Pre-Existing Immunity to Tyrosinase-Related Protein (TRP)-2, a New TRP-2 Isoform, and the NY-ESO-1 Melanoma Antigen in a Patient with a Dramatic Response to Immunotherapy

Patient

MM is a 63-year-old female who underwent a wide local excision of a primary melanoma on her back in 1981. In May 1997, she developed a s.c. metastasis on her left chest wall, which was resected, and she was started on a chemoimmunotherapy regimen consisting of cisplatin, vinblastine, dacarbazine, IL-2, and IFN-γ. Because of disease progression, she was referred to the Surgery Branch of the National Cancer Institute (Bethesda, MD) in June 1998. At that time, she had developed metastatic disease in multiple sites including the lungs, liver, intrapelvic area, left abdominal wall, and left thigh. She was started on a four-peptide vaccination protocol using 1 mg each of gp100: 209–217 (210 M), gp100: 280–288 (288V), MART-1: 27–35, and tyrosinase: 368–376 in IFA s.c. every 3 wk. Most of her tumors completely regressed after 2 cycles of treatment (day 45), including complete resolution of a large tumor in her left thigh, an intrapelvic mass, a liver lesion, and most of the nodules in her lungs. She completed a total of six cycles of vaccinations in October 1998. A year later (October 1999), she developed a frontal lobe metastatic brain lesion and a s.c. nodule on her right chest wall, both of which were resected. Bulk TIL 1790 used in this study was grown from the chest wall lesion. In March 2000, she underwent a second right temporal craniotomy for resection of recurrent disease at the prior resection site, followed by whole brain irradiation. The patient is doing well at this time, with disease limited to three small lung lesions (~1 × 1 cm) that are slow growing.

Cell lines

Melanoma-reactive CTL were derived from bulk TIL cultures grown in IMDM (Life Technologies, Gaithersburg, MD) containing 6000 IU/ml human rIL-2 (Chiron, Emeryville, CA) as previously described (23). CTL clones were derived from bulk TIL cultures by limiting dilution with the addition of anti-CD3 Ab (OKT-3; Ortho-McNeil Pharmaceuticals, Raritan, NJ). Briefly, 5 × 10⁴ irradiated (3000–3400 cG) PBMC from three allogeneic donors were plated in round-bottom 96-well plates with 0.5–90 T cells per well. Cells were cultured in RPMI 1640 medium (Life Technologies) containing 20% heat-inactivated FBS (Life Technologies) and 30 ng/ml OKT-3 Ab with 300 IU/ml IL-2. The same dose of IL-2 was added on day 7, and clones were tested for recognition of HLA-A2⁺ vs HLA-A2⁻ melanoma cell lines 14 days after stimulation. After testing, the remainder of T cells were expanded by plating them in a T25 flask with 2.5 × 10⁷ irradiated PBMC from three allogeneic donors in 25 ml of RPMI 1640 medium containing 10% FBS and 30 ng/ml OKT-3 Ab. Subsequent expansion was similarly done, but with 1–2 × 10⁶ CTL and 2.5 × 10⁸ allogeneic PBMC plated in an upright T162 flask in 150 ml of medium.

The melanoma cell lines 624–28, 624–38, 938, 526, 888, 888A2, F002S, and F002R were established in the Surgery Branch (National Cancer Institute). 624–28 and 624–38 cell lines were derived from the same parental cell line, 624-mel, and share a similar pattern of Ag and HLA allele expression, except for HLA-A0201 Ag, which was expressed by 624–38 cells but not by 624–28 cells because of aberrant pre-mRNA splicing (24). 888A2-mel cell line was obtained by stable transfection of 888-mel with HLA-A2 cDNA (pCDNA3 plasmid; Invitrogen, San Diego, CA). F002S cell line is deficient in gp100 expression. F002R cell line was derived from F002S cell line by in vitro immunoselection for loss of MART-1 expression (22). T2 cells, deficient in transporter-associated protein, were used to test HLA-A2-restricted peptides for CTL activity. 293 cell line was derived from primary human embryonal kidney cells and was used for transfection purposes. 293A2 cell line was obtained by stable transfection of 293 cells with HLA-A2 cDNA. All cell lines were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 10 mM HEPES buffer, 100 U/ml penicillin-streptomycin (Biofluids, Rockville, MD), 2 mM L-glutamine (Biofluids). This medium is referred to as complete medium (CM) in this paper.

