CA2158446C - Nucleic acid coding for a tumor rejection antigen precursor - Google Patents
Nucleic acid coding for a tumor rejection antigen precursor Download PDFInfo
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- CA2158446C CA2158446C CA002158446A CA2158446A CA2158446C CA 2158446 C CA2158446 C CA 2158446C CA 002158446 A CA002158446 A CA 002158446A CA 2158446 A CA2158446 A CA 2158446A CA 2158446 C CA2158446 C CA 2158446C
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- nucleic acid
- tumor rejection
- rejection antigen
- hla
- acid molecule
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention relates to nucleic acid molecules coding for a tumor rejection antigen precursor. Specifically, the tumor rejection antigen precursor, or "TRAP", is processed into at least one tumor rejection antigen, which is presented by HLA-A2 molecules. Ramifications of the discovery are also set forth.
Description
' ~O 94/21126 2158446 PCTIUS94/02487 N[JC:LEIC ACID CODING FOR A TpMOR
REJEX7i'ION ANfl'ICEN PRECURSOR
FIELD OF THE INVENTION
This invention relates to a nucleic acid molecule which codes for a tumor rejection antigen precursor. More particularly, the invention concerns a gene, whose tumor rejection antigen precursor is processed, inter alia, into at least one tumor rejection antigen that is presented by HLA-A2 molecules on cell surfaces.
BACKGROUND AND PRIOR ART
The process by which the mammalian immune system recognizes and reacts to foreign or alien materials is a complex one. An important facet of the system is the T cell response. This response requires that T cells recognize and interact with complexes of cell surface molecules, referred to as human leukocyte antigens ("HLA"), or major histocompatibility complexes ("MHCs"), and peptides. The peptides are derived from larger molecules which are processed by the cells which also present the HLA/MHC molecule. See in this regard Male et al., Advanced Immunology (J.P. Lipincott Company, 1987), especially chapters 6-10. The interaction of T cell and complexes of HLA/peptide is restricted, requiring a T cell specific for a particular combination of an FiLA
molecule and a peptide. If a specific T cell is not present, there is no T cell response even if its partner complex is present. Similarly, there is no response if the specific complex is absent, but the T cell is present. This mechanism is involved in the immune system's response to foreign materials, in autoimmune pathologies, and in responses to cellular abnormalities. Recently, much work has focused on the mechanisms by which proteins are processed into the HLA
binding peptides. See, in this regard, Barinaga, Science 257:
880 (1992); Fremont et al., Science 257: 919 (1992); Matsumura et al., Science 257: 927 (1992); Latron et al., Science 257:
964 (1992).
~,=-----r-- - -- _-'ftl 8446 The mechanism by which T cells recognize cellular abnormalities has also been implicated in cancer. For example, in PCT applicati.on PCT/US92/04354, filed May 22, 1992, published on November 26, 1992, a family of genes is disclosed, which are processed into peptides which, in t'.urn, are expressed'on cell surfaces, which can lead to lysis of the tumor cells by specific CTLs. The genes are said to code for "tumor rejection antigen precursors" or "TRAP" molecules, and the peptides derived therefrom are referred to as "tumor rejection antigens" or "TRAs". See Traversari et al., Immunogenetics 35: 145 (1992); van der Bruggen et al., Science 254: 1643 (1991), for further information on th:Ls fam=ily of genes.
In Canadian Patent Application S.N. 2,143,335, filed August 30, 1993, nonapeptides are taught which are presented by thi=_ HLA-Al molecule. The Application teaches that given the known specificity of particular peptides for particular HLA molecules, one should expect a particular peptide to bind one HLA molecule, but not others. This is important, because different individuals possess different HLA phenotypes. As a result, while identification of a particular peptide as being a partner for a specific HLA molecule has diagnostic and therapeutic ramifications, these are only relevant for individuals with that particular HLA phenotype. There is a need for further work in the area, because cellular abnormalities are not restricted to one particular HLA
phenotype, and targeted therapy requires some knowledge of the phenotype of the abnormal cells at issue.
2a In Canadian Patent Application S.N. 2,154,468, filed January 18, 1994, the fact that the MAGE-1 expression product is processed to a second TRA is disclosed. This second TRA is presented by HLA-C10-molecules. The disclosure shows that a given TRAP can yield a plurality df TRAs.
In Canadian Patent Application S.N. 2,152,266, filed December 14, 1993õ
"O 94/21126 PCT/US94/02487 tyrosinase is described as a tumor rejection antigen precursor. This reference discloses that a molecule which is produced by some normal cells (e.g., melanocytes), is processed in tumor cells to yield a tumor rejection antigen that is presented by HLA-A2 molecules.
It has now been found that another nucleic acid molecule codes for a tumor rejection antigen precursor which differs from those described previously. The TRAP of the invention is processed to at least one tumor rejection antigen that is presented by HLA-A2 molecules; however sequence analysis indicates that the TRAP of the invention is not, nor is it related to, tyrosinase. Thus the invention relates to a nucleic acid molecule which codes for a tumor rejection antigen precursor, or "TRAP" molecule. This "TRAP" molecule is not tyrosinase. Further, the TRAP of the invention is processed to at least one tumor rejection antigen, or "TRA", which is presented by HLA-A2 molecules. The TRA is not tyrosinase related, and other TRAs derived from the TRAPs of the invention may be presented by other HLA molecules.
The invention and various aspects thereof will be elaborated upon in the disclosure which follows. , BRIEF DESCRIPTION OF THE FIGURES
Figure 1A presents results of cell lysis experiments using CTL clone 1/95 against LB39-MEL, K562, and LB39 blasts.
Figure 1B shows lysis using CTL clone 1/95 against SK23-MEL and SK29-MEL.
Figure 2 sets forth results of a TNF release assay using various cell lines with CTL 1/95.
Figure 3A shows TNF release induced by different cell lines, including transfectants, when tested with CTL clone 1/95.
Figure 3B presents TNF release data using CTL clone IVSB.
Figure 3C shows TNF release using CTL clone 10/196.
Figure 4 presents a panel of tissues, cell lines, and tumors tested for expression of the gene AaGlcl24 using polymerase chain reaction (PCR) using oligonucleotide probes 'ft 15844 6 derived from the nucleic acid molecule described herein.
DETAILED DEBCRIPTION OF PREFERRED EHEODIMENTB
Examp1e 1 A melanoma cell line, "LB-39-MEL" was established from melanoma cells taken f'rom patient LB39, using standard methodologies. Once the cell line was established, a sample thereof was irradiated, so as to render it non-proliferative.
These irradiated cells were then used to isolate cytolytic T
cells ("CTLs") specific thereto.
A sample of peripheral blood mononuclear cells ("PBMCs") was taken from patient LB39, and contacted to the irradiated melanoma cells. The mixture was observed for lysis .of the melanoma cells, which indicated that CTLs specific for a complex of peptide and HLA molecule presented by the melanoma cells were present in the sample.
The lysis zissay employed was a chromium release assay following Herin et al., Int.J.Cancer 39:390-396 (1987). The assay is described herein. The target melanoma cells were grown in v t , and then resuspended at 107 cells/ml in DMEM, supplemented with 10 mM HEPES and 30$ FCS, and incubated for 45 minutes at 37 C with 200 Ci/ml of Na ("Cr) 04. Labelled cells were washed three times with DMEM, supplemented with 10 mM Hepes. These were then resuspended in DMEM supplemented with 10 mM Hepes and 10% FCS, after which 100 ul aliquots containing 103 cells, were distributed into 96 well microplates. Samples of PBLs were added in 100 ul of the same medium, and assays were carried out in duplicate. Plates were centrifuge.: for 4 minutes at 100g, and incubated for four hours at 37 C in a 80% of COi atmosphere.
