AN ANAPLASTIC LYMPHOMA KINASE (ALK) CANCER VACCINE AND METHODS OF USE
20230020894 · 2023-01-19
Assignee
Inventors
Cpc classification
A61K2039/6093
HUMAN NECESSITIES
A61K39/001196
HUMAN NECESSITIES
A61K31/4439
HUMAN NECESSITIES
A61K39/001196
HUMAN NECESSITIES
A61K31/4439
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
C12N9/12
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
A61K39/00
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
Provided herein are isolated anaplastic lymphoma kinase (ALK) peptides that are fragments of the cytoplasmic portion of an ALK protein shared by cancers having an ALK rearrangement and cancers expressing the ALK protein, that bind a human leukocyte antigen (HLA), and elicit an immune response against one or more ALK-positive cancers. Also provided are isolated ALK peptides that are modified with an amphiphilic conjugate to increase T-cell expansion and greatly enhance anti-tumor efficacy. The invention also provides polynucleotides encoding isolated ALK peptides, vaccines comprising an isolated ALK peptide or polynucleotide, immunogenic compositions thereof, and kits for administering the same. Methods of treatment and methods of generating an immune response in a subject by administering the ALK-specific peptide antigens, immunogens, vaccines, or immunogenic compositions thereof are provided.
Claims
1. An isolated anaplastic lymphoma kinase (ALK) peptide, wherein the peptide: i) is a fragment of the cytoplasmic portion of an ALK protein shared by cancers having an ALK rearrangement and cancers expressing the ALK protein; ii) is capable of binding a human leukocyte antigen (HLA); and iii) is capable of generating an immune response against one or more ALK-positive cancers, wherein the peptide does not include the amino acid sequence AMLDLLHVA.
2. An isolated anaplastic lymphoma kinase (ALK) peptide capable of generating an immune response against one or more ALK-positive cancers, wherein the ALK peptide comprises the amino acid sequence RPRPSQPSSL.
4. An isolated anaplastic lymphoma kinase (ALK) peptide capable of generating an immune response against one or more ALK-positive cancers, wherein the ALK peptide comprises the amino acid sequence IVRCIGVSL.
5. An isolated synthetic anaplastic lymphoma kinase (ALK) peptide capable of generating an immune response against one or more ALK-positive cancers, wherein the ALK peptide comprises the amino acid sequence VPRKNITLI.
6. An isolated synthetic anaplastic lymphoma kinase (ALK) peptide capable of generating an immune response against one or more ALK-positive cancers, wherein the ALK peptide comprises the amino acid sequence TAAEVSVRV.
7. The peptide of claim 1, wherein the peptide comprises an amino acid sequence that has at least about 95% identity to a sequence listed in Table 3, Table 4, or Table 5.
8. The peptide of claim 1, wherein the peptide comprises or consists of an amino acid sequence selected from those listed in Table 3, Table 4, or Table 5.
9. The peptide of claim 1, wherein the peptide comprises an amino acid sequence that has at least about 95% identity to an amino acid sequence selected from the group consisting of RPRPSQPSSL; IVRCIGVSL; VPRKNITLI; and TAAEVSVRV.
10. The peptide of any one of claims 1-9, wherein the peptide is conjugated to an amphiphile.
11. The peptide of claim 10, wherein the amphiphile is N-hydroxy succinimidyl ester-end-functionalized poly(ethylene glycol)-lipid (NHS-PEG2KDa-DSPE).
12. The peptide of any one of claims 1-11, wherein the one or more ALK-positive cancers is selected from the group consisting of non-small cell lung cancer (NSCLC), anaplastic large cell lymphoma (ALCL), neuroblastoma, B-cell lymphoma, thyroid cancer, colon cancer, breast cancer, inflammatory myofibroblastic tumors (IMT), renal carcinoma, esophageal cancer, and melanoma.
13. The peptide of claim 12, wherein the one or more ALK-positive cancers is non-small cell lung cancer (NSCLC).
14. The peptide of claim 12, wherein the one or more ALK-positive cancers is anaplastic large cell lymphoma (ALCL).
15. The peptide of claim 1, wherein the HLA is encoded by a HLA class I allele.
16. The peptide of claim 15, wherein the HLA class I allele is selected from those listed in Table 1, Table 2, or Table 3.
17. The peptide of any one of claim 15 or 16, wherein the HLA class I allele is HLA A*02:01, HLA A*68:02, HLA B*07:02, or HLA C*07:02.
18. The peptide of claim 1, wherein the ALK protein is a full-length human ALK protein.
19. The peptide of claim 1, wherein the ALK rearrangement is a nucleophosmin-ALK rearrangement (NPM-ALK) or an echinoderm microtubule-associate protein-like 4-ALK rearrangement (EML4-ALK).
20. A polynucleotide encoding the ALK peptide of any one of claims 1-19.
21. A vaccine comprising the polynucleotide of claim 20.
22. A vaccine comprising the ALK peptide of any one of claims 1-19.
23. A vaccine comprising two or more isolated synthetic anaplastic lymphoma kinase (ALK) peptides capable of generating an immune response against one or more ALK-positive cancers, wherein the two or more isolated ALK peptides are selected from those listed in Table 3, Table 4, or Table 5.
24. The vaccine of claim 23, wherein the two or more ALK peptides comprise two or more amino acid sequences selected from the following: AMLDLLHVA; RPRPSQPSSL; IVRCIGVSL; VPRKNITLI; and/or TAAEVSVRV.
25. The vaccine of any one of claim 23 or 24, wherein at least one of the two or more ALK peptides is conjugated to an amphiphile.
26. The vaccine of claim 25, wherein the amphiphile is N-hydroxy succinimidyl ester-end-functionalized poly(ethylene glycol)-lipid (NHS-PEG2KDa-DSPE).
27. An immunogenic composition comprising the vaccine of any one of claims 21-26 and a pharmaceutically acceptable carrier, diluent, or excipient.