Cytokine release assays

CTL cells (5 × 10⁴) were plated with 1 × 10⁵ target cells in 96-well round-bottom plates in 200 μl of CM. After 18–24 h of incubation at 37°C, the supernatant was harvested for detection of IFN-γ release using ELISA kits (Endogen, Cambridge, MA). For the MHC blocking assays, target cells were incubated with the appropriate mAb at a final concentration of 50 μg/ml (except for BB7.2, which was used at 25% final volume) for 1 h at 37°C before the addition of CTLs. W6/32 (anti-HLA class I) was obtained from the American Type Culture Collection (Manassas, VA). TU39 (anti-HLA class II) and L243 (anti-HLA-DR) mAbs were purchased from BD PharMingen (San Diego, CA). Anti-HLA-A2 mAbs, SB00-60 and BB7.2, were a kind gift from Dr. F. Marincola (Department of Transfusion Medicine, National Cancer Institute) and Dr. E. Bergmann-Leitner (Department of Immunology, Walter Reed Army Institute of Research, Silver Spring, MD), respectively. For the 12-day in vitro sensitization assay, PBMC were plated at 3 × 10⁶ cells in 24-well plates in 2 ml of IMDM, prepared as the CM but with 10% heat-inactivated human AB serum. On day 0, peptides were added at a final concentration of 1 μM and the plates were incubated at 37°C. On day 1 and every 3–4 days thereafter, 300 IU/ml human rIL-2 was added. On day 12, cells were harvested for testing. Targets were T2 cells that had been pulsed with peptides at 1 μM for 2 h at 37°C and washed once. Detection of IFN-γ release was done as described above, but with 1 × 10⁵ CTL instead of 5 × 10⁴.

cDNA library screening

624-mel cDNA expression library was kindly prepared by Dr. R.-F. Wang (Baylor College of Medicine, Houston, TX). Briefly, total RNA was extracted from 624-mel cells using TRIzol reagent (Life Technologies). Poly(A) RNA was purified from total RNA by the poly(A) tract isolation system (Promega, Madison, WI) and converted to cDNA using an oligo(dT) primer. The cDNA was ligated to BstXI adapters and digested with NotI, then ligated to the expression vector pEAK 8. The cDNA was electroporated into DH10B cells (Life Technologies). Pools containing ~100 cDNA clones were prepared from bacteria. The plasmid DNA (200 ng) were transfected into 293A2 cells with Effectene reagent (Qiagen, Valencia, CA). Briefly, 4 × 10⁴ cells per well were plated in flat-bottom 96-well plates in complete medium the day before transfection. On the day of transfection, plasmid DNA (200 ng) from each pool were diluted in 29 μl of buffer EC. Next, 0.8 μl of Enhancer was added and mixed. The mixture was incubated at room temperature for 5 min. After the incubation, 5 μl of Effectene reagent was diluted in 17.5 μl of buffer EC and then added to and mixed with the DNA-Enhancer mixture. The mixture was incubated for 10 min at room temperature. During this time, old medium from 293A2 plates was removed and replaced with 100 μl of fresh CM. After the incubation, the DNA-Enhancer-Effectene mixture was added drop-wise onto the cells. The plates were then incubated at 37° C/5% CO₂ for 24 h. The following day, the old medium was removed and replaced with 100 μl of fresh CM. A total of 5 × 10⁴ CTL in 100 μl of CM were added to each well, and the plates were incubated at 37°C for 24 h. ELISA for IFN-γ was performed as described.