Plates were centrifuged again, and 100 ul aliquots of supernatant were collected and counted. Percentage of s'Cr release was calculated as follows:
-t s'Cr release = (ER-SR) x 100 (MR-SR) where ER is observed, experimental s'Cr release, SR is spontaneous release measured by incubating 103 labeled cells in 200 ul of medium alone, and MR is_maximum release, obtained by adding 100 ui 0.3% Triton X-100 f (Tracletna.rk) to target cells.
Those mononuclear blood samples which showed high CTL
activity were expanded and cloned via limiting dilution, and 5 were screened again, using the same methodology. The CTL
clone LB39-CTL 1/95 was thus isolated.
The same method was used to test target K562 cells,, as well as autologous, PHA induced T cell blasts. These results, presented in Figure lA, show that this CTL clone recognizes and lyses the melanoma cell line, but neither of K562 or the T cell blasts. The CTL, LB39-CTL 1/95, was then tested against melanoma cell lines SK23-MEL and SK29 MEL, in the same manner described, suPra. Cells from both of these lines were also lysed. These lines were both isolated from patients who were typed as HLA-A2, as was LB39. This suggested that the CTL clone LB39-CTL 1/95 recognized an antigen presented by HLA-A2.
Example 2 Further studies were carried out to determine if LB39-CTL
1/95 also produced tumor necrosis factor ("TNF") when contacted with target cells. The method used was that described by Traversari et al., Immunogenetics 35: 145-152 (1992). Briefly, samples of the CTL line were combined with samples of a target cell of interest, in culture medium.
After 24 hours, supernatant from the cultures was removed, and then tested on TNF sensitive WEHI cells. In addition to LB39-MEL and SK23-MEL, described supra, another HLA-A2 line, i.e., SK29-MEL.1, an HLA-A2 loss variant, i.e., SEK29-MEL1.22, and a non HLA-A2 line, i.e., MZ2-MEL, which is HLA-Al, were tested.
The results, presented in terms of the percentage of WEHI
cells which died upon exposure to the supernatant, are shown in Figure 2. These results show that the HLA-A2 loss variant SK 29-MEL.1.22 is no longer capable of stimulating the CTL
clone, thus confirming that the antigen recognized by LB39-CTL-I/95 is presented by HLA-A2.
Examnle 3 The results from Example 2 indicated that SK MEL 29.1 presented the target antigen of interest. As such, it was used as a source of total'mRNA to prepare a cDNA library.
Total RNA was isolated from the cell line. The mRNA was 5 isolated using an oligo-dT binding kit, following well recognized techniques. Once the mRNA was secured, it was transcribed into cDNA, again using standard methodologies.
The cDNA was then ligated to EcoRI adaptors and cloned into the EcoRI site of plasmid pcDNA-I/Amp, in accordance with manufacturer's instructions. The recombinant plasmids were then electroporated into JM101 E. co i(electroporation conditions: l pulse at 25 farads, 2500 V).
The transfected bacteria were selected with ampicillin (50 g/ml), and then divided into 800 pools of 100 clones each. Each pool represented about 50 different cDNAs, as analysis showed that about 50% of plasmids contained an insert. Each pool was amplified to saturation, and plasmid DNA was isolated via alkaline lysis, potassium acetate precipitation without phenol extraction, following Maniatis et al., in Molecular Cloning:' A Laboratory Manual (Cold Spring Harbor, N.Y., 1982).
Example 4 Following preparation of the library described in Example 3, the cDNA was transfected into eukaryotic cells. The transfections, described herein, were carried out in duplicate. Samples of COS-7 cells were seeded, at 15,000 cells/well into tissue culture flat bottom microwells, in Dulbeco's modified Eagles Medium ("DMEM") supplemented with 10t fetal calf serum. The cells were incubated overnight at 37 C, medium was removed and then replaced by 30 ul/well of DMEM medium containing 10% NU-SERUM (Trade-mark), 400 g/ml DEAE-dextran, 100 pM chloroquine, 100 ng of plasmid pcDNA-I/Amp-A2 and 100 ng of DNA of a pool of the cDNA library described supra. Plasmid pcDNA-I/amp-A2 contains the HLA-A2 gene from SK29-MEL. Following four hours of incubation at 37 C, the medium was removed, and replaced by 50 l of PBS
containing 10% DMSO. This medium was removed after two minutes and replaced by 200 l of DMEM supplemented with 10% of FCS.
Following this change in medium, COS cells were incubated for 48 hours at 370C. Medium was then discarded, and 1000 cells of CTL 1/95 were added, in 100 l of Iscove medium containing 10% pooled human serum, supplemented with 25 U/ml of IL-2. Supernatant was removed after 24 hours, and TNF
content was determined in the assay on WEHI cells, as described by Traversari et al., supra.
Of the 800 pools tested, 99% stimulated TNF release, to a concentration of from 3-6 pg/ml in the supernatant. Two pools gave yields over 8 pg/ml, with a duplicate well also yielding over 8 pg/ml.
Examp e 5 The two pools showing high yields of TNF in the supernatant were selected for further study. Specifically, the bacteria were cloned, and 800 bacteria were tested from each pool. Plasmid DNA was extracted therefrom, transfected into a new sample of COS cells in the same manner as described supra, and the cells were again tested for stimulation of LB39-CTL clone 1/95. One positive clone was found, referred to as AaGlcl24. Convincing evidence that the transfected cells were recognized by CTLs was obtained by carrying out a comparative test of COS cells transfected with cDNA from the positive clone and the HLA-A2 gene, COS cells transfected only with HLA-A2, and line SK29-MEL. TNF release in CTL
supernatant was measured by testing it on WEHI cells, as referred to supra. The optical density of the surviving WEHI
cells was measured using MTT. Figure 3A showa the results obtained with CTL clone 1/95.
Further tests showed that the peptide presented by HLA-A2 in the transfected cells was different from that observed previously, i.e., a tyrosinase derived peptide. CTL clone IVSB is specific to complexes of tyrosinase derived peptide and HLA-A2. When this CTL clone was contacted to cells transfected with AaGlcl24 and HLA-A2, TNF release was minimal, as shown in Figure 3B.
~., WO 94/21126 ~+ ~ ~' PCT/US94/02487 Example 6 The cDNA from the positive clone was removed, and sequenced following art known techniques. A sequence search revealed that the plasmid insert showed no homology to known genes or proteins. SEQUENCE ID NO: 1 presents cDNA nucleotide information, showing a large, open reading frame from positions 75 to 431, corresponding to a protein product of 119 amino acids. Sequence ID NO: 2 sets forth the extended sequence of which SEQ ID NO: 1 is a part.
Example 7 In the same manner that CTL clone LB39-CTL 1/95 was isolated, a sample of PBMCs and a melanoma cell line developed from patient SK29(AV) were used to isolate CTL clone SK29-CTL
10/196. This new cell line was tested in the same manner as is set forth in Example 5. The results of the assays, depicted in Figure 3C, show that the tumor rejection antigen coded for by AaGlcl24 (referred to as antigen "LB39-Aa"
hereafter), is also recognized by this CTL clone. These experiments indicate that other patients can, and in fact do, generate CTLs specific for this antigen.
Oligonucleotide probes were derived from the described sequences, and were used in standard polymerase chain reaction methodologies to determine expression of the gene in normal tissues, tumors, and tumor cell lines. These results are presented in figure 4, and show that among normal tissues tested, only melanocytes expressed the gene. Note the expression in all tumor samples and/or melanoma cell lines tested.
The foregoing experiments describe a newly isolated nucleic acid sequence coding for a tumor rejection antigen precursor, a "TRAP" molecule. The molecule is processed intracellularly in a manner which leads to production of at least one tumor rejection antigen, or "TRA", which is presented by HLA-A2 molecules. While it has been observed previously that HLA-A2 molecules present peptides derived from tyrosinase, the nucleic acid sequences of the invention do not code for tyrosinase, and the TRAs are not tyrosinase derived.