28. The immunogenic composition of claim 27, further comprising an adjuvant.
29. The immunogenic composition of claim 28, wherein the adjuvant is a synthetic complex of carboxymethylcellulose, polyinosinic-polycytidylic acid, and double-stranded RNA (poly ICLC) or CpG oligonucleotides.
30. The immunogenic composition of claim 28, wherein the adjuvant is conjugated to an amphiphile.
31. The immunogenic composition of claim 30, wherein the amphiphile conjugated to the adjuvant is N-hydroxy succinimidyl ester-end-functionalized poly(ethylene glycol)-lipid (NHS-PEG2KDa-DSPE).
32. A method of treating an ALK-positive cancer in a subject, the method comprising administering to the subject an effective amount of the ALK peptide of any one of claims 1-11, the vaccine of any one of claims 21-26, or the immunogenic composition of any one of claims 27-31.
33. A method of treating a subject with one or more ALK-positive cancers, the method comprising administering to the subject an effective amount of the ALK peptide of any one of claims 1-11, the vaccine of any one of claims 21-26, or the immunogenic composition of any one of claims 27-31.
34. The method of any one of claim 32 or 33, further comprising administering, simultaneously or sequentially, to the subject an effective amount of one or more of an ALK inhibitor, immune checkpoint inhibitor, and/or tyrosine kinase inhibitor (TKI).
35. The method of claim 34, wherein the immune checkpoint inhibitor is selected from one or more of a programmed cell death protein 1 (PD-1) inhibitor, programmed death-ligand 1 (PD-L1) inhibitor, or cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) inhibitor.
36. The method of claim 35, wherein the PD-1 inhibitor is an anti-PD1 antibody.
37. The method of claim 34, wherein the ALK inhibitor is lorlatinib.
38. The method of claim 35, wherein the anti-PD1 antibody is administered in combination with lorlatinib.
39. The method of any one of claims 32-38, wherein an adjuvant is concomitantly administered to the subject.
40. The method of any one of claims 32-39, wherein the ALK-positive cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), anaplastic large cell lymphoma (ALCL), neuroblastoma, B-cell lymphoma, thyroid cancer, colon cancer, breast cancer, inflammatory myofibroblastic tumors (IMT), renal carcinoma, esophageal cancer, and melanoma.
41. The method of claim 40, wherein the ALK-positive cancer is non-small cell lung cancer (NSCLC).
42. The method of claim 40, wherein the ALK-positive cancer is anaplastic large cell lymphoma (ALCL).
43. The method of any one of claims 32-42, wherein the subject had at least one prior treatment with at least one tyrosine kinase inhibitor (TKI).
44. The method of any one of claims 32-43, wherein the step of administering comprises one or more doses of the ALK peptide, the vaccine, or the immunogenic composition.
45. The method of any one of claims 32-44, wherein the step of administering comprises at least six (6) prime doses and at least two (2) booster doses of the ALK peptide, the vaccine, or the immunogenic composition.
46. A method of generating an immune response in a subject comprising administering to the subject an effective amount of the ALK peptide of any one of claims 1-11, the vaccine of any one of claims 21-26, or the immunogenic composition of any one of claims 27-31.
47. The method of claim 46, wherein the immune response comprises producing T-lymphocytes.
48. A kit comprising an agent for administration to a subject with one or more ALK-positive cancers, wherein the agent comprises the ALK peptide of any one of claims 1-11, the vaccine of any one of claims 21-26, or the immunogenic composition of any one of claims 27-31.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0105] Lung cancer is the most common cause of cancer-related death worldwide, and the annual incidence of anaplastic lymphoma kinase (ALK) expressing non-small cell lung cancer (NSCLC) in the U.S. is about 8,000 cases. In these patients, treatment with ALK tyrosine kinase inhibitors (TKIs) fails to induce durable remissions. In this context, a successful ALK vaccine could lead to durable responses and greatly improve survival and quality of life for NSCLC patients. ALK represents an attractive target for vaccine development because of its oncogenicity, its immunogenicity, and its restricted expression to tumor tissue rather than healthy adult tissue. Importantly, use of a therapeutic ALK peptide vaccine could potentially be extended to many other cancer types which are driven by ALK rearrangements or activating mutations (i.e., ALK-positive cancers), such as anaplastic large cell lymphoma (ALCL), neuroblastoma, B-cell lymphoma, thyroid cancer, colon cancer, breast cancer, inflammatory myofibroblastic tumors (IMT), renal carcinoma, esophageal cancer, and melanoma. Therefore, a vaccine as described herein generated against the rearranged portion of ALK can both prevent the development of ALK-positive tumors and more effectively treat patients diagnosed with ALK-positive tumors.
[0106] As described below, the present invention features isolated ALK-specific immunogenic antigens, e.g., peptide antigens, derived from ALK-positive cell lines and immune cells from patients with ALK-positive cancers. Such immunogenic antigens are also referred to as “immunogens” herein. The ALK-specific immunogenic antigens elicit a potent immune response, e.g., in the form of reactive T-lymphocytes, following administration or delivery to, or introduction into, a subject, particularly, a human subject. The isolated ALK-specific immunogenic antigens may be used in methods to treat and/or reduce disease caused by oncogenic ALK gene fusions, rearrangements, duplications or mutations. The isolated ALK-specific immunogenic antigens may be conjugated to an amphiphilic tail in order to significantly increase T-cell expansion and greatly enhance anti-tumor efficacy.
[0107] The immunogenic ALK antigens described herein may be used in immunogenic compositions (e.g., ALK-specific vaccines) that treat ALK-positive cancers caused by oncogenic ALK gene fusions, rearrangements, duplications or mutations in a subject, particularly a human subject, to whom the immunogenic composition or vaccine, is administered. The vaccine elicits a potent ALK protein-specific T cell response that treats and/or protects against ALK-positive cancers in a subject. The antigens, immunogens, immunogenic compositions and vaccines, and pharmaceutical compositions thereof, of the invention provide an additional treatment option for patients that have either become resistant to or have failed to respond to prior and traditional therapies for ALK-positive cancers.
