Transfection and expression of a gene coding for a human melanoma antigen recognized by autologous cytolytic T lymphocytes

Human melanoma line MZ2-MEL expresses several antigens recognized by autologous cytolytic T lymphocytes (CTL). As a first step towards the cloning of the gene coding for one of these antigens, we tried to obtain transfectants expressing the antigen. The DNA recipient cell was a variant of MZ2-MEL which had been selected with a CTL clone for the loss of antigen E. It was cotransfected with genomic DNA of the original melanoma line and with selective plasmid pSVtkneo. Geneticin-resistant transfectants were obtained at a frequency of 2 × 10^–4. These transfectants were then screened for their ability to stimulate the production of tumor necrosis factor by the anti-E CTL clone. One transfectant expressing antigen E was identified among 70 000 drug-resistant transfectants. Its sensitivity to lysis by the anti-E CTL was equal to that of the original melanoma cell line. When this transfectant was submitted to immunoselection with the anti-E CTL clone, the resulting antigen-loss variants were found to have lost several of the transfected pSVtkneo sequences. This indicated that the gene coding for the antigen had been integrated in the vicinity of pSVtkneo sequences, as expected for cotransfected DNA. Address correspondence and offprint requests to: T. Boon.     

A new gene coding for an antigen recognized by autologous cytolytic T lymphocytes on a human renal carcinoma

Previous reports have described antigens that are recognized on human melanoma cells by autologous cytolytic T lymphocytes (CTL). The genes coding for a number of these antigens have been identified. Here we report the cloning of a gene that codes for an antigen recognized by autologous CTL on a human renal carcinoma cell line. This antigen is presented byHLA-B7 and is encoded by a new gene that we have namedRAGE1. No expression ofRAGE1 was found in normal tissues other than retina. RAGE1 expression was found in only one of 57 renal cell carcinoma samples, and also in some sarcomas, infiltrating bladder carcinomas, and melanomas. This represents the first identification of an antigen recognized by autologous CTL on a renal tumor.     

A HLA-DQ5 restricted Melan-A/MART-1 epitope presented by melanoma tumor cells to CD4+ T lymphocytes

Melan-A/MART1 is a melanocytic differentiation antigen expressed by tumor cells of the majority of melanoma patients and, as such, is considered as a good target for melanoma immunotherapy. Nonetheless, the number of class I and II restricted Melan-A epitopes identified so far remains limited. Here we describe a new Melan-A/MART-1 epitope recognized in the context of HLA-DQa1*0101 and HLA-DQb1*0501, -DQb1*0502 or -DQb1*0504 molecules by a CD4+ T cell clone. This clone was obtained by in vitro stimulation of PBMC from a healthy donor by the Melan-A51-73 peptide previously reported to contain a HLA-DR4 epitope. The Melan-A51-73 peptide, therefore contains both HLA-DR4 and HLA-DQ5 restricted epitope. We further show that Melan-A51-63 is the minimal peptide optimally recognized by the HLA-DQ5 restricted CD4+ clone. Importantly, this clone specifically recognizes and kills tumor cell lines expressing Melan-A and either HLA-DQb1*0501, -DQb1*0504 or -DQb1*0502 molecules. Moreover, we could detect CD4+ T cells secreting IFN-gamma in response to Melan-A51-63 and Melan-A51-73 peptides among tumor infiltrating and blood lymphocytes from HLA-DQ5+ patients. This suggests that spontaneous CD4+ T cell responses against this HLA-DQ5 epitope occur in vivo. Together these data significantly increase the fraction of melanoma patients susceptible to benefit from a Melan-A class II restricted vaccine approach.     

Multiple HLA class II-restricted melanocyte differentiation antigens are recognized by tumor-infiltrating lymphocytes from a patient with melanoma

Dramatic clinical responses were observed in patient 888 following the adoptive transfer of autologous tumor-infiltrating lymphocytes (TIL). Previously, extensive analysis of the specificity of class I-restricted T cells from patient 888 TIL has revealed that these T cells recognize a mutated, as well as several nonmutated tumor Ags. Additional studies that were conducted on TIL from patient 888 indicated that they contained CD4-positive T cells that recognized the autologous tumor that had been induced to express HLA class II molecules. Tumor-reactive CD4-positive T cell clones were isolated from TIL and tested for their ability to react with Ags that are recognized by HLA class I-restricted, melanoma-reactive T cells. Using this approach, T cell clones were identified that recognized an epitope expressed in both the tyrosinase-related protein 1 and tyrosinase-related protein 2 Ags in the context of the HLA-DRbeta1*1502 class II gene product. Additional clones were found to recognize an epitope of gp100 in the context of the same HLA-DR restriction element. These observations provide an impetus to develop strategies directed toward generating HLA class II-restricted tumor-reactive T cells.     