REJEX7i'ION ANfl'ICEN PRECURSOR
FIELD OF THE INVENTION
This invention relates to a nucleic acid molecule which codes for a tumor rejection antigen precursor. More particularly, the invention concerns a gene, whose tumor rejection antigen precursor is processed, inter alia, into at least one tumor rejection antigen that is presented by HLA-A2 molecules on cell surfaces.
BACKGROUND AND PRIOR ART
The process by which the mammalian immune system recognizes and reacts to foreign or alien materials is a complex one. An important facet of the system is the T cell response. This response requires that T cells recognize and interact with complexes of cell surface molecules, referred to as human leukocyte antigens ("HLA"), or major histocompatibility complexes ("MHCs"), and peptides. The peptides are derived from larger molecules which are processed by the cells which also present the HLA/MHC molecule. See in this regard Male et al., Advanced Immunology (J.P. Lipincott Company, 1987), especially chapters 6-10. The interaction of T cell and complexes of HLA/peptide is restricted, requiring a T cell specific for a particular combination of an FiLA
molecule and a peptide. If a specific T cell is not present, there is no T cell response even if its partner complex is present. Similarly, there is no response if the specific complex is absent, but the T cell is present. This mechanism is involved in the immune system's response to foreign materials, in autoimmune pathologies, and in responses to cellular abnormalities. Recently, much work has focused on the mechanisms by which proteins are processed into the HLA
binding peptides. See, in this regard, Barinaga, Science 257:
880 (1992); Fremont et al., Science 257: 919 (1992); Matsumura et al., Science 257: 927 (1992); Latron et al., Science 257:
964 (1992).
~,=-----r-- - -- _-'ftl 8446 The mechanism by which T cells recognize cellular abnormalities has also been implicated in cancer. For example, in PCT applicati.on PCT/US92/04354, filed May 22, 1992, published on November 26, 1992, a family of genes is disclosed, which are processed into peptides which, in t'.urn, are expressed'on cell surfaces, which can lead to lysis of the tumor cells by specific CTLs. The genes are said to code for "tumor rejection antigen precursors" or "TRAP" molecules, and the peptides derived therefrom are referred to as "tumor rejection antigens" or "TRAs". See Traversari et al., Immunogenetics 35: 145 (1992); van der Bruggen et al., Science 254: 1643 (1991), for further information on th:Ls fam=ily of genes.
In Canadian Patent Application S.N. 2,143,335, filed August 30, 1993, nonapeptides are taught which are presented by thi=_ HLA-Al molecule. The Application teaches that given the known specificity of particular peptides for particular HLA molecules, one should expect a particular peptide to bind one HLA molecule, but not others. This is important, because different individuals possess different HLA phenotypes. As a result, while identification of a particular peptide as being a partner for a specific HLA molecule has diagnostic and therapeutic ramifications, these are only relevant for individuals with that particular HLA phenotype. There is a need for further work in the area, because cellular abnormalities are not restricted to one particular HLA
phenotype, and targeted therapy requires some knowledge of the phenotype of the abnormal cells at issue.
2a In Canadian Patent Application S.N. 2,154,468, filed January 18, 1994, the fact that the MAGE-1 expression product is processed to a second TRA is disclosed. This second TRA is presented by HLA-C10-molecules. The disclosure shows that a given TRAP can yield a plurality df TRAs.
In Canadian Patent Application S.N. 2,152,266, filed December 14, 1993õ
"O 94/21126 PCT/US94/02487 tyrosinase is described as a tumor rejection antigen precursor. This reference discloses that a molecule which is produced by some normal cells (e.g., melanocytes), is processed in tumor cells to yield a tumor rejection antigen that is presented by HLA-A2 molecules.
It has now been found that another nucleic acid molecule codes for a tumor rejection antigen precursor which differs from those described previously. The TRAP of the invention is processed to at least one tumor rejection antigen that is presented by HLA-A2 molecules; however sequence analysis indicates that the TRAP of the invention is not, nor is it related to, tyrosinase. Thus the invention relates to a nucleic acid molecule which codes for a tumor rejection antigen precursor, or "TRAP" molecule. This "TRAP" molecule is not tyrosinase. Further, the TRAP of the invention is processed to at least one tumor rejection antigen, or "TRA", which is presented by HLA-A2 molecules. The TRA is not tyrosinase related, and other TRAs derived from the TRAPs of the invention may be presented by other HLA molecules.
The invention and various aspects thereof will be elaborated upon in the disclosure which follows. , BRIEF DESCRIPTION OF THE FIGURES
Figure 1A presents results of cell lysis experiments using CTL clone 1/95 against LB39-MEL, K562, and LB39 blasts.
Figure 1B shows lysis using CTL clone 1/95 against SK23-MEL and SK29-MEL.
Figure 2 sets forth results of a TNF release assay using various cell lines with CTL 1/95.
Figure 3A shows TNF release induced by different cell lines, including transfectants, when tested with CTL clone 1/95.
Figure 3B presents TNF release data using CTL clone IVSB.
Figure 3C shows TNF release using CTL clone 10/196.
Figure 4 presents a panel of tissues, cell lines, and tumors tested for expression of the gene AaGlcl24 using polymerase chain reaction (PCR) using oligonucleotide probes 'ft 15844 6 derived from the nucleic acid molecule described herein.
DETAILED DEBCRIPTION OF PREFERRED EHEODIMENTB
Examp1e 1 A melanoma cell line, "LB-39-MEL" was established from melanoma cells taken f'rom patient LB39, using standard methodologies. Once the cell line was established, a sample thereof was irradiated, so as to render it non-proliferative.
These irradiated cells were then used to isolate cytolytic T
cells ("CTLs") specific thereto.
A sample of peripheral blood mononuclear cells ("PBMCs") was taken from patient LB39, and contacted to the irradiated melanoma cells. The mixture was observed for lysis .of the melanoma cells, which indicated that CTLs specific for a complex of peptide and HLA molecule presented by the melanoma cells were present in the sample.
The lysis zissay employed was a chromium release assay following Herin et al., Int.J.Cancer 39:390-396 (1987). The assay is described herein. The target melanoma cells were grown in v t , and then resuspended at 107 cells/ml in DMEM, supplemented with 10 mM HEPES and 30$ FCS, and incubated for 45 minutes at 37 C with 200 Ci/ml of Na ("Cr) 04. Labelled cells were washed three times with DMEM, supplemented with 10 mM Hepes. These were then resuspended in DMEM supplemented with 10 mM Hepes and 10% FCS, after which 100 ul aliquots containing 103 cells, were distributed into 96 well microplates. Samples of PBLs were added in 100 ul of the same medium, and assays were carried out in duplicate. Plates were centrifuge.: for 4 minutes at 100g, and incubated for four hours at 37 C in a 80% of COi atmosphere.
Plates were centrifuged again, and 100 ul aliquots of supernatant were collected and counted. Percentage of s'Cr release was calculated as follows:
-t s'Cr release = (ER-SR) x 100 (MR-SR) where ER is observed, experimental s'Cr release, SR is spontaneous release measured by incubating 103 labeled cells in 200 ul of medium alone, and MR is_maximum release, obtained by adding 100 ui 0.3% Triton X-100 f (Tracletna.rk) to target cells.
Those mononuclear blood samples which showed high CTL
activity were expanded and cloned via limiting dilution, and 5 were screened again, using the same methodology. The CTL
clone LB39-CTL 1/95 was thus isolated.