DNA sequencing

Sequencing of the isolated cDNA clone was performed with an ABI Prism 310 automated capillary electrophoresis instrument (PerkinElmer, Foster City, CA) using the Dye Terminator Cycle Sequencing Ready Reaction kit (PerkinElmer). Searches for sequence homology were done with the Gen-Bank database using the basic local alignment search tool algorithm.

Peptide synthesis

Peptides were synthesized using a solid-phase method based on standard F-moc chemistry on a multiple peptide synthesizer (Gilson, Worthington, OH). Peptide identity was verified by laser desorption mass spectrometry (Biosynthesis, Lewisville, TX). Lyophilized peptides were solubilized in DMSO at a 10 mg/ml concentration.

Characterization of TIL 1790

The 1790 TIL line was isolated from a metastatic s.c. lesion in the right chest wall of patient MM, who was HLA-typed as A0201, A0301, B15, B35, C3, and C4. The 1790 TIL line, in four separate experiments, recognized a panel of melanoma cell lines matched for HLA-A2 but did not recognize non-HLA-A2 cell lines (Table I). In addition, TIL 1790 was tested against peptide-pulsed T2 cells and was found to recognize MART-1 27–35 but not the tyrosinase or two gp100 peptides against which patient MM was immunized. Of interest, TIL 1790 recognized both MART-1 peptide and F002R cells, a melanoma cell line that does not express MART-1.

The lack of MART-1 expression on F002R cells was confirmed in the same experiment by the lack of recognition of F002R cells by a control anti-MART-1 CTL clone (clone V2C8) (data not shown). This demonstrated that besides MART-1, 1790 TIL line recognized at least one other melanoma Ag.

Ag specificity of TIL clones MR7, MB4, and M8

In an attempt to identify the Ag(s) recognized by TIL 1790, a number of CTL clones were generated from this TIL line by limiting dilution. CTL clones MR7, MB4, and M8 were selected for further study based on their ability to specifically release cytokine (IFN-γ) in response to HLA-A2 melanoma cell lines but not to cell lines that did not express HLA-A2 (Table II), and based on the lack of recognition of T2 cells pulsed with peptides from MART-1, gp-100, and tyrosinase (data not shown). The fact that non-HLA-A2 melanoma cell lines such as 888 stimulated cytokine release from clones MR7 and MB4 after being stably transfected with the cDNA encoding HLA-A2 Ag indicated that they expressed the Ag(s) of interest and that MR7 and MB4 recognized a melanoma Ag restricted by HLA-A2. Clone M8 recognized neither 888 nor 888A2; however, it reacted with 624–38, but not the HLA-A2 negative variant, 624–28. This suggested that M8 also recognized a melanoma Ag restricted by HLA-A2. This HLA-A2 restriction was confirmed with Ab blocking studies performed on clone MR7. Anti-class I Ab (W6/32) caused a 100% reduction in the activity of clone MR7, whereas anti-class II and anti-DR Abs had a minimal effect (6 and 13%, respectively). In addition, anti-HLA-A2 Abs, SB00-60 and BB7.2, blocked the activity of clone MR7 at 99 and 77% efficiency, respectively. These same Abs exerted a minimal effect (20 and 0% reduction, respectively) on a control, HLA-A24-restricted CTL clone (clone G5G10), which was specific for a mutated β-catenin epitope. Clones MR7, M8, and MB4 were tested for recognition of previously described Ags in a coculture assay using 293A2 cells transfected with cDNA encoding these Ags (Table III). Clone MR7 recognized TRP-2, and clone M8 recognized NY-ESO-1, whereas clone MB4 did not recognize any of the known Ags tested. In addition, a cDNA clone coding for TRP-2 was isolated and sequenced when CTL clone MR7 was used to screen the 624-mel library (data not shown). The nonapeptide TRP-2: 180–188, and the overlapping NY-ESO-1 peptides NY-ESO-1: 157–167, NY-ESO-1: 157–165, and NY-ESO-1: 155–163 were found to be the epitopes recognized by CTL MR7 and CTL M8, respectively (Table IV).