Identification of ALK Proteins
[0108] The use of computational algorithms has been successfully applied in recent studies to identify T-cell neoantigens in both human and mice. (Carreno B M, et al. Cancer immunotherapy. A dendritic cell vaccine increases the breadth and diversity of melanoma neoantigen-specific T-cells. Science. 2015; 348(6236):803-808; Gubin M M, et al. Checkpoint blockade cancer immunotherapy targets tumour-specific tumor antigens. Nature. 2014; 515(7528):577-581). However, these algorithms are almost exclusively based on the affinities of synthetic peptides, and do not necessarily consider other parameters such as antigen expression levels, intracellular processing, and the transport of the peptides prior to HLA binding. Altogether, these factors can significantly limit the accuracy of the current algorithms in predicting genuine T-cell epitopes.
[0109] By using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the peptides presented on the surface of HLA monoallelic cells, only 26% of the peptides that actually bind common HLA alleles were predicted by Immune Epitope Database (IEDB) algorithms, one of the most commonly used algorithms (www.iedb.org). (Abelin J G, et al. Mass Spectrometry Profiling of HLA-Associated Peptidomes in Mono-allelic Cells Enables More Accurate Epitope Prediction. Immunity. 2017; 46(2):315-326). The level of accuracy drops down to 0% for rare HLA alleles. Thus, current algorithms used to predict antigenic peptides are generally inaccurate and misleading as to which antigens are actually presented on tumor cell surfaces. (See Abelin J G, et al. Mass Spectrometry Profiling of HLA-Associated Peptidomes in Mono-allelic Cells Enables More Accurate Epitope Prediction. Immunity. 2017; 46(2):315-326).
[0110] For direct identification of ALK antigenic peptides effectively presented on the cell surface of tumor cells in the most common HLA haplotypes, the HLA monoallelic cell system and algorithms as provided in Abelin J G, et al. (Mass Spectrometry Profiling of HLA-Associated Peptidomes in Mono-allelic Cells Enables More Accurate Epitope Prediction. Immunity. 2017; 46(2):315-326), which is incorporated herein in its entirety, were adapted. To directly identify ALK peptides actually presented on the surface of ALK-expressing tumor cells, HLA-peptide complexes were pulled from ALK-expressing cell lines lysates and the HLA-bound peptides were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
[0111] The invention provides for a method for identifying the ALK-specific peptides provided herein as described in Example 1. In some embodiments, the ALK-expressing cell lines for use in identifying the ALK antigenic peptides provided herein encode specific HLA-alleles (e.g., HLA class I alleles) and may express or may be transduced with a construct to express an ALK fusion protein. In some embodiments, the ALK-expressing cells lines are generated as described in Abelin J G, et al. (Mass Spectrometry Profiling of HLA-Associated Peptidomes in Mono-allelic Cells Enables More Accurate Epitope Prediction. Immunity. 2017; 46(2):315-326), which is incorporated herein in its entirety. In some embodiments, the HLA is encoded by a HLA class I allele. Nonlimiting examples of HLA class I alleles expressed by ALK-expressing cell lines are provided in Table 1, Table 2 or Table 3. In some embodiments, the HLA class I allele is HLA A*02:01, HLA A*68:02, HLA B*07:02, or HLA C*07:02.
[0112] In some embodiments, the fusion protein is an ALK protein fused to an echinoderm microtubule-associated protein-like 4 (EML4) protein (ELM4-ALK). In some embodiments, the ELM4-ALK fusion protein is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a ELM4-ALK fusion protein in Homo Sapiens or a variant thereof. In some embodiments, the ELM4-ALK fusion protein is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary ELM4-ALK fusion protein amino acid sequence from Homo Sapiens (GenBank: BAM37627.1) as provided below:
TABLE-US-00012 1 mdgfagsldd sisaastsdv qdrlsalesr vqqqedeitv lkaaladvlr rlaisedhva 61 svkksvsskg qpspravipm scitngsgan rkpshtsavs iagketlssa aksgtekkke 121 kpqgqrekke eshsndqspq iraspspqps sqplqihrqt pesknatptk sikrpspaek 181 shnswensdd srnklskips tpklipkvtk tadkhkdvii nqegeyikmf mrgrpitmfi 241 psdvdnyddi rtelppeklk lewaygyrgk dcranvyllp tgeivyfias vvvlfnyeer 301 tqrhylghtd cvkclaihpd kiriatgqia gvdkdgrplq phvrvwdsvt lstlqiiglg 361 tfergvgcld fskadsgvhl cviddsnehm ltvwdwqrka kgaeikttne vvlavefhpt 421 dantiitcgk shiffwtwsg nsltrkqgif gkyekpkfvq claflgngdv ltgdsggvml 481 iwskttvept pgkgpkgvyq iskqikahdg svftlcqmrn gmlltgggkd rkiilwdhdl 541 npereiefsa srarlpghva adhppavyrr khqelqamqm elqspeykls klrtstimtd 601 ynpnycfagk tssisdlkev prknitlirg lghgafgevy egqvsgmpnd psplqvavkt 661 lpevcseqde ldflmealii skfnhqnivr cigvslqslp rfillelmag gdlksflret 721 rprpsqpssl amldllhvar diacgcqyle enhfihrdia arnclltcpg pgrvakigdf 781 gmardiyras yyrkggcaml pvkwmppeaf megiftsktd twsfgvllwe ifslgympyp 841 sksnqevlef vtsggrmdpp kncpgpvyri mtqcwqhqpe drpnfaiile rieyctqdpd 901 vintalpiey gplveeeekv pvrpkdpegv ppllvsqqak reeerspaap pplpttssgk 961 aakkptaaei svrvprgpav egghvnmafs qsnppselhk vhgsrnkpts lwnptygswf 1021 tekptkknnp iakkephdrg nlglegsctv ppnvatgrlp gaslllepss ltanmkevpl 1081 frlrhfpcgn vnygyqqqgl pleaatapga ghyedtilks knsmnqpgp
[0113] In some embodiments, the ELM4-ALK fusion protein is encoded by a nucleic acid sequence from Homo Sapiens that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to GenBank: AB274722.1 as provided below:
TABLE-US-00013 1 ggcggcgcgg cgcggcgctc gcggctgctg cctgggaggg aggccgggca ggcggctgag 61 cggcgcggct ctcaacgtga cggggaagtg gttcgggcgg ccgcggctta ctaccccagg 121 gcgaacggac ggacgacgga ggcgggagcc ggtagccgag ccgggcgacc tagagaacga 181 gcgggtcagg ctcagcgtcg gccactctgt cggtccgctg aatgaagtgc ccgcccctct 241 gagcccggag cccggcgctt tccccgcaag atggacggtt tcgccggcag tctcgatgat 301 agtatttctg ctgcaagtac ttctgatgtt caagatcgcc tgtcagctct tgagtcacga 361 gttcagcaac aagaagatga aatcactgtg ctaaaggcgg ctttggctga tgttttgagg 421 cgtcttgcaa tctctgaaga tcatgtggcc tcagtgaaaa aatcagtctc aagtaaaggc 481 caaccaagcc ctcgagcagt tattcccatg tcctgtataa ccaatggaag tggtgcaaac 541 agaaaaccaa gtcataccag tgctgtctca attgcaggaa aagaaactct ttcatctgct 601 gctaaaagtg gtacagaaaa aaagaaagaa aaaccacaag gacagagaga aaaaaaagag 661 gaatctcatt ctaatgatca aagtccacaa attcgagcat caccttctcc ccagccctct 721 tcacaacctc tccaaataca cagacaaact ccagaaagca agaatgctac tcccaccaaa 781 agcataaaac gaccatcacc agctgaaaag tcacataatt cttgggaaaa ttcagatgat 841 agccgtaata aattgtcgaa aataccttca acacccaaat taataccaaa agttaccaaa 901 actgcagaca agcataaaga tgtcatcatc aaccaagaag gagaatatat taaaatgttt 961 atgcgcggtc ggccaattac catgttcatt ccttccgatg ttgacaacta tgatgacatc 1021 agaacggaac tgcctcctga gaagctcaaa ctggagtggg catatggtta tcgaggaaag 1081 gactgtagag ctaatgttta ccttcttccg accggggaaa tagtttattt cattgcatca 1141 gtagtagtac tatttaatta tgaggagaga actcagcgac actacctggg ccatacagac 1201 tgtgtgaaat gccttgctat acatcctgac aaaattagga ttgcaactgg acagatagct 1261 ggcgtggata aagatggaag gcctctacaa ccccacgtca gagtgtggga ttctgttact 1321 ctatccacac tgcagattat tggacttggc acttttgagc gtggagtagg atgcctggat 1381 ttttcaaaag cagattcagg tgttcattta tgtgttattg atgactccaa tgagcatatg 1441 cttactgtat gggactggca gaagaaagca aaaggagcag aaataaagac aacaaatgaa 1501 gttgttttgg ctgtggagtt tcacccaaca gatgcaaata ccataattac atgcggtaaa 1561 tctcatattt tcttctggac ctggagcggc aattcactaa caagaaaaca gggaattttt 1621 gggaaatatg aaaagccaaa atttgtgcag tgtttagcat tcttggggaa tggagatgtt 1681 cttactggag actcaggtgg agtcatgctt atatggagca aaactactgt agagcccaca 1741 cctgggaaag gacctaaagt gtaccgccgg aagcaccagg agctgcaagc catgcagatg 1801 gagctgcaga gccctgagta caagctgagc aagctccgca cctcgaccat catgaccgac 1861 tacaacccca actactgctt tgctggcaag acctcctcca tcagtgacct gaaggaggtg 1921 ccgcggaaaa acatcaccct cattcggggt ctgggccatg gagcctttgg ggaggtgtat 1981 gaaggccagg tgtccggaat gcccaacgac ccaagccccc tgcaagtggc tgtgaagacg 2041 ctgcctgaag tgtgctctga acaggacgaa ctggatttcc tcatggaagc cctgatcatc 2101 agcaaattca accaccagaa cattgttcgc tgcattgggg tgagcctgca atccctgccc 2161 cggttcatcc tgctggagct catggcgggg ggagacctca agtccttcct ccgagagacc 2221 cgccctcgcc cgagccagcc ctcctccctg gccatgctgg accttctgca cgtggctcgg 2281 gacattgcct gtggctgtca gtatttggag gaaaaccact tcatccaccg agacattgct 2341 gccagaaact gcctcttgac ctgtccaggc cctggaagag tggccaagat tggagacttc 2401 gggatggccc gagacatcta cagggcgagc tactatagaa agggaggctg tgccatgctg 2461 ccagttaagt ggatgccccc agaggccttc atggaaggaa tattcacttc taaaacagac 2521 acatggtcct ttggagtgct gctatgggaa atcttttctc ttggatatat gccatacccc 2581 agcaaaagca accaggaagt tctggagttt gtcaccagtg gaggccggat ggacccaccc 2641 aagaactgcc ctgggcctgt ataccggata atgactcagt gctggcaaca tcagcctgaa 2701 gacaggccca actttgccat cattttggag aggattgaat