MART-1+ melanoma cells 526 (Fig. 3D), 501A, and 624.38 (data not shown) but not to 1938 melanoma cells, which are HLA-A201+ but do not express gp100 and MART-1, or to G-43 melanoma cells, which are HLA-A2– but express gp100 and MART-1 (data not shown).

These results show the ability of the TCR-like Fab-PE38KDEL fusion molecules to bind the authentic endogenously derived MHC-peptide complex when at a limited density on the surface of the tumor cells.

Internalization of TCR-like antibodies. Fab 2F1-PE38KDEL fusion protein was labeled with FITC and tested for its binding and internalization on JY cells pulsed with the appropriate gp100-derived G9-280 peptide. As shown with unlabeled Fab in Fig. 3B, the FITC-labeled Fab 2F1-PE38KDEL molecule binds specifically to G9-280 peptide-pulsed JY cells but not to cells pulsed with a control peptide G9-209. Internalization was monitored by incubating cells with FITC-labeled Fab 2F1-PE38KDEL at 4°C for 1 h and transferred to 37°C. Membranous binding was observed immediately after the cells were transferred to 37°C (time 0). No fluorescence was observed on the negative control (JY cells + peptide G9-209; data not shown). After 15 min, the majority of stain intensity was mainly on the surface of the cell (Fig. 4A ) and dense areas of fluorescence were detected, which may indicate processes of microcapping of MHC-peptide complexes. After 30 min, fluorescence was concentrated in small vesicles (Fig. 4B). After 1 h, the FITC-labeled antibody was detected in larger vesicles (Fig. 4C). After 6 h, intense staining was observed around the nucleus in the ER-Golgi compartment (Fig. 4D). Cells kept on ice for 3 h showed membranous staining (data not shown).


Figure 4
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  		Figure 4. Cellular internalization of 2F1-PE38-FITC. JY cells were loaded with the specific G9-280 peptide and incubated at 4°C in the presence of 2F1-PE38-FITC. Cells were then transferred to 37°C and monitored for the immunotoxin internalization at indicated time points: 15 min (A), 30 min (B), 1 h (C), and 6 h (D). Propidium iodide was used to detect the nucleus.

 
These results show that TCR-like antibodies fused to toxin rapidly internalize after binding to cell surface peptide-MHC complexes.

The TCR-like Fab-PE38KDEL immunotoxins are a potent killer of APCs. The ability of the Fab-PE38KDEL immunotoxins to inhibit protein synthesis of JY cells was used as a measure of their cytotoxic effect. As shown in Fig. 5A , cytotoxicity by 2F1 Fab-PE38KDEL was observed only when JY cells were loaded with the gp100 G9-280 peptide with an IC50 of ~0.5 ng/mL (black dots). No cytotoxic activity was observed on JY cells that were either unloaded or loaded with other HLA-A201–restricted peptides such as these from TAX or CMV. Similar results were observed with the RMAS-HHD cell line (data not shown). We then analyzed whether the toxins can also show cytotoxic activity toward melanoma cells such as FM3D, which express 20-fold fewer sites on their surface (1 x 104 molecules per cell) than JY cells (1.5 x 105 to 2 x 105 molecules per cell; ref. 19). As shown in Fig. 5B, 2F1 Fab-PE38KDEL induced killing in FM3D cells pulsed with the gp100 G9-280 peptide but not with other HLA-A201–restricted control peptides. Furthermore, it did not kill the HLA-A201– melanoma G-43 cells pulsed with G9-280 or control peptides. The IC50 for G9-280–pulsed FM3D was ~200 ng/mL, possibly reflecting the lower number of HLA-A201/G9-280 sites compared with G9-280–pulsed JY cells. Similar results were observed with the MART-1–specific CLA12 Fab-PE38KDEL fusion proteins (Supplementary Fig. S4B).


Figure 5
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  		Figure 5. Cytotoxic activity of different immunotoxins toward peptide-loaded APCs and melanoma cell lines. A, cytotoxic activity of recombinant Fab 2F1-PE38KDEL toward JY cells loaded with G9-280 peptide or with other control HLA-A201–restricted peptides. Cells were incubated for 24 h with recombinant Fab 2F1-PE38KDEL. [3H]leucine incorporation into cellular protein was measured. The results are expressed as a percentage of control where no immunotoxin was added. B, same assay as in A was done on HLA-A201+ FM3D cells and HLA-A201– G-43 cells loaded with the specific G9-280 or control HLA-A201–restricted peptides. C and D, cytotoxic activity of 2F1-PE38KDEL (C) and CLA12-PE38KDEL (D) toward melanoma cell lines Mel526 (Figure 5), Mel624.38 (Figure 5), Mel501A (Figure 5), G-43 (Figure 5), and 1938 (Figure 5) after 24 h of incubation. [3H]leucine incorporation into cellular protein was measured. The results are expressed as a percentage of control where no immunotoxin was added.