Identification of a viral antigen recognized by H-2-restricted cytolytic T lymphocytes on a murine leukemia virus-induced tumor

Monoclonal antibodies were produced against protein p30, a structural protein of murine leukemia viruses (MuLV) coded by the gag gene of MuLV. Three monoclonal antibodies of different isotypes (i.e., IgG-1, IgG-2a, and IgG-2b) were chosen for extensive analysis. These three antibodies bound to mouse tumor cells induced by Friend, Moloney, Rauscher, and Gross MuLV, but not to noninfected normal mouse spleen cells. The ability of these monoclonal antibodies to inhibit cytolytic T lymphocyte (CTL) activity by masking the antigens recognized by CTL on the target cell surface was studied in various CTL systems. It was found that the only CTL that were consistently inhibited in their lytic activity came from BALB.B (H-2b) mice immunized against syngeneic Gross MuLV-induced B.GV cells. These results thus showed that a subpopulation of BALB.B anti-Gross MuLV CTL recognized a Gross MuLV gag gene product expressed on the surface of B.GV cells.     

Proteasome-assisted identification of a SSX-2-derived epitope recognized by tumor-reactive CTL infiltrating metastatic melanoma

The tumor Ag SSX-2 (HOM-MEL-40) was found by serological identification of Ags by recombinant expression cloning and was shown to be a cancer/testis Ag expressed in a wide variety of tumors. It may therefore represent a source of CD8(+) T cell epitopes useful for specific immunotherapy of cancer. To identify potential SSX-2-derived epitopes that can be recognized by CD8(+) T cells, we used an approach that combined: 1) the in vitro proteasomal digestion of precursor peptides overlapping the complete SSX-2 sequence; 2) the prediction of SSX-2-derived peptides with an appropriate HLA-A2 binding score; and 3) the analysis of a tumor-infiltrated lymph node cell population from an HLA-A2(+) melanoma patient with detectable anti-SSX-2 serum Abs. This strategy allowed us to identify peptide SSX-2(41-49) as an HLA-A2-restricted epitope. SSX2(41-49)-specific CD8(+) T cells were readily detectable in the tumor-infiltrated lymph node population by multimer staining, and CTL clones isolated by multimer-guided cell sorting were able to lyse HLA-A2(+) tumor cells expressing SSX-2.     

Lysis by interleukin 2-stimulated tumor-infiltrating lymphocytes of autologous and allogeneic tumor target cells

Summary Stepwise counterflow centrifugal elutriation of leukapheresed human mononuclear cells (MNC) in a Beckman JE-6B rotor and J6-M/E centrifuge yielded a population highly enriched in natural killer (NK) cells (70–75% large granular lymphocytes with 10–13 times greater NK activity) at a flow rate of 38–44 ml/min using a fixed rotor speed of 3000 rpm at 27° C. However, the mean cell recovery was <1%. To obtain sufficient numbers of purified NK cells for adoptive immunotherapy, a strategy combining counterflow centrifugal elutriation with adherence of recombinant interleukin-2(rIL-2)-activated NK cells to plastic was developed. First, MNC were elutriated to give a twofold enrichment in NK cells, containing 22% Leu19⁺ cells, 18% large granular lymphocytes and 51 lytic units of activity against K562 targets as opposed to the unfractionated MNC containing 10% Leu19⁺ cells, 7% large granular lymphocytes and 26 lytic units of activity. The mean recovery was 80±15% (n=10). Further enrichment was obtained by isolation of the elutriated cells that adhered to plastic after culture for 24 h in the presence of 1000 U/ml rIL-2. The initial adherent lymphokine-activated killer (A-LAK) cells represented 1–4% of total MNC, but their subsequent expansion was at least 10–22-fold during 8–14 days in culture with 1000 U/ml rIL-2. Using this strategy, 2 × 10⁹ normal MNC, obtained by leukapheresis, yielded 5 × 10⁸ A-LAK cells with a total of 5.7 × 10⁵ lytic units of cytotoxicity against K562 and a total of 3.3 × 10⁵ lytic units against Daudi targets. This enrichment method has yielded sufficient numbers of A-LAK cells to form the basis for a phase I clinical trial of adoptive immunotherapy in patients with advanced cancer.     