The same method was used to test target K562 cells,, as well as autologous, PHA induced T cell blasts. These results, presented in Figure lA, show that this CTL clone recognizes and lyses the melanoma cell line, but neither of K562 or the T cell blasts. The CTL, LB39-CTL 1/95, was then tested against melanoma cell lines SK23-MEL and SK29 MEL, in the same manner described, suPra. Cells from both of these lines were also lysed. These lines were both isolated from patients who were typed as HLA-A2, as was LB39. This suggested that the CTL clone LB39-CTL 1/95 recognized an antigen presented by HLA-A2.
Example 2 Further studies were carried out to determine if LB39-CTL
1/95 also produced tumor necrosis factor ("TNF") when contacted with target cells. The method used was that described by Traversari et al., Immunogenetics 35: 145-152 (1992). Briefly, samples of the CTL line were combined with samples of a target cell of interest, in culture medium.
After 24 hours, supernatant from the cultures was removed, and then tested on TNF sensitive WEHI cells. In addition to LB39-MEL and SK23-MEL, described supra, another HLA-A2 line, i.e., SK29-MEL.1, an HLA-A2 loss variant, i.e., SEK29-MEL1.22, and a non HLA-A2 line, i.e., MZ2-MEL, which is HLA-Al, were tested.
The results, presented in terms of the percentage of WEHI
cells which died upon exposure to the supernatant, are shown in Figure 2. These results show that the HLA-A2 loss variant SK 29-MEL.1.22 is no longer capable of stimulating the CTL
clone, thus confirming that the antigen recognized by LB39-CTL-I/95 is presented by HLA-A2.
Examnle 3 The results from Example 2 indicated that SK MEL 29.1 presented the target antigen of interest. As such, it was used as a source of total'mRNA to prepare a cDNA library.
Total RNA was isolated from the cell line. The mRNA was 5 isolated using an oligo-dT binding kit, following well recognized techniques. Once the mRNA was secured, it was transcribed into cDNA, again using standard methodologies.
The cDNA was then ligated to EcoRI adaptors and cloned into the EcoRI site of plasmid pcDNA-I/Amp, in accordance with manufacturer's instructions. The recombinant plasmids were then electroporated into JM101 E. co i(electroporation conditions: l pulse at 25 farads, 2500 V).
The transfected bacteria were selected with ampicillin (50 g/ml), and then divided into 800 pools of 100 clones each. Each pool represented about 50 different cDNAs, as analysis showed that about 50% of plasmids contained an insert. Each pool was amplified to saturation, and plasmid DNA was isolated via alkaline lysis, potassium acetate precipitation without phenol extraction, following Maniatis et al., in Molecular Cloning:' A Laboratory Manual (Cold Spring Harbor, N.Y., 1982).
Example 4 Following preparation of the library described in Example 3, the cDNA was transfected into eukaryotic cells. The transfections, described herein, were carried out in duplicate. Samples of COS-7 cells were seeded, at 15,000 cells/well into tissue culture flat bottom microwells, in Dulbeco's modified Eagles Medium ("DMEM") supplemented with 10t fetal calf serum. The cells were incubated overnight at 37 C, medium was removed and then replaced by 30 ul/well of DMEM medium containing 10% NU-SERUM (Trade-mark), 400 g/ml DEAE-dextran, 100 pM chloroquine, 100 ng of plasmid pcDNA-I/Amp-A2 and 100 ng of DNA of a pool of the cDNA library described supra. Plasmid pcDNA-I/amp-A2 contains the HLA-A2 gene from SK29-MEL. Following four hours of incubation at 37 C, the medium was removed, and replaced by 50 l of PBS
containing 10% DMSO. This medium was removed after two minutes and replaced by 200 l of DMEM supplemented with 10% of FCS.
Following this change in medium, COS cells were incubated for 48 hours at 370C. Medium was then discarded, and 1000 cells of CTL 1/95 were added, in 100 l of Iscove medium containing 10% pooled human serum, supplemented with 25 U/ml of IL-2. Supernatant was removed after 24 hours, and TNF
content was determined in the assay on WEHI cells, as described by Traversari et al., supra.
Of the 800 pools tested, 99% stimulated TNF release, to a concentration of from 3-6 pg/ml in the supernatant. Two pools gave yields over 8 pg/ml, with a duplicate well also yielding over 8 pg/ml.
Examp e 5 The two pools showing high yields of TNF in the supernatant were selected for further study. Specifically, the bacteria were cloned, and 800 bacteria were tested from each pool. Plasmid DNA was extracted therefrom, transfected into a new sample of COS cells in the same manner as described supra, and the cells were again tested for stimulation of LB39-CTL clone 1/95. One positive clone was found, referred to as AaGlcl24. Convincing evidence that the transfected cells were recognized by CTLs was obtained by carrying out a comparative test of COS cells transfected with cDNA from the positive clone and the HLA-A2 gene, COS cells transfected only with HLA-A2, and line SK29-MEL. TNF release in CTL
supernatant was measured by testing it on WEHI cells, as referred to supra. The optical density of the surviving WEHI
cells was measured using MTT. Figure 3A showa the results obtained with CTL clone 1/95.
Further tests showed that the peptide presented by HLA-A2 in the transfected cells was different from that observed previously, i.e., a tyrosinase derived peptide. CTL clone IVSB is specific to complexes of tyrosinase derived peptide and HLA-A2. When this CTL clone was contacted to cells transfected with AaGlcl24 and HLA-A2, TNF release was minimal, as shown in Figure 3B.
~., WO 94/21126 ~+ ~ ~' PCT/US94/02487 Example 6 The cDNA from the positive clone was removed, and sequenced following art known techniques. A sequence search revealed that the plasmid insert showed no homology to known genes or proteins. SEQUENCE ID NO: 1 presents cDNA nucleotide information, showing a large, open reading frame from positions 75 to 431, corresponding to a protein product of 119 amino acids. Sequence ID NO: 2 sets forth the extended sequence of which SEQ ID NO: 1 is a part.
Example 7 In the same manner that CTL clone LB39-CTL 1/95 was isolated, a sample of PBMCs and a melanoma cell line developed from patient SK29(AV) were used to isolate CTL clone SK29-CTL
10/196. This new cell line was tested in the same manner as is set forth in Example 5. The results of the assays, depicted in Figure 3C, show that the tumor rejection antigen coded for by AaGlcl24 (referred to as antigen "LB39-Aa"
hereafter), is also recognized by this CTL clone. These experiments indicate that other patients can, and in fact do, generate CTLs specific for this antigen.
Oligonucleotide probes were derived from the described sequences, and were used in standard polymerase chain reaction methodologies to determine expression of the gene in normal tissues, tumors, and tumor cell lines. These results are presented in figure 4, and show that among normal tissues tested, only melanocytes expressed the gene. Note the expression in all tumor samples and/or melanoma cell lines tested.
The foregoing experiments describe a newly isolated nucleic acid sequence coding for a tumor rejection antigen precursor, a "TRAP" molecule. The molecule is processed intracellularly in a manner which leads to production of at least one tumor rejection antigen, or "TRA", which is presented by HLA-A2 molecules. While it has been observed previously that HLA-A2 molecules present peptides derived from tyrosinase, the nucleic acid sequences of the invention do not code for tyrosinase, and the TRAs are not tyrosinase derived.
The invention thus involves an isolated nucleic acid molecule which codes for a tumor rejection antigen precursor, or "TRAP", with the proviso that the TRAP is not tyrosinase.
The TRAP coded for is one which is processed to at least one tumor rejection antigen, or TRA, which is presented by HLA-A2 molecules on cell surfaces. The nucleic acid molecules of the invention may be, e.g., genomic DNA, ("gDNA"), complementary DNA ("cDNA"), or a form of RNA. The invention also involves isolated nucleic acid molecules which are complementary to the molecules described above. An especially preferred form of the invention is a molecule which contains the sequence set forth in SEQ ID NO: 1.