actgcaccca ggacccggat 2761 gtaatcaaca ccgctttgcc gatagaatat ggtccacttg tggaagagga agagaaagtg 2821 cctgtgaggc ccaaggaccc tgagggggtt cctcctctcc tggtctctca acaggcaaaa 2881 cgggaggagg agcgcagccc agctgcccca ccacctctgc ctaccacctc ctctggcaag 2941 gctgcaaaga aacccacagc tgcagaggtc tctgttcgag tccctagagg gccggccgtg 3001 gaagggggac acgtgaatat ggcattctct cagtccaacc ctccttcgga gttgcacagg 3061 gtccacggat ccagaaacaa gcccaccagc ttgtggaacc caacgtacgg ctcctggttt 3121 acagagaaac ccaccaaaaa gaataatcct atagcaaaga aggagccaca cgagaggggt 3181 aacctggggc tggagggaag ctgtactgtc ccacctaacg ttgcaactgg gagacttccg 3241 ggggcctcac tgctcctaga gccctcttcg ctgactgcca atatgaagga ggtacctctg 3301 ttcaggctac gtcacttccc ttgtgggaat gtcaattacg gctaccagca acagggcttg 3361 cccttagaag ccgctactgc ccctggagct ggtcattacg aggataccat tctgaaaagc 3421 aagaatagca tgaaccagcc tgggccctga gctcggtcac acactcactt ctcttccttg 3481 ggatccctaa gaccgtggag gagagagagg caatcaatgg ctccttcaca aaccagagac 3541 caaatgtcac gttttgtttt gtgccaacct attttgaagt accaccaaaa aagctgtatt 3601 ttgaaaatgc tttagaaagg ttttgagcat gggttcatcc tattctttcg aaagaagaaa 3661 atatcataaa aatgagtgat aaatacaagg cccagatgtg gttgcataag gtttttatgc 3721 atgtttgttg tatacttcct tatgcttctt ttaaattgtg tgtgctctgc ttcaatgtag 3781 tcagaattag ctgcttctat gtttcatagt tggggtcata gatgtttcct tgccttgttg 3841 atgtggacat gagccatttg aggggagagg gaacggaaat aaaggagtta tttgtaatga 3901 aaaaaaaaaa aaaaaaaaaa aaaaaa
[0114] In some embodiments, the ALK-expressing cell line may include the B721.221 human lymphoblastic cell line, which does not express endogenous HLA class I (A, B and C) due to gamma-ray-induced mutations in the HLA complex. (Shimizu Y, DeMars R. Production of human cells expressing individual transferred HLA-A, -B, -C genes using an HLA-A, -B, -C null human cell line. J. Immunol. 1989; 142(9):3320-3328). In some embodiments, the B721.221 cell line is transduced with a construct encoding an EML4-ALK fusion protein. In some embodiments, the construct encodes EML4-ALK variant 1, the most frequent EML4-ALK fusion protein (Lin J J, et al. Impact of EML4-ALK Variant on Resistance Mechanisms and Clinical Outcomes in ALK-Positive Lung Cancer. J. Clin. Oncol. 2018: JCO2017762294). In one embodiment, the ELM4-ALK variant 1 fusion protein has an amino acid sequence from Homo Sapiens that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to GenBank: BAF73611.1 as provided below:
TABLE-US-00014 1 mdgfagsldd sisaastsdv qdrlsalesr vqqqedeitv lkaaladvlr rlaisedhva 61 svkksvsskg qpspravipm scitngsgan rkpshtsavs iagketlssa aksgtekkke 121 kpqgqrekke eshsndqspq iraspspqps sqplqihrqt pesknatptk sikrpspaek 181 shnswensdd srnklskips tpklipkvtk tadkhkdvii nqegeyikmf mrgrpitmfi 241 psdvdnyddi rtelppeklk lewaygyrgk dcranvyllp tgeivyfias vvvlfnyeer 301 tqrhylghtd cvkclaihpd kiriatgqia gvdkdgrplq phvrvwdsvt lstlqiiglg 361 tfergvgcld fskadsgvhl cviddsnehm ltvwdwqkka kgaeikttne vvlavefhpt 421 dantiitcgk shiffwtwsg nsltrkqgif gkyekpkfvq claflgngdv ltgdsggvml 481 iwskttvept pgkgpkvyrr khqelqamqm elqspeykls klrtstimtd ynpnycfagk 541 tssisdlkev prknitlirg lghgafgevy egqvsgmpnd psplqvavkt lpevcseqde 601 ldflmealii skfnhqnivr cigvslqslp rfillelmag gdlksflret rprpsqpssl 661 amldllhvar diacgcqyle enhfihrdia arnclltcpg pgrvakigdf gmardiyras 721 yyrkggcaml pvkwmppeaf megiftsktd twsfgvllwe ifslgympyp sksnqevlef 781 vtsggrmdpp kncpgpvyri mtqcwqhqpe drpnfaiile rieyctqdpd vintalpiey 841 gplveeeekv pvrpkdpegv ppllvsqqak reeerspaap pplpttssgk aakkptaaev 901 svrvprgpav egghvnmafs qsnppselhr vhgsrnkpts lwnptygswf tekptkknnp 961 iakkepherg nlglegsctv ppnvatgrlp gaslllepss ltanmkevpl frlrhfpcgn 1021 vnygyqqqgl pleaatapga ghyedtilks knsmnqpgp
[0115] In some embodiments, the fusion protein is an ALK protein fused to a nucleophosmin (NPM) protein (NPM-ALK). In some embodiments, ALK-expressing cell lines may include anaplastic large cell lymphoma (ALCL) cell lines encoding frequent HLA-alleles (e.g., Karpas-299, DEL, and SR-786). These ALCL cell lines express high levels of the NPM-ALK fusion protein. In some embodiments, the NPM-ALK fusion protein is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a NPM-ALK fusion protein in Homo Sapiens. In some embodiments, the NPM-ALK fusion protein is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary NPM-ALK fusion protein amino acid sequence from Homo Sapiens as provided below (ALK cytoplasmic portion in bold font):