 
These results show that Fab-PE38KDEL fusion proteins kill cells bearing specific peptide-MHC complexes.

Cytotoxic activity of TCR-like Fab-PE38KDEL immunotoxins toward melanoma cells expressing endogenous gp100 and MART-1. Naturally expressed target antigens, HLA-A201+ melanoma cells that express gp100 and MART-1 were used to evaluate the activity of Fab-PE38KDEL fusion molecules. The gp100 G9-280–specific 2F1 Fab-PE38KDEL fusion protein exhibited cytotoxic activity on gp100+, HLA-A201+ melanoma cells Mel526, 501A, and 624.38. However, it does not show cytotoxic activity on HLA-A201–, -/gp100+ G-43 cells or on HLA-A201+, gp100– 1938 cells (Fig. 5C). Similarly, MART-1–specific CLA12 Fab-PE38KDEL fusion protein exhibited cytotoxic activity toward MART-1+, HLA-A201+ melanoma cells Mel526 and Mel624.38 but it does not show cytotoxic activity on HLA-A201–, gp100+ G-43 cells (Fig. 5D). The IC50 of the immunotoxin molecules 2F1 Fab-PE38KDEL and CLA12 Fab-PE38KDEL to antigen+ and HLA-A201+ cells was approximately 20 to 100 ng/mL, depending on the cell type and the target antigen (Fig. 5C and D). Thus, these results indicate that TCR-like antibodies fused to PE38KDEL display specific cytotoxic activity on melanoma cells that express natural endogenous differentiation antigens gp100 and MART-1.

In vivo antitumor activity of TCR-like Fab-PE38KDEL fusion proteins. Based on the in vitro cytotoxic activity of 2F1 Fab-PE38KDEL and CLA12 Fab-PE38KDEL, we analyzed their ability to alter the development of human melanoma in irradiated NOD-SCID β2M-deficient mice. Mice, inoculated with 107 Mel526 tumor cells, were randomly assigned to treatment groups, with four mice in each group. Treatment began on day 10 after inoculation when the tumor size reached ~55 mm2 and four i.v. doses of CLA12-PE38KDEL at either 0.05 or 0.125 mg/kg were administered at 48-h intervals. Injection of PBS was used as a control. The fifth and sixth injections were given on days 20 and 24, respectively. Tumor volumes were recorded for 34 days. By day 34, tumors in mice receiving PBS diluent grew to a size averaging 530 ± 127 mm3. Treatment with 0.05 mg/kg CLA12-PE38KDEL delayed tumor development compared with control, and by day 34, tumor size reached 259 ± 92 mm3 (P = 0.0006; Fig. 6A ). The effect on tumor was dose dependent because treatment with 0.125 mg/kg CLA12-PE38KDEL delayed tumor development to a greater extent, reaching ~25% (135 ± 56 mm3) of the size of the tumors in the control group on day 34 (P = 0.0002; Fig. 6B). To further assess the specificity of 2F1 Fab-PE38KDEL and CLA12 Fab-PE38KDEL, a nonrelevant immunotoxin recognizing the complex of CMV peptide p65 bound to HLA-A201 (H9-PE38KDEL) was used as a control (Fig. 6C and D). Mice bearing a 55 mm2 melanoma tumor were injected using the same protocol with 0.125 mg/kg of the gp100-specific or MART-1–specific immunotoxins 2F1-PE38KDEL or CLA12-PE38KDEL, respectively. PBS or CMV-specific immunotoxin H9-PE38KDEL was used as a control. By day 35, tumor size reached 791 ± 161 mm3. Treatment with control immunotoxin (H9 Fab-PE38KDEL) had no significant effect on tumor growth that had reached 721 ± 120 mm3 by day 35. Treatment with either 2F1 Fab-PE38KDEL or CLA12 Fab-PE38KDEL, however, considerably and consistently delayed tumor growth. By day 35, the tumor size reached 164.5 ± 22 mm3 and 162.5 ± 27 mm3, respectively.


Figure 6
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  		Figure 6. Antitumor activity of different immunotoxins on subcutaneous human melanoma tumor in SCID mice. Groups of animals were injected with 10 x 106 Mel526 melanoma cells on day 0. When tumor reached 55 mm3, animals were administered to 48-h intervals with four i.v. injections of 0.05 mg/kg (A) and 0.125 mg/kg (B) of CLA12 Fab-PE38KDEL in PBS. The fifth and sixth injections were given 4 and 8 d later. Control group received diluent alone. C and D, animals were administered to 48-h intervals with four i.v. injections of 0.125 mg/kg of CLA12 Fab-PE38KDEL (C) or 2F1 Fab-PE38KDEL (D) in PBS. The fifth and sixth injections were given 4 and 8 d later. Control groups received diluent alone or nonbinding immunotoxin CMV-specific H9 Fab-PE38KDEL. No cytotoxicity was observed at these doses. Comparison of tumor size between treated and control groups gave P < 0.0006. Points, mean (n = 4); bars, SD.