Also encompassed by the invention are vectors which contain the nucleic acid molecules of the invention, operably linked to a promoter. The vectors may also include a molecule coding for HLA-A2. As these two molecules, i.e., HLA-A2 and the TRAP are necessary to generate a cytolytic T cell response, the invention also encompasses expression systems where nucleic acid molecules coding for TRAP and for HLA-A2 are presented as separate portions in, e.g., a kit. The invention also encompasses cell lines transfected by the vectors described herein, be these prokaryotic cells, such as E. coli, or eukaryotic cells, such as Chinese hamster ovary ("CHO") or COS cells.
As indicated , the complexes of TRA and HLA-A2 provoke a cytolytic T cell response, and as such isolated complexes of the tumor rejection antigen and an HLA-A2 molecule are also encompassed by the invention, as are isolated tumor rejection antigen precursors coded for by the previously described nucleic acid sequences.
The invention also involves an isolated nucleic acid molecule which codes for or is complementary to a nucleic acid molecule which codes for a tumor rejection antigen precursor 9a which is processed to a tumor rejection antigen presented by a human leukocyte antigen A2 (HLA-A2) molecule, wherein said tumor rejection antigen precursor comprises the amino acid sequence set forth in SEQ ID NO:1.
Also encompassed by the invention is a recombinant expression vector comprising an isolated nucleic acid molecule which codes for a tumor rejection antigen precursor which is processed to a tumor rejection antigen presented by a human leukocyte antigen A2 (HLA-A2) molecule, operably linked to a promoter, wherein said tumor rejection antigen precursor comprises the amino acid sequence set forth in SEQ ID N0:1.
The invention further provides an isolated tumor rejection antigen precursor molecule which is not a tyrosinase, and which comprises an amino acid sequence corresponding to a peptide which forms a complex with a human leukocyte antigen A2 (HLA-A2) molecule, said isolated tumor rejection antigen precursor molecule having an amino acid sequence encoded by the nucleotide sequence set forth in SEQ
ID NO:1.
Further encompassed by the invention is a polyclonal antibody which specifically binds to a tumor rejection antigen precursor wherein said tumor rejection antigen precursor comprises the amino acid sequence encoded by an isolated nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:1.
The invention as described herein has a number of uses, some of which are described herein. First, the identification of a tumor rejection antigen which is specifically presented by HLA-A2 molecules, as well as a nucleic acid molecule coding for its parallel tumor rejection antigen precursor permits the artisan to diagnose a disorder characterized by expression of the TRAP. These methods involve determining expression of the TRAP gene, and/or TRAs derived therefrom, such as TRA
presented by HLA-A2. Other TRAs may also be derived from the TRAPs of the invention and presented by different HLA
molecules. In the former situation, such determinations can 5 be carried out via any standard nucleic acid determination assay, including the polymerase chain reaction, or assaying with labelled hybridization probes. In the latter situation, assaying with binding partners for complexes of TRA and HLA, such as antibodies, is especially preferred.
The TRAP coded for is one which is processed to at least one tumor rejection antigen, or TRA, which is presented by HLA-A2 molecules on cell surfaces. The nucleic acid molecules of the invention may be, e.g., genomic DNA, ("gDNA"), complementary DNA ("cDNA"), or a form of RNA. The invention also involves isolated nucleic acid molecules which are complementary to the molecules described above. An especially preferred form of the invention is a molecule which contains the sequence set forth in SEQ ID NO: 1.
Also encompassed by the invention are vectors which contain the nucleic acid molecules of the invention, operably linked to a promoter. The vectors may also include a molecule coding for HLA-A2. As these two molecules, i.e., HLA-A2 and the TRAP are necessary to generate a cytolytic T cell response, the invention also encompasses expression systems where nucleic acid molecules coding for TRAP and for HLA-A2 are presented as separate portions in, e.g., a kit. The invention also encompasses cell lines transfected by the vectors described herein, be these prokaryotic cells, such as E. coli, or eukaryotic cells, such as Chinese hamster ovary ("CHO") or COS cells.
As indicated , the complexes of TRA and HLA-A2 provoke a cytolytic T cell response, and as such isolated complexes of the tumor rejection antigen and an HLA-A2 molecule are also encompassed by the invention, as are isolated tumor rejection antigen precursors coded for by the previously described nucleic acid sequences.
The invention also involves an isolated nucleic acid molecule which codes for or is complementary to a nucleic acid molecule which codes for a tumor rejection antigen precursor 9a which is processed to a tumor rejection antigen presented by a human leukocyte antigen A2 (HLA-A2) molecule, wherein said tumor rejection antigen precursor comprises the amino acid sequence set forth in SEQ ID NO:1.
Also encompassed by the invention is a recombinant expression vector comprising an isolated nucleic acid molecule which codes for a tumor rejection antigen precursor which is processed to a tumor rejection antigen presented by a human leukocyte antigen A2 (HLA-A2) molecule, operably linked to a promoter, wherein said tumor rejection antigen precursor comprises the amino acid sequence set forth in SEQ ID N0:1.
The invention further provides an isolated tumor rejection antigen precursor molecule which is not a tyrosinase, and which comprises an amino acid sequence corresponding to a peptide which forms a complex with a human leukocyte antigen A2 (HLA-A2) molecule, said isolated tumor rejection antigen precursor molecule having an amino acid sequence encoded by the nucleotide sequence set forth in SEQ
ID NO:1.
Further encompassed by the invention is a polyclonal antibody which specifically binds to a tumor rejection antigen precursor wherein said tumor rejection antigen precursor comprises the amino acid sequence encoded by an isolated nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:1.
The invention as described herein has a number of uses, some of which are described herein. First, the identification of a tumor rejection antigen which is specifically presented by HLA-A2 molecules, as well as a nucleic acid molecule coding for its parallel tumor rejection antigen precursor permits the artisan to diagnose a disorder characterized by expression of the TRAP. These methods involve determining expression of the TRAP gene, and/or TRAs derived therefrom, such as TRA
presented by HLA-A2. Other TRAs may also be derived from the TRAPs of the invention and presented by different HLA
molecules. In the former situation, such determinations can 5 be carried out via any standard nucleic acid determination assay, including the polymerase chain reaction, or assaying with labelled hybridization probes. In the latter situation, assaying with binding partners for complexes of TRA and HLA, such as antibodies, is especially preferred.
10 The isolation of the TRAP gene also makes it possible to isolate the TRAP molecule itself, especially TRAP molecules containing the amino acid sequence of SEQ ID NO: 1. These isolated molecules when presented as the TRA, or as complexes of TRA and HLA, such as HLA-A2, may be combined with materials such as adjuvants to produce vaccines useful in treating disorders characterized by expression of the TRAP molecule.
In addition, vaccines can be prepared from cells which present the TRA/HLA complexes on their surface, such as non-proliferative cancer cells, non-proliferative transfectants, etcetera. In all cases where cells are used as a vaccine, these can be cells transfected with coding sequences for one or both of the components necessary to prove a CTL response, or be cells which express both molecules without transfection.
Further, the TRAP molecule, its associated TRAs, as well as complexes of TRA and HLA, may be used to produce antibodies, using standard techniques well known to the art.
When "disorder" is used herein, it refers to any pathological condition where the tumor rejection antigen precursor is expressed. An example of such a disorder is cancer melanoma in particular.
Therapeutic approaches based upon the disclosure are premised on a response by a subject's immune system, leading to lysis of TRA presenting cells, such as HLA-A2 cells. One such approach is the administration of CTLs specific to the complex to a subject with abnormal cells of the phenotype at issue. it is within the skill of the artisan to develop such CTLs in vitro. Specifically, a sample of cells, such as blood -'O 94/21126 2158 ~ ~ PCT/US94/02487 , cells, are contacted to a cell presenting the complex and capable of provoking a specific CTL to proliferate. The target cell can be: a transfectant, such as a COS cell of the type described supra. These transfectants present the desired complex on their surface and, when combined with a CTL of interest, stimulate its proliferation. COS cells, such as those used herein are widely available, as are other suitable host cells.