Augmentation of interleukin-2-induced activation of human melanoma tumor-infiltrating lymphocytes by heteroconjugate antibody

Summary Heteroconjugate (HC) antibody (anti-CD3 mAb × anti-p97 melanoma mAb) or monomeric anti-CD3 mAb by itself did not induce proliferation of uncultured melanoma tumor-infiltrating lymphocytes (TILs). They also failed to induce IL-2 production in uncultured TILs, although anti-CD3 mAb, but not HC antibody, stimulated IL-2 production in peripheral blood mononuclear cells (PBMCs). Sequential treatment of uncultured TILs from p97-antigen-positive (p97⁺) melanomas with HC antibody, followed by washing and incubation with interleukin-2 (IL-2), induced significantly higher proliferation than incubation with IL-2 alone. HC antibody pretreatment led to significantly greater results than with anti-CD3 mAb at a 1 ng/ml level in IL-2-induced proliferation of TILs from p97⁺ melanomas, similar to those with anti-CD3 mAb at a level of 100 ng/ml. HC antibody (1 ng/ml) pretretment did not enhance IL-2-induced proliferation of either TILs from p97^– melanomas or PBMCs, while anti-CD3 mAb enhanced the proliferation of TILs from some p97^– melanomas and PBMCs. Regardless of the pretreatment of uncultured TILs with HC antibody or anti-CD3 mAb, IL-2-activated TILs were cytotoxic primarily only to autologous tumor cells, and their phenotypes remained the same. Thus, HC antibody can augment IL-2-induced activation of TILs only from p97⁺ melanomas, without altering their pattern of cytotoxicity or phenotype. The findings were consistent with observations at the clonal level. In contrast to anti-CD3 mAb, HC pretreatment of uncultured TILs from only p97⁺ melanoma prior to limiting-dilution analysis increased the number of proliferating TIL clones, including autologous tumor-specific cytotoxic T lymphocyte clones. These results suggest that use of HC antibody in vivo would be more advantageous than anti-CD3 mAb, with regard to augmentation of IL-2-induced TIL activation.This work was supported in part by grants CA47 891, CA09 599, and RR5511-27 from the National Institutes of Health     

Identification of five new HLA-B*3501-restricted epitopes derived from common melanoma-associated antigens, spontaneously recognized by tumor-infiltrating lymphocytes

We previously described HLA-B35-restricted melanoma tumor-infiltrating lymphocyte responses to frequently expressed melanoma-associated Ags: tyrosinase, Melan-A/MART-1, gp100, MAGE-A3/MAGE-A6, and NY-ESO-1. Using clones derived from these TIL, we identified in this study the corresponding epitopes. We show that five of these epitopes are new and that melanoma cells naturally present all the six epitopes. Interestingly, five of these epitopes correspond to or encompass melanoma-associated Ag epitopes presented in other HLA contexts, such as A2, A1, B51, and Cw3. In particular, the HLA-B35-restricted Melan-A epitope is mimicked by the peptide 26-35, already known as the most immunodominant melanoma epitope in the HLA-A*0201 context. Because this peptide lacked adequate anchor amino acid residues for efficient binding to HLA-B35, modified peptides were designed. Two of these analogues were found to induce higher PBL- and tumor-infiltrating lymphocyte-specific responses than the parental peptide, suggesting that they could be more immunogenic in HLA-B*3501 melanoma patients. These data have important implications for the formulation of polypeptide-based vaccines as well as for the monitoring of melanoma-specific CTL response in HLA-B*3501 melanoma patients.     

The identification of an Onchocerca-specific recombinant antigen containing a T cell epitope.

Recombinant Onchocerca volvulus Ag have been derived from expression libraries and examined for their ability to stimulate PBMC from patients infected with O. volvulus. Ten clones producing recombinant Ag were selected and plaque purified; lysogens were produced and found to express beta-galactosidase fusion proteins ranging in molecular mass from 115 to 138 kDa. When ammonium sulfate-precipitated lysates of these recombinant phage clones were examined for their ability to stimulate PBMC from a patient with onchocerciasis, all 10 recombinants produced stimulation above that to nonrecombinant phage. When individual fusion proteins, affinity purified on anti-beta-galactosidase linked to agarose, were used to stimulate PBMC from patients with onchocerciasis, only one of the recombinant Ag induced PBMC proliferation (stimulation index greater than 4) above that to Ag from nonrecombinant phage. Characterization of the DNA coding for this Ag showed it to be 1.2 kb in length with a small (90 bp) open reading frame; furthermore, it appears to be Onchocerca specific (on genomic dot blots) and single copy. Using overlapping peptides encompassing the entire open reading frame, one T cell epitope has been localized.     