TABLE-US-00015 MEDSMDMDMSPLRPQNYLFGCELKADKDYHFKVDNDENEHQLSLRTVSLG AGAKDELHIVEAEAMNYEGSPIKVTLATLKMSVQPTVSLGGFEITPPVVL RLKCGSGPVHISGQHLVVYRRKHQELQAMQMELQSPEYKLSKLRTSTIMT DYNPNYCFAGKTSSISDLKEVPRKNNTLIRGLGHGAFGEVYEGQVSGMPN DPSPLQVAVKTLPEVCSEQDELDFLMEALIISKFNHQNIVRCIGVSLQSL PRFILLELMAGGDLKSFLRETRPRPSQPSSLAMLDLLHVARDIACGCQYL EENHFIHRDIAARNCLLTCPGPGRVAKIGDFGMARDIYRASYYRKGGCAM LPVKWMPPEAFMEGIFTSKTDTWSFGVLLWEIFSLGYMPYPSKSNQEVLE FVTSGGRMDPPKNCPGPVYRIMTQCWQHQPEDRPNFAIILERIEYCTQDP DVINTALPIEYGPLVEEEEKVPVRPKDPEGVPPLLVSQQAKREEERSPAA PPPLPTTSSGKAAKKPTAAEVSVRVPRGPAVEGGHVNMAFSQSNPPSELH RVHGSRNKPTSLWNPTYGSWFTEKPTKKNNPIAKKEPHERGNLGLEGSCT VPPNVATGRLPGASLLLEPSSLTANMKEVPLFRLRHFPCGNVNYGYQQQG LPLEAATAPGAGHYEDTILKSKNSMNQPGP
[0116] In some embodiments, the NPM-ALK fusion protein is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary NPM-ALK fusion protein amino acid sequence from Homo Sapiens (GenBank: AAA58698.1) as provided below:
TABLE-US-00016 1 medsmdmdms plrpqnylfg celkadkdyh fkvdndeneh qlslrtvslg agakdelhiv 61 eaeamnyegs pikvtlatlk msvqptvslg gfeitppvvl rlkcgsgpvh isgqhlvvyr 121 rkhqelqamq melqspeykl sklrtstimt dynpnycfag ktssisdlke vprknitlir 181 glghgafgev yegqvsgmpn dpsplqvavk tlpevcseqd eldflmeali iskfnhqniv 241 rcigvslqsl prfillelma ggdlksflre trprpsqpss lamldllhva rdiacgcqyl 301 eenhfihrdi aarnclltcp gpgrvakigd fgmardiyra syyrkggcam lpvkwmppea 361 fmegiftskt dtwsfgvllw eifslgympy psksnqevle fvtsggrmdp pkncpgpvyr 421 imtqcwqhqp edrpnfaiil erieyctqdp dvintalpie ygplveeeek vpvrpkdpeg 481 vppllvsqqa kreeerspaa ppplpttssg kaakkptaae vsvrvprgpa vegghvnmaf 541 sqsnppselh kvhgsrnkpt slwnptygsw ftekptkknn piakkephdr gnlglegsct 601 vppnvatgrl pgasllleps sltanmkevp lfrlrhfpcg nvnygyqqqg lpleaatapg 661 aghyedtilk sknsmnqpgp
[0117] In some embodiments, the NPM-ALK fusion protein is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary NPM-ALK fusion protein amino acid sequence from Homo Sapiens as provided below:
TABLE-US-00017 M E D S M D M D M S P L R P Q N Y L F G C E L K A D K D Y H F K V D N D E N E H Q L S L R T V S L G A G A K D E L H I V E A E A M N Y E G S P I K V T L A T L K M S V Q P T V S L G G F E I T P P V V L R L K C G S G P V H I S G Q H L V V Y R R K H Q E L Q A M Q M E L Q S P E Y K L S K L R T S T I M T D Y N P N Y C F A G K T S S I S D L K E V P R K N I T L I R G L G H G A F G E V Y E G Q V S G M P N D P S P L Q V A V K T L P E V C S E Q D E L D F L M E A L I I S K F N H Q N I V R C I G V S L Q S L P R F I L L E L M A G G D L K S F L R E T R P R P S Q P S S L A M L D L L H V A R D I A C G C Q Y L E E N H F I H R D I A A R N C L L T C P G P G R V A K I G D F G M A R D I Y R A S Y Y R K G G C A M L P V K W M P P E A F M E G I F T S K T D T W S F G V L L W E I F S L G Y M P Y P S K S N Q E V L E F V T S G G R M D P P K N C P G P V Y R I M T Q C W Q H Q P E D R P N F A I I L E R I E Y C T Q D P D V I N T A L P I E Y G P L V E E E E K V P V R P K D P E G V P P L L V S Q Q A K R E E E R S P A A P P P L P T T S S G K A A K K P T A A E V S V R V P R G P A V E G G H V N M A F S Q S N P P S E L H K V H G S R N K P T S L W N P T Y G S W F T E K P T K K N N P I A K K E P H D R G N L G L E G S C T V P P N V A T G R L P G A S L L L E P S S L T A N M K E V P L F R L R H F P C G N V N Y G Y Q Q Q G L P L E A A T A P G A G H Y E D T I L K S K N S M N Q P G P *
[0118] In some embodiments, an exemplary NPM-ALK fusion protein is encoded by a nucleic acid sequence from Homo Sapiens that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the sequence provided below:
TABLE-US-00018 1 atggaagattcgatggacatggacatgagccccctgaggccccagaactatcttttcggt 60 61 tgtgaactaaaggccgacaaagattatcactttaaggtggataatgatgaaaatgagcac 120 121 cagttatctttaagaacggtcagtttaggggctggtgcaaaggatgagttgcacattgtt 180 181 gaagcagaggcaatgaattacgaaggcagtccaattaaagtaacactggcaactttgaaa 240 241 atgtctgtacagccaacggtttcccttgggggctttgaaataacaccaccagtggtctta 300 301 aggttgaagtgtggttcagggccagtgcatattagtggacagcacttagtagtgtaccgc 360 361 cggaagcaccaggagctgcaagccatgcagatggagctgcagagccctgagtacaagctg 420 421 agcaagctccgcacctcgaccatcatgaccgactacaaccccaactactgctttgctggc 480 481 aagacctcctccatcagtgacctgaaggaggtgccgcggaaaaacatcaccctcattcgg 540 541 ggtctgggccatggcgcctttggggaggtgtatgaaggccaggtgtccggaatgcccaac 600 601 gacccaagccccctgcaagtggctgtgaagacgctgcctgaagtgtgctctgaacaggac 660 661 gaactggatttcctcatggaagccctgatcatcagcaaattcaaccaccagaacattgtt 720 721 cgctgcattggggtgagcctgcaatccctgccccggttcatcctgctggagctcatggcg 