To detail the therapeutic methodology, referred to as adoptive transfer (Greenberg, J. Immunol. 136 (5) : 1917 (1986) ;
Reddel et al., Science 257: 238 (7-10-92); Lynch et al., Eur.
J. Immunol. 21: 1403-1410 (1991); Kast et al., Cell 59: 603-614 (11-17-89)), cells presenting the desired complex are combined with CT:Ls leading to proliferation of the CTLs specific thereto. The proliferated CTLs are then administered to a subject with a cellular abnormality which is characterized by certain of the abnormal cells presenting the particular complex. The CTLs then lyse the abnormal cells, thereby achieving the desired therapeutic goal.
The foregoincj therapy assumes that at least some of the subject's abnormal cells present the HLA/TRA complex. This can be determined very easily, as the art is very familiar with methods for identifying cells which present a particular HLA molecule, as well as how to identify cells expressing DNA
containing the indlicated:sequences. Once isolated, such cells can be used with a sample of a subject's abnormal cells to determine lysis jiz vitro. If lysis is observed, then the use of specific CTLs in such a therapy may alleviate the condition associated with the abnormal cells. A less involved methodology examines the abnormal cells for HLA phenotyping, using standard assays, and determines expression via amplification using, e.g., PCR.
Adoptive transfer is not the only form of therapy that is available in accordance with the invention. CTLs can also be provoked in vivo, using a number of approaches. One approach, i.e., the use of non-proliferative cells expressing the complex, has been. elaborated upon supra. The cells used in I
WO 94/21126 ~~ PCT/US94/02487 this approach may be those that normally express the complex, such as irradiated melanoma cells or cells transfected with one or both of the genes necessary for presentation of the complex. Chen et al., Proc. Natl. Acad. Sci. USA 88: 110-114 (January, 1991) exemplifies this approach, showing the use of transfected cells expressing HPVE7 peptides in a therapeutic regime. Various cell types may be used. Similarly, vectors carrying one or both of the genes of interest may be used.
Viral or bacterial vectors are especially preferred. In these systems, the gene of interest is carried by, e.g., a Vaccinia virus or the bacteria BCG, and the materials de facto "infect"
host cells. The cells which result present the complex of interest, and are recognized by autologous CTLs, which then proliferate. A similar effect can be achieved by combining the tumor rejection antigen or the precursor itself with an adjuvant to facilitate incorporation into HLA-A2 presenting cells which present the HLA molecule of interest. The TRAP is processed to yield the peptide partner of the HLA molecule while the TRA is presented without the need for further processing.
Other aspects of the invention will be clear to the skilled artisan and need not be repeated here.
The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention.
'70 94/21126 PCT/US94/02487 ~ 5 8 4 4 (1) GENERAL INFORMi%TION:
(i) APPLICAN'I'S: Brichard, Vincent, Van Pel, Aline, Traversari, Catia, Wolfel, Thomas, Boon-Falleur, Thierry, De Plaen, Etienne (ii) TITLE OF INVENTION: ISOLATED NUCLEIC ACID SEQUENCE CODING
FOR A TUMOR REJECTION ANTIGEN PRECURSOR PROCESSED TO AT LEAST ONE
(iii) NUMBER O:F SEQUENCES: 2 (iv) CORRESPON'DENCE ADDRESS:
(A) ADDR:ESSEE: Felfe & Lynch (B) STREET: 805 Third Avenue (C) CITY: New York City (D) STATE: New York (E) COUN'TRY : USA
(F) ZIP: 10022 (v) COMPUTER :READABLE FORM:
(A) MEDI'UM TYPE: Diskette, 5.25 inch, 360 kb storage (B) COMP'UTER: IBM PS/2 (C) OPERATING SYSTEM: PC-DOS
(D) SOFTWARE: Wordperfect (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/032,978 (B) FILING DATE: 18-Mar-1993 (vii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Hanson., Norman D.
(B) REGISTRATION NUMBER: 30,946 (C) REFERENCE/DOCKET NUMBER: LUD 309 (viii) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212) 688-9200 (B) TELEFAX: (212) 838-3884 21596,1113 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 354 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOCY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Met Pro Arg Glu Asp Ala His Phe Ile Tyr Gly Tyr Pro Lys Lys Gly His Gly His Ser Tyr Thr Thr Ala Glu Glu Ala Ala Gly Ile Gly Ile Leu Thr Val Ile Leu Gly Val Leu Leu leu Ile Gly Cys Trp Tyr Cys Arg Arg Arg Asn Gly Tyr Arg Ala Leu Met Asp Lys Ser Leu His Val Gly Thr Gln Cys Ala Leu Thr Arg Arg Cys Pro Gln Glu Gly Phe Asp His Arg Asp Ser Lys Val Ser Leu Gln Glu Lys Asn Cys Glu Pro Val Val Pro Asn Ala Pro Pro Ala Tyr Glu Lys Leu Ser Ala Glu Gln Ser Pro Pro Pro Tyr Ser Pro "10 94/21126 8 4 4 PCT/US94/02487 (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 676 base pairs (B) TYPE: nucleic acid 5 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
AGCCATCTCT AATAATAAG'.C CAGTGTTAAA ATTTTAGTAG GTCCGCTAGC AGTACTAATC 600
In addition, vaccines can be prepared from cells which present the TRA/HLA complexes on their surface, such as non-proliferative cancer cells, non-proliferative transfectants, etcetera. In all cases where cells are used as a vaccine, these can be cells transfected with coding sequences for one or both of the components necessary to prove a CTL response, or be cells which express both molecules without transfection.
Further, the TRAP molecule, its associated TRAs, as well as complexes of TRA and HLA, may be used to produce antibodies, using standard techniques well known to the art.
When "disorder" is used herein, it refers to any pathological condition where the tumor rejection antigen precursor is expressed. An example of such a disorder is cancer melanoma in particular.
Therapeutic approaches based upon the disclosure are premised on a response by a subject's immune system, leading to lysis of TRA presenting cells, such as HLA-A2 cells. One such approach is the administration of CTLs specific to the complex to a subject with abnormal cells of the phenotype at issue. it is within the skill of the artisan to develop such CTLs in vitro. Specifically, a sample of cells, such as blood -'O 94/21126 2158 ~ ~ PCT/US94/02487 , cells, are contacted to a cell presenting the complex and capable of provoking a specific CTL to proliferate. The target cell can be: a transfectant, such as a COS cell of the type described supra. These transfectants present the desired complex on their surface and, when combined with a CTL of interest, stimulate its proliferation. COS cells, such as those used herein are widely available, as are other suitable host cells.
To detail the therapeutic methodology, referred to as adoptive transfer (Greenberg, J. Immunol. 136 (5) : 1917 (1986) ;
Reddel et al., Science 257: 238 (7-10-92); Lynch et al., Eur.
J. Immunol. 21: 1403-1410 (1991); Kast et al., Cell 59: 603-614 (11-17-89)), cells presenting the desired complex are combined with CT:Ls leading to proliferation of the CTLs specific thereto. The proliferated CTLs are then administered to a subject with a cellular abnormality which is characterized by certain of the abnormal cells presenting the particular complex. The CTLs then lyse the abnormal cells, thereby achieving the desired therapeutic goal.
The foregoincj therapy assumes that at least some of the subject's abnormal cells present the HLA/TRA complex. This can be determined very easily, as the art is very familiar with methods for identifying cells which present a particular HLA molecule, as well as how to identify cells expressing DNA
containing the indlicated:sequences. Once isolated, such cells can be used with a sample of a subject's abnormal cells to determine lysis jiz vitro. If lysis is observed, then the use of specific CTLs in such a therapy may alleviate the condition associated with the abnormal cells. A less involved methodology examines the abnormal cells for HLA phenotyping, using standard assays, and determines expression via amplification using, e.g., PCR.