 
We therefore conclude that 2F1-PE38KDEL and CLA12-PE38KDEL are specific and effective in slowing the growth of HLA-A201+gp100+MART-1+ tumors at nontoxic doses.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
Here, we report the isolation of two novel human recombinant antibody fragments Fab-CAG10 and Fab-CLA12 that recognize HLA-A201/MART-126-37 complex. They possess a similar affinity as previously reported for Fab 2F1 and Fab G2D12 that bind to HLA-A201/gp100280-288 and HLA-A201/gp100154-162 complexes, respectively (11). Differences in binding of these antibody fragments to melanoma cell lines revealed the differential expression of the various specific peptide-HLA-A201 complexes. MART-1–specific and gp100 G9-280–specific HLA-A201 complexes express higher levels in melanoma cell lines compared with G9-154 epitopes of the gp100 protein (data not shown).

Our extended panel of melanoma-specific antibodies with TCR-like specificity was used for targeting toxin to cells that express specific peptide-MHC complex in vitro and in vivo. We constructed different human antibody-toxin hybrid molecules (immunotoxins), in which a human Fab antibody is fused to the Pseudomonas exotoxin derivative PE38KDEL.

All the recombinant immunotoxins we have analyzed show high binding specificity to APCs and melanoma cells expressing the specific peptide-HLA-A201 complex. The immunotoxin 2F1-PE38KDEL has a high rate of internalization (within 30 min) and accumulation in the cytosol of the cell (at 6 h) where it exerts its function. When tested in vitro, Fab-PE38KDEL immunotoxins kill APCs and melanoma cells in a peptide-dependent MHC-restricted manner. Immunotoxin G2D12-PE38KDEL was not as potent in killing melanoma cell lines compared with 2F1-PE38KDEL, CAG10-PE38KDEL, and CLA12-PE38KDEL (data not shown), and this order correlates with lower expression of p154/HLA-A201 complexes on the surface of the melanoma cell line. Furthermore, CLA12-PE38KDEL and 2F1-PE38KDEL have specific antitumor activity in a mouse xenograft model for human melanoma. It is therefore able to penetrate solid tumor and substantially delay tumor growth in mice at doses that do not produce animal toxicity.

Having constructed immunotoxin against several melanoma epitopes, we aimed at increasing the probability of eradicating tumor cell variants. As tumor cells tend to mutate, they may lack the target antigen entirely or express it at levels too low for effective immunotoxin-mediated killing. Such mutant cells could be eradicated with cocktails of two or more immunotoxins recognizing different target antigens (20). In our in vitro study, we did not observe a significantly higher cytotoxic effect when using more than one immunotoxin. This may be due to relatively homogenous expression of the target antigen in melanoma cell lines. However, in patients carrying a large tumor mass, mutations are abundant and using a cocktail of two or more immunotoxins may show a beneficial effect.

The only immunotoxin that is directed to tumor cells and currently being evaluated for treatment of metastatic melanoma is composed of ricin A chain conjugated to a murine mAb directed against high molecular weight melanoma antigens. In this case, the use of murine antibody may induce the development of anti-immunotoxin antibodies, which will result in decreased efficacy and limit repetitive dosing with an immunotoxin (21–23). Thus, we propose the evaluation of a different class of toxin target for melanoma and the use of a human antibody fragment that is less immunogenic than a mouse antibody and of smaller size, which should allow a better tumor penetration and repeated administrations. In addition, strategies to reduce the toxicity and the immunogenicity of the toxin, such as a chemical modification with polyethylene glycol (24), will be further required to maximize the toxin therapeutic potency in human.

The data presented here are a proof of principle that specific MHC complexes can be used in humans as target therapy for melanoma. Moreover, the delivery of TCR-like antibodies, PE38KDEL, could serve as a first-line therapy to debulk tumor mass and prevent further rigorous growth and may be a general approach that can be readily extended to known immunodominant peptides for other HLA (25, 26) and different cancer types.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: Israel Science Foundation grant 229/05 (Y. Reiter), NIH grant RO-1 CA115550 (Y. Reiter), Baylor Health Care Systems Foundation, Falk Foundation, and NIH grants RO-1 CA78846, RO-1 CA85540, PO-1 CA84512, and U-19 AI-57234 (J. Banchereau). J. Banchereau holds the W.W. Caruth, Jr. Chair for Transplantation Immunology Research. K. Palucka holds the Ramsay Chair for Cancer Immunology.

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

We thank Dr. Malka Epel, Dr. Oryan Makler, Dr. Roy Noy, and Kfir Oved for help; Dina Segal (Technion) and Amanda Cobb and Dr. Yanying Cao (Baylor Institute for Immunology Research) for technical support; and Drs. Gerard Zurawski and Jonathan Sohnis for critical reading and discussion.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 3/13/08. Revised 4/ 7/08. Accepted 6/ 4/08.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
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