780 781 gggggagacctcaagtccttcctccgagagacccgccctcgcccgagccagccctcctcc 840 841 ctggccatgctggaccttctgcacgtggctcgggacattgcctgtggctgtcagtatttg 900 901 gaggaaaaccacttcatccaccgagacattgctgccagaaactgcctcttgacctgtcca 960 961 ggccctggaagagtggccaagattggagacttcgggatggcccgagacatctacagggcg 1020 1021 agctactatagaaagggaggctgtgccatgctgccagttaagtggatgcccccagaggcc 1080 1081 ttcatggaaggaatattcacttctaaaacagacacatggtcctttggagtgctgctatgg 1140 1141 gaaatcttttctcttggatatatgccataccccagcaaaagcaaccaggaagttctggag 1200 1201 tttgtcaccagtggaggccggatggacccacccaagaactgccctgggcctgtataccgg 1260 1261 ataatgactcagtgctggcaacatcagcctgaagacaggcccaactttgccatcattttg 1320 1321 gagaggattgaatactgcacccaggacccggatgtaatcaacaccgctttgccgatagaa 1380 1381 tatggtccacttgtggaagaggaagagaaagtgcctgtgaggcccaaggaccctgagggg 1440 1441 gttcctcctctcctggtctctcaacaggcaaaacgggaggaggagcgcagcccagctgcc 1500 1501 ccaccacctctgcctaccacctcctctggcaaggctgcaaagaaacccacagctgcagag 1560 1561 gtctctgttcgagtccctagagggccggccgtggaagggggacacgtgaatatggcattc 1620 1621 tctcagtccaaccctccttcggagttgcacaaggtccacggatccagaaacaagcccacc 1680 1681 agcttgtggaacccaacgtacggctcctggtttacagagaaacccaccaaaaagaataat 1740 1741 cctatagcaaagaaggagccacacgacaggggtaacctggggctggagggaagctgtact 1800 1801 gtcccacctaacgttgcaactgggagacttccgggggcctcactgctcctagagccctct 1860 1861 tcgctgactgccaatatgaaggaggtacctctgttcaggctacgtcacttcccttgtggg 1920 1921 aatgtcaattacggctaccagcaacagggcttgcccttagaagccgctactgcccctgga 1980 1981 gctggtcattacgaggataccattctgaaaagcaagaatagcatgaaccagcctgggccc 2040 2041 tga 2043
ALK Immunogenic Polypeptides
[0119] The present invention features the identification of ALK antigens and immunogenic polypeptides (immunogens) with the ability to generate an immune response so as to treat a disease and its symptoms, either prophylactically or therapeutically, caused by oncogenic ALK gene fusions, rearrangements, duplications or mutations (e.g., ALK-positive cancers) following administration and delivery to a susceptible subject. It will also be appreciated that the isolated ALK antigen proteins as described herein and used as immunogens elicit an immune response, e.g., producing T-lymphocytes, in a subject. The ALK antigens and immunogens of the invention may be incorporated into a pharmaceutical composition, immunogenic composition, or vaccine as provided herein.
[0120] In some embodiments, the isolated ALK antigen protein elicits a protective immune response against at least one, more than one, or all types of ALK-positive cancers.
[0121] In some embodiments, the disease caused by oncogenic ALK gene fusions, rearrangements, duplications or mutations is an ALK-positive cancer. Nonlimiting examples of ALK-positive cancers include non-small cell lung cancer (NSCLC), anaplastic large cell lymphoma (ALCL), neuroblastoma, B-cell lymphoma, thyroid cancer, colon cancer, breast cancer, inflammatory myofibroblastic tumors (IMT), renal carcinoma, esophageal cancer, melanoma, or a combination thereof. In some embodiments, the ALK-positive cancer is non-small cell lung cancer (NSCLC). In some embodiments, the ALK-positive cancer is anaplastic large cell lymphoma (ALCL).
ALK Antigens and Immunogens
[0122] The present invention provides herein ALK antigens and immunogens capable of generating an immune response against one or more diseases caused by oncogenic ALK gene fusions, rearrangements, duplications or mutations (e.g., ALK-positive cancers). An ALK antigen or immunogen as described herein is a polypeptide, peptide, or antibody-binding portion thereof. In some embodiments, the ALK antigen or immunogen is a protein or fragment thereof having an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an anaplastic lymphoma kinase (ALK) capable of inducing an immune response in an immunized subject. In some embodiments, the ALK antigen or immunogen is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the ALK protein in Homo Sapiens. In some embodiments, the ALK antigen or immunogen is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the full-length amino acid sequence from Homo Sapiens as provided below (ALK cytoplasmic portion in bold font):