A new tyrosinase epitope recognized in the HLA-B*4002 context by CTL from melanoma patients

Melanoma reactive CTL were obtained by stimulating PBL from a melanoma patient in remission since 1994 following adjuvant TIL immunotherapy, with the autologous melanoma cell line. They were cloned by limiting dilution. One CTL clone recognized melanoma cell lines expressing tyrosinase and the B*4002 molecule, either spontaneously or upon transfection. We demonstrated that this clone recognizes the tyrosinase-derived nonapeptide 316-324 (ADVEFCLSL) and the overlapping decapeptide 315–324 (SADVEFCLSL). We derived two distinct additional specific CTL clones from this same patient that were also reactive against B*4002 melanoma cell lines, suggesting a relative diversity of this specific repertoire in this patient. Stimulating PBMC derived from four additional B*4002 melanoma patients with the tyrosinase 316–324 nonapeptide induced the growth of specific cells for two of the patients, demonstrating the immunogenicity of this new epitope. Our data show that this nonapeptide is a new tool that could be used to generate melanoma-specific T cells for adoptive immunotherapy or serve as a peptide vaccine for HLA-B*4002 melanoma patients.     

Minor structural changes in a mutated human melanoma antigen correspond to dramatically enhanced stimulation of a CD4+ tumor-infiltrating lymphocyte line

While most immunotherapies for cancer have focused on eliciting specific CD8+ cytotoxic T lymphocyte killing of tumor cells, a mounting body of evidence suggests that stimulation of anti-tumor CD4+ T cell help may be required for highly effective therapy. Several MHC class II-restricted tumor antigens that specifically activate such CD4+ helper T lymphocytes have now been identified, including one from a melanoma tumor that is caused by a single base-pair mutation in the glycolytic enzyme triosephosphate isomerase. This mutation results in the conversion of a threonine residue to isoleucine within the antigenic epitope, concomitant with a greater than five log-fold increase in stimulation of a CD4+ tumor-infiltrating lymphocyte line. Here, we present the crystal structures of HLA-DR1 in complex with both wild-type and mutant TPI peptide antigens, the first structures of tumor peptide antigen/MHC class II complexes recognized by CD4+ T cells to be reported. These structures show that very minor changes in the binding surface for T cell receptor correspond to the dramatic differences in T cell stimulation. Defining the structural basis by which CD4+ T cell help is invoked in an anti-tumor immune response will likely aid the design of more effective cancer immunotherapies.     

Characterization of discontinuous epitope of prion protein recognized by the monoclonal antibody T2

The anti-prion protein (PrP) monoclonal antibody T2 has previously been prepared using PrP-knockout mice immunized with mouse recombinant PrP residues 121-231, however its interaction mechanism to PrP antigen has not been cleared. Here we identified and characterized the epitope of T2 antibody. The competitive ELISA with 20-mer synthetic peptides derived from PrP121-231 showed that T2 antibody had no affinity for these peptides. The analysis with deletion mutants of PrP revealed that 10 amino acids in the N terminus and 66 amino acids in the C terminus of PrP121-231 were necessary for reactivity with T2. Two far regions are necessary for complete affinity of the T2 antibody for PrP; either region alone is not sufficient to retain the affinity. The epitope recognized by T2 antibody is discontinuous and conformational. We examined the effect of disulfide bond and salt bridges. Alkylation of cysteine residues in C terminus of PrP121-231, which breaks a disulfide bond and disrupts the structure, had diminished the reactivity. Mutations induced in the PrP121-231 to break the disulfide bond or salt bridges, markedly had reduced the reactivity with T2 antibody. It suggests that T2 antibody recognized the structure maintained by the disulfide bond and salt bridges.     

Cytolytic T lymphocytes recognize an antigen encoded by MAGE-A10 on a human melanoma.