Adoptive transfer is not the only form of therapy that is available in accordance with the invention. CTLs can also be provoked in vivo, using a number of approaches. One approach, i.e., the use of non-proliferative cells expressing the complex, has been. elaborated upon supra. The cells used in I
WO 94/21126 ~~ PCT/US94/02487 this approach may be those that normally express the complex, such as irradiated melanoma cells or cells transfected with one or both of the genes necessary for presentation of the complex. Chen et al., Proc. Natl. Acad. Sci. USA 88: 110-114 (January, 1991) exemplifies this approach, showing the use of transfected cells expressing HPVE7 peptides in a therapeutic regime. Various cell types may be used. Similarly, vectors carrying one or both of the genes of interest may be used.
Viral or bacterial vectors are especially preferred. In these systems, the gene of interest is carried by, e.g., a Vaccinia virus or the bacteria BCG, and the materials de facto "infect"
host cells. The cells which result present the complex of interest, and are recognized by autologous CTLs, which then proliferate. A similar effect can be achieved by combining the tumor rejection antigen or the precursor itself with an adjuvant to facilitate incorporation into HLA-A2 presenting cells which present the HLA molecule of interest. The TRAP is processed to yield the peptide partner of the HLA molecule while the TRA is presented without the need for further processing.
Other aspects of the invention will be clear to the skilled artisan and need not be repeated here.
The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention.
'70 94/21126 PCT/US94/02487 ~ 5 8 4 4 (1) GENERAL INFORMi%TION:
(i) APPLICAN'I'S: Brichard, Vincent, Van Pel, Aline, Traversari, Catia, Wolfel, Thomas, Boon-Falleur, Thierry, De Plaen, Etienne (ii) TITLE OF INVENTION: ISOLATED NUCLEIC ACID SEQUENCE CODING
FOR A TUMOR REJECTION ANTIGEN PRECURSOR PROCESSED TO AT LEAST ONE
(iii) NUMBER O:F SEQUENCES: 2 (iv) CORRESPON'DENCE ADDRESS:
(A) ADDR:ESSEE: Felfe & Lynch (B) STREET: 805 Third Avenue (C) CITY: New York City (D) STATE: New York (E) COUN'TRY : USA
(F) ZIP: 10022 (v) COMPUTER :READABLE FORM:
(A) MEDI'UM TYPE: Diskette, 5.25 inch, 360 kb storage (B) COMP'UTER: IBM PS/2 (C) OPERATING SYSTEM: PC-DOS
(D) SOFTWARE: Wordperfect (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/032,978 (B) FILING DATE: 18-Mar-1993 (vii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Hanson., Norman D.
(B) REGISTRATION NUMBER: 30,946 (C) REFERENCE/DOCKET NUMBER: LUD 309 (viii) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212) 688-9200 (B) TELEFAX: (212) 838-3884 21596,1113 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 354 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOCY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Met Pro Arg Glu Asp Ala His Phe Ile Tyr Gly Tyr Pro Lys Lys Gly His Gly His Ser Tyr Thr Thr Ala Glu Glu Ala Ala Gly Ile Gly Ile Leu Thr Val Ile Leu Gly Val Leu Leu leu Ile Gly Cys Trp Tyr Cys Arg Arg Arg Asn Gly Tyr Arg Ala Leu Met Asp Lys Ser Leu His Val Gly Thr Gln Cys Ala Leu Thr Arg Arg Cys Pro Gln Glu Gly Phe Asp His Arg Asp Ser Lys Val Ser Leu Gln Glu Lys Asn Cys Glu Pro Val Val Pro Asn Ala Pro Pro Ala Tyr Glu Lys Leu Ser Ala Glu Gln Ser Pro Pro Pro Tyr Ser Pro "10 94/21126 8 4 4 PCT/US94/02487 (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 676 base pairs (B) TYPE: nucleic acid 5 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
AGCCATCTCT AATAATAAG'.C CAGTGTTAAA ATTTTAGTAG GTCCGCTAGC AGTACTAATC 600
Claims (16)
1. An isolated nucleic acid molecule which codes for or is complementary to a nucleic acid molecule which codes for a tumor rejection antigen precursor which is processed to a tumor rejection antigen presented by a human leukocyte antigen A2 (HLA-A2) molecule, wherein said tumor rejection antigen precursor comprises the amino acid sequence set forth in SEQ
ID NO:1.
ID NO:1.
2. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid molecule codes for said tumor rejection antigen precursor.
3. The isolated nucleic acid molecule of claim 2, wherein said nucleic acid molecule is DNA.
4. The isolated nucleic acid molecule of claim 3, wherein said DNA is cDNA.
5. The isolated nucleic acid molecule of claim 4, comprising the nucleotide sequence of SEQ ID NO: 1.
6. Recombinant expression vector comprising an isolated nucleic acid molecule which codes for a tumor rejection antigen precursor which is processed to a tumor rejection antigen presented by a human leukocyte antigen A2 (HLA-A2) molecule, operably linked to a promoter, wherein said tumor rejection antigen precursor comprises the amino acid sequence set forth in SEQ ID NO:l.
7. Recombinant expression vector comprising the nucleotide sequence of SEQ ID NO:1.
8. Prokaryotic cell or eukaryote cell transformed or transfected with the isolated nucleic acid molecule of claim 1.
9. Prokaryotic cell or eukaryotic cell transformed or transfected with the recombinant expression vector of claim 6.
10. Prokaryotic cell or eukaryotic cell transformed or transfected with the recombinant expression vector of claim 7.
11. The prokaryotic cell or eukaryotic cell of claim 8, transfected with a nucleic acid molecule which codes for HLA-A2.
12. The recombinant expression vector of claim 6, further comprising a nucleic acid molecule which codes for HLA-A2.
13. Isolated tumor rejection antigen precursor coded for by the nucleic acid molecule of claim 2.
14. An isolated tumor rejection antigen precursor molecule which is not a tyrosinase, and which comprises an amino acid sequence corresponding to a peptide which forms a complex with a human leukocyte antigen A2 (HLA-A2) molecule, said isolated tumor rejection antigen precursor molecule having an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:1.
15. Composition of matter comprising the isolated tumor rejection antigen precursor of claim 14, and an adjuvant.
16. A polyclonal antibody which specifically binds to a tumor rejection antigen precursor wherein said tumor rejection antigen precursor comprises the amino acid sequence encoded by an isolated nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:1.