Isolation of a cDNA clone recognized by TIL clone MB4

A cDNA library prepared from 624-mel cells was next used to screen for a gene encoding reactivity with CTL MB4. Plasmid DNA from pools containing ~100 cDNA clones was transiently transfected into 293A2 cells, a human embryonal kidney cell line transfected with the cDNA encoding HLA-A2 Ag. From 400 pools, the cDNA in one pool stimulated strong IFN-γ release from CTL MB4. By subcloning the positive pool, one cDNA clone (cDNA 198) was identified which induced a high level of IFN-γ secretion from TIL clone MB4 (Table V).

Sequence determination

The sequence of cDNA 198 was identical to that of the TRP-2 gene with the exception of a 99-nt insert between exon 6 and exon 7 of the TRP-2 gene (Fig. 1). This extra insert was composed of two separate sequences, named 6b and 6c, and each showed complete homology with two noncontiguous sequences in intron 6 of the TRP-2 gene. The deduced amino acid sequence was translated in-frame with the TRP-2 product. The two polypeptides encoded by cDNA 198 and the cDNA encoding the previously described TRP-2 were identical except for an in-frame insert of a 33-aa sequence following amino acid 393. 6b and 6c were coding sequences and their flanking regions conformed to the intron consensus motif GT-AG (data not shown); therefore, 6b and 6c were identified as novel exons, alternatively spliced from intron 6 of TRP-2. In addition to the insertion in the coding region, the sequence of cDNA 198 differed from TRP-2 in the noncoding regions. The initial 122 bp (of 414 bp) of the 5′ untranslating region (5′ UTR) of TRP-2 was deleted in cDNA 198. However, the 3′ UTR of TRP-2 was further extended with a 10-bp sequence in cDNA 198. The protein encoded by cDNA 198 was named TRP-2-6b.

Identification of the T cell epitope in TRP-2-6b that was recognized by TIL clone MB4

The fact that CTL MB4 did not recognize 293A2 cells transfected with TRP-2 cDNA suggested that the antigenic epitope may be encoded by the 99-bp sequence of the new exons 6b and 6c. Using a peptide-HLA motif search program, 22 peptides were generated with predicted HLA-A2 binding, from the new 33-aa sequence (encoded by exons 6b and 6c) and 10 amino acids upstream and downstream of it. These peptides were pulsed on T2 cells and tested for recognition by CTL MB4. Two peptides, ATTNILEHV and NATTNILEHV, designated TRP-2-6b: 403–411 and TRP-2-6b: 402–411, respectively, were recognized by CTL MB4 when pulsed on T2 cells (Table 6). The decapeptide TRP-2-6b: 402–411 was able to sensitize target cells for recognition by CTL MB4 at 5 ng/ml, whereas the nonapeptide TRP-2-6b: 403–411 was recognized at 50 pg/ml (Fig. 2).

Immunologic reactivity against NY-ESO-1, TRP-2, TRP-2-6b, and the four peptides (MART-1, two gp100 peptides, and tyrosinase) used for immunization of patient MM, before and during the course of treatment

Patient MM’s PBMC collected before and during the course of treatment were tested for reactivity against NY-ESO-1, TRP-2, TRP-2-6b, and the four peptides that patient MM was immunized with, by stimulating the PBMC in vitro for 12 days with the un-modified epitopes from these Ags (Fig. 3). The nonapeptide from TRP-2-6b was used in this experiment. HLA-A2-restricted telomerase: 540–548 was used as an irrelevant peptide control. The patient’s pretreatment PBMC (day 0) exhibited strong reactivity against NY-ESO-1 and the two TRP-2 isoforms, none of which she had been vaccinated against (Fig. 3). The reactivity against the TRP-2 Ags increased and persisted over time. In contrast, the immune responses directed against MART-1, gp100: 209–217, gp100: 280–288, and tyrosinase, the peptides she had been vaccinated with, were not present in the prevaccination PBMC (Fig. 3).