From melanoma patient LB1751, cytolytic T lymphocytes (CTL) were generated that lysed specifically autologous tumor cells. To establish whether these CTL recognized one of the Ags that had previously been defined, a CTL clone was stimulated with cells expressing various MAGE genes. It produced TNF upon stimulation with target cells expressing MAGE-A10. The Ag was found to be nonapeptide GLYDGMEHL (codons 254-262), which is presented by HLA-A2.1. This is the first report on the generation of anti-MAGE CTL by autologous mixed lymphocyte-tumor cell culture (MLTC) from a melanoma patient other than patient MZ2, from whom the first MAGE gene was identified. MAGE genes are expressed in many tumors but not by normal tissues except male germline cells and placenta, which do not express HLA molecules. Therefore, the identification of an antigenic peptide derived from MAGE-A10 adds to the repertoire of tumor-specific shared Ags available for anti-tumoral vaccination trials.     

T lymphocytes can mediate lysis of autologous melanoma cells by multiple mechanisms: Evidence with a single T cell clone

Summary The specificity analysis of a CD3⁺, WT31⁺, CD8⁺ cytotoxic T lymphocyte (CTL) clone (CTL 49), isolated from peripheral blood lymphocytes of a melanoma patient (no. 665) after mixed lymphocyte culture with an HLA-A2⁺ allogeneic lymphoblastoid cell line (VSKB-LCL), revealed that CTL 49 could lyse, in addition to HLA-A2⁺ lines, autologous HLA-A2^– melanoma (Me665/2) and K562 targets. Killing of VSKB-LCL, but not of Me665/2, could be inhibited by anti-CD3 and by anti-HLA-A2 antibodies or by modulation of the CD3 complex. Cold-target competition studies showed that K562, but not VSKB-LCL, could compete with Me665/2 for lysis by CTL 49. However, unlike K562, Me665/2 could be lysed by CTL 49 in a Ca²⁺-independent fashion in 4 h and 18 h assays. CTL 49 expressed mRNA specific for tumor necrosis factor (TNF) and, to a lesser extent, for lymphotoxin (TNF). Exposure of the clone to anti-CD3 antibodies induced the expression of interferon(IFN)--specific and the up-regulation of TNF- and TNF-specific mRNA. Antibodies to TNF, TNF and IFN reduced the lysis of Me665/2, but not of K562, by CTL 49 in 18-h cytotoxic assays. Antibodies to TNF and to IFN almost completely inhibited the lysis seen on Me665/2 (but not on K562), in 96-h assays, by supernatants isolated from VSKB-LCL- or anti-CD3-stimulated CTL 49 cells. Taken together, these data indicate that major-histocompatibility-complex-independent lysis of autologous tumor cells and of natural killer reference targets by the same alloreactive T cell clone are activities related at the level of target recognition but distinct at the level of the lytic hit. Thus, efficient lysis of autologous tumor cells results from a complex mechanism based upon direct effector-target interaction as well as on cytokine-mediated cytolytic effects.     

Clonal analysis of cytotoxic T lymphocytes (CTL) against autologous melanoma

Summary This study investigates the nature and specificity of cytotoxic T lymphocytes (CTL) in patients with melanoma which are able to kill autologous melanoma cells. Interleukin 2 (IL2)-dependent T cell clones from two melanoma patients and a normal subject were generated in mixed lymphocyte cultures (MLC) or mixed lymphocyte tumor cell cultures (MLTC) and propagated for prolonged periods in tissue culture. Analysis of their phenotype by a wide range of monoclonal antibodies (M.Abs) revealed two main phenotypes which depended on whether they expressed Fc receptors detected by Leu 11 M.Abs or not. Leu 11^– T cells (referred to as Type 1) were inhibited by M.Abs to T3, T8, and a common HLA, ABC antigen. Conversely Leu 11⁺ T cells (referred to as Type 2) were inhibited by M.Ab to Leu 11 but not by M.Ab to T3, T8 and the HLA, ABC antigen. Subtypes among Type 1 cells were recognized which depended on their specificity. The most restricted were CTL [Type 1(a)] clones generated only in MLTC which recognized the autologous melanoma cell plus 1 of 11 other melanoma target cells. Type 1(b) CTL clones recognized a larger proportion (approximately 50%) of the melanoma cells. A third category [Type 1(c)] recognized antigens on melanoma cells shared with that on the EBV-transformed B cells used as stimulators in the MLC. Type 2 CTL clones had broad specificity to melanoma and nonmelanoma cells, characteristic of that described for lymphokine activated killer (LAK) cells. The latter were MHC unrestricted but further studies are required to clarify whether the Type 1 CTL clones are MHC restricted or not. The CTL activity of all clones was inhibited by M.Ab to the sheep red blood cell receptor and to the T10 antigens. It is suggested that recognition of these different types of CTL clones may assist future studies on the immune response against melanoma and the nature of antigens recognized by CTL.