Applications Claiming Priority (3)
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|---|---|---|---|
| US08/032,978 | 1993-03-18 | ||
| US08/032,978 US5620886A (en) | 1993-03-18 | 1993-03-18 | Isolated nucleic acid sequence coding for a tumor rejection antigen precursor processed to at least one tumor rejection antigen presented by HLA-A2 |
| PCT/US1994/002487 WO1994021126A1 (en) | 1993-03-18 | 1994-03-09 | Nucleic acid coding for a tumor rejection antigen precursor |
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| CA2158446A1 CA2158446A1 (en) | 1994-09-29 |
| CA2158446C true CA2158446C (en) | 2009-01-06 |
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|---|---|---|---|
| CA002158446A Expired - Lifetime CA2158446C (en) | 1993-03-18 | 1994-03-09 | Nucleic acid coding for a tumor rejection antigen precursor |
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| US (2) | US5620886A (en) |
| EP (1) | EP0690675B1 (en) |
| JP (1) | JP3433322B2 (en) |
| CN (1) | CN1072719C (en) |
| AT (1) | ATE258379T1 (en) |
| AU (1) | AU675707B2 (en) |
| CA (1) | CA2158446C (en) |
| DE (2) | DE69433518D1 (en) |
| DK (1) | DK0690675T5 (en) |
| ES (1) | ES2210252T3 (en) |
| FI (1) | FI954360A0 (en) |
| NO (1) | NO953601L (en) |
| NZ (1) | NZ263348A (en) |
| PT (1) | PT690675E (en) |
| TW (1) | TW442568B (en) |
| WO (1) | WO1994021126A1 (en) |
| ZA (1) | ZA941812B (en) |
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| US5856091A (en) * | 1993-03-18 | 1999-01-05 | Ludwig Institute For Cancer Research | Isolated nucleic acid sequence coding for a tumor rejection antigen precursor processed to at least one tumor rejection antigen presented by HLA-A2 |
| EP1630229B1 (en) * | 1994-04-22 | 2013-04-03 | THE UNITED STATES OF AMERICA, as represented by the Secretary of the Department of Health and Human Services | Melanoma antigens |
| US5874560A (en) * | 1994-04-22 | 1999-02-23 | The United States Of America As Represented By The Department Of Health And Human Services | Melanoma antigens and their use in diagnostic and therapeutic methods |
| US5997870A (en) * | 1994-06-03 | 1999-12-07 | Ludwig Institute For Cancer Research | Isolated peptides which bind to HLA-B44 Molecules |
| US5821122A (en) * | 1995-06-07 | 1998-10-13 | Inserm (Institute Nat'l De La Sante Et De La Recherche . .) | Isolated nucleic acid molecules, peptides which form complexes with MHC molecule HLA-A2 and uses thereof |
| UA68327C2 (en) * | 1995-07-04 | 2004-08-16 | Gsf Forschungszentrum Fur Unwe | A recombinant mva virus, an isolated eukaryotic cell, infected with recombinant mva virus, a method for production in vitro of polypeptides with use of said cell, a method for production in vitro of virus parts (variants), vaccine containing the recombinant mva virus, a method for immunization of animals |
| US6096313A (en) * | 1996-02-09 | 2000-08-01 | Ludwig Institute For Cancer Research | Compositions containing immunogenic molecules and granulocyte-macrophage colony stimulating factor, as an adjuvant |
| FR2746110B1 (en) * | 1996-03-14 | 1998-04-17 | METHOD OF TREATING GENE THERAPY OF HUMAN TUMORS AND CORRESPONDING RECOMBINANT VIRUSES | |
| US5674749A (en) * | 1996-03-26 | 1997-10-07 | Ludwig Institute For Cancer Research | Monoclonal antibodies which bind to tumor rejection antigen precursor melan-A, and uses thereof |
| AU727028B2 (en) * | 1996-06-25 | 2000-11-30 | Ludwig Institute For Cancer Research | Brain glycogen phosphorylase cancer antigen |
| DE69839765D1 (en) | 1997-01-27 | 2008-09-04 | Ludwig Inst Cancer Res | LOCATION-1 TUMOR ASSOCIATED NUCLEIC ACIDS |
| US5879892A (en) * | 1997-04-25 | 1999-03-09 | Ludwig Institute For Cancer Research | Leukemia associated genes |
| JP2002500891A (en) * | 1998-01-26 | 2002-01-15 | ジェンザイム・コーポレーション | Antigen-specific cells, methods for producing these cells and uses thereof |
| US6245525B1 (en) | 1998-07-27 | 2001-06-12 | Ludwig Institute For Cancer Research | Tumor associated nucleic acids and uses therefor |
| US7001999B1 (en) | 1998-04-15 | 2006-02-21 | Ludwig Institute For Cancer Research | Tumor associated nucleic acids and uses therefor |
| US6770456B1 (en) | 1998-07-29 | 2004-08-03 | Ludwig Institute For Cancer Research | Endogenous retrovirus tumor associated nucleic acids and antigens |
| DE19913707A1 (en) * | 1999-03-26 | 2000-10-05 | Privates Inst Bioserv Gmbh | Immunosorbent for sepsis therapy |
| KR20020047249A (en) | 1999-10-19 | 2002-06-21 | 에드워드 에이. 맥더모트, 주니어 | MAGE-A12 Antigenic Peptides and Uses Thereof |
| US20030113919A1 (en) * | 2001-08-17 | 2003-06-19 | Aventis Pasteur, Ltd. | Immunogenic targets for melanoma |
| EP3020413A1 (en) | 2002-01-30 | 2016-05-18 | The Brigham and Women's Hospital, Inc. | Compositions and methods related to TIM-3, a TH1-specific cell surface molecule |
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| US5342774A (en) * | 1991-05-23 | 1994-08-30 | Ludwig Institute For Cancer Research | Nucleotide sequence encoding the tumor rejection antigen precursor, MAGE-1 |
| US5487974A (en) * | 1992-12-22 | 1996-01-30 | Ludwig Institute For Cancer-Research | Method for detecting complexes containing human leukocyte antigen A2 (HLA-A2) molecules and a tyrosinase drived peptide on abnormal cells |
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1994
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- 1994-03-09 ES ES94911498T patent/ES2210252T3/en not_active Expired - Lifetime
- 1994-03-09 PT PT94911498T patent/PT690675E/en unknown
- 1994-03-09 CA CA002158446A patent/CA2158446C/en not_active Expired - Lifetime
- 1994-03-09 AU AU63993/94A patent/AU675707B2/en not_active Expired
- 1994-03-09 DK DK94911498T patent/DK0690675T5/en active
- 1994-03-09 DE DE69433518A patent/DE69433518D1/en not_active Expired - Lifetime
- 1994-03-09 JP JP52108894A patent/JP3433322B2/en not_active Expired - Lifetime
- 1994-03-09 DE DE69433518T patent/DE69433518T4/en not_active Expired - Lifetime
- 1994-03-09 NZ NZ263348A patent/NZ263348A/en not_active IP Right Cessation
- 1994-03-09 AT AT94911498T patent/ATE258379T1/en active
- 1994-03-15 ZA ZA941812A patent/ZA941812B/en unknown
- 1994-03-18 CN CN94103425A patent/CN1072719C/en not_active Expired - Lifetime
- 1994-07-18 TW TW083106543A patent/TW442568B/en not_active IP Right Cessation
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1995
- 1995-03-03 US US08/398,409 patent/US5854203A/en not_active Expired - Lifetime
- 1995-09-13 NO NO953601A patent/NO953601L/en not_active Application Discontinuation
- 1995-09-15 FI FI954360A patent/FI954360A0/en unknown
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| AU675707B2 (en) | 1997-02-13 |
| CA2158446A1 (en) | 1994-09-29 |
| NO953601D0 (en) | 1995-09-13 |
| ZA941812B (en) | 1994-10-13 |
| JPH08507693A (en) | 1996-08-20 |
| WO1994021126A1 (en) | 1994-09-29 |
| FI954360A (en) | 1995-09-15 |
| NO953601L (en) | 1995-10-31 |
| AU6399394A (en) | 1994-10-11 |
| TW442568B (en) | 2001-06-23 |
| DK0690675T5 (en) | 2008-11-17 |
| NZ263348A (en) | 1996-06-25 |
| EP0690675B1 (en) | 2004-01-28 |
| US5854203A (en) | 1998-12-29 |
| PT690675E (en) | 2004-06-30 |
| CN1093406A (en) | 1994-10-12 |
| EP0690675A4 (en) | 1998-12-23 |
| DK0690675T3 (en) | 2004-06-01 |
| CN1072719C (en) | 2001-10-10 |
| ATE258379T1 (en) | 2004-02-15 |
| JP3433322B2 (en) | 2003-08-04 |
| DE69433518T2 (en) | 2004-06-24 |
| ES2210252T3 (en) | 2004-07-01 |
| DE69433518D1 (en) | 2004-03-04 |
| US5620886A (en) | 1997-04-15 |
| EP0690675A1 (en) | 1996-01-10 |
| FI954360A0 (en) | 1995-09-15 |
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