TABLE-US-00019 TASSGGMGAIGLLWLLPLLLSTAAVGSGMGTGQRAGSPAAGPPLQPREP LSYSRLQRKSLAVDEVVPSLFRVYARDLLLPPSSSELKAGRPEARGSLA LDCAPLLRLLGPAPGVSWTAGSPAPAEARTLSRVLKGGSVRKLRRAKQL VLELGEEAILEGCVGPPGEAAVGLLQFNLSELFSWWIRQGEGRLRIRLM PEKKASEVGREGRLSAAIRASQPRLLFQIFGTGHSSLESPTNMPSPSPD YFTWNLTWIMKDSFPFLSHRSRYGLECSFDFPCELEYSPPLHDLRNQSW SWRRIPSEEASQMDLLDGPGAERSKEMPRGSFLLLNTSADSKHTILSPW MRSSSEHCTLAVSVHRHLQPSGRYIAQLLPHNEAAREILLMPTPGKHGW TVLQGRIGRPDNPFRVALEYISSGNRSLSAVDFFALKNCSEGTSPGSKM ALQSSFTCWNGTVLQLGQACDFHQDCAQGEDESQMCRKLPVGFYCNFED GFCGWTQGTLSPHTPQWQVRTLKDARFQDHQDHALLLSTTDVPASESAT VTSATFPAPIKSSPCELRMSWLIRGVLRGNVSLVLVENKTGKEQGRMVW HVAAYEGLSLWQWMVLPLLDVSDRFWLQMVAWWGQGSRAIVAFDNISIS LDCYLTISGEDKILQNTAPKSRNLFERNPNKELKPGENSPRQTPIFDPT VHWLFTTCGASGPHGPTQAQCNNAYQNSNLSVEVGSEGPLKGIQIWKVP ATDTYSISGYGAAGGKGGKNTMMRSHGVSVLGIFNLEKDDMLYILVGQQ GEDACPSTNQLIQKVCIGENNVIEEEIRVNRSVHEWAGGGGGGGGATYV FKMKDGVPVPLIIAAGGGGRAYGAKTDTFHPERLENNSSVLGLNGNSGA AGGGGGWNDNTSLLWAGKSLQEGATGGHSCPQAMKKWGWETRGGFGGGG GGCSSGGGGGGYIGGNAASNNDPEMDGEDGVSFISPLGILYTPALKVME GHGEVNIKHYLNCSHCEVDECHMDPESHKVICFCDHGTVLAEDGVSCIV SPTPEPHLPLSLILSVVTSALVAALVLAFSGIMIVYRRKHQELQAMQME LQSPEYKLSKLRTSTIMTDYNPNYCFAGKTSSISDLKEVPRKNITLIRG LGHGAFGEVYEGQVSGMPNDPSPLQVAVKTLPEVCSEQDELDFLMEALI ISKFNHQNIVRCIGVSLQSLPRFILLELMAGGDLKSFLRETRPRPSQPS SLAMLDLLHVARDIACGCQYLEENHFIHRDIAARNCLLTCPGPGRVAKI GDFGMARDIYRASYYRKGGCAMLPVKWMPPEAFMEGIFTSKTDTWSFGV LLWEIFSLGYMPYPSKSNQEVLEFVTSGGRMDPPKNCPGPVYRIMTQCW QHQPEDRPNFAIILERIEYCTQDPDVINTALPIEYGPLVEEEEKVPVRP KDPEGVPPLLVSQQAKREEERSPAAPPPLPTTSSGKAAKKPTAAEISVR VPRGPAVEGGHVNMAFSQSNPPSELHKVHGSRNKPTSLWNPTYGSWFTE KPTKKNNPIAKKEPHDRGNLGLEGSCTVPPNVATGRLPGASLLLEPSSL TANMKEVPLFRLRHFPCGNVNYGYQQQGLPLEAATAPGAGHYEDTILKS KNSMNQPGP
[0123] In some embodiments, the ALK antigen or immunogen is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the full-length ALK amino acid sequence from Homo Sapiens as provided below:
TABLE-US-00020 G A A A V V A A G T S R R L C S E G R G A P R C F P A A L W S A T Q S R G R * * W V R R G R Q D F G R P C P E R P Q L L P P G P L Q C L R T L R S R G A G E S K D A A N L R S A G A G I H A Q K F S R Q T V R S L P A A E R * L E G A Q D G S L R P R F P P R P G R R A W R S Q K E R K R R P G Q R A A A G S R R S Q P * K L Q R L E A A P R G D R P Q L R L R G A G E D G T Q L P P P F N H S S S S V P S A A S Y R R G R G T R R G E R E A Q G P S Q * A Q C A * V S L D S P L S F Q V C F I * T P A R L R A V G G K Q E T C A H A Q S S G D Q V E G A A G Y Q G L F R A S S H L G E S E G * G W A R R A V * T A S S G G M G A I G L L W L L P L L L S T A A V G S G M G T G Q R A G S P A A G P P L Q P R E P L S Y S R L Q R K S L A V D F V V P S L F R V Y A R D L L L P P S S S E L K A G R P E A R G S L A L D C A P L L R L L G P A P G V S W T A G S P A P A E A R T L S R V L K G G S V R K L R R A K Q L V L E L G E E A I L E G C V G P P G E A A V G L L Q F N L S E L F S W W I R Q G E G R L R I R L M P E K K A S E V G R E G R L S A A I R A S Q P R L L F Q I F G T G H S S L E S P T N M P S P S P D Y F T W N L T W I M K D S F P F L S H R S R Y G L E C S F D F P C E L E Y S P P L H D L R N Q S W S W R R I P S E E A S Q M D L L D G P G A E R S K E M P R G S F L L L N T S A D S K H T I L S P W M R S S S E H C T L A V S V H R H L Q P S G R Y I A Q L L P H N E A A R E I L L M P T P G K H G W T V L Q G R I G R P D N P F R V A L E Y I S S G N R S L S A V D F F A L K N C S E G T S P G S K M A L Q S S F T C W N G T V L Q L G Q A C D F H Q D C A Q G E D E S Q M C R K L P V G F Y C N F E D G F C G W T Q G T L S P H T P Q W Q V R T L K D A R F Q D H Q D H A L L L S T T D V P A S E S A T V T S A T F P A P I K S S P C E L R M S W L I R G V L R G N V S L V L V E N K T G K E Q G R M V W H V A A Y E G L S L W Q W M V L P L L D V S D R F W L Q M V A W W G Q G S R A I V A F D N I S I S L D C Y L T I S G E D K I L Q N T A P K S R N L F E R N P N K E L K P G E N S P R Q T P I F D P T V H W L F T T C G A S G P H G P T Q A Q C N N A Y Q N S N L S V E V G S E G P L K G I Q I W K V P A T D T Y S I S G Y G A A G G K G G K N T M M R S H G V S V L G I F N L E K D D M L Y I L V G Q Q G E D A C P S T N Q L I Q K V C I G E N N V I E E E I R V N R S V H E W A G G G G G G G G A T Y V F K M K D G V P V P L I I A A G G G G R A Y G A K T D T F H P E R L E N N S S V L G L N G N S G A A G G G G G W N D N T S L L W A G K S L Q E G A T G G H S C P Q A M K K W G W E T R G G F G G G G G G C S S G G G G G G Y I G G N A A S N N D P E M D G E D G V S F I S P L G I L Y T P A L K V M E G H G E V N I K H Y L N C S H C E V D E C H M D P E S H K V I C F C D H G T V L A E D G V S C I V S P T P E P H L P L S L I L S V V T S A L V A A L V L A F S G I M I V Y R R K H Q E L Q A M Q M E L Q S P E Y K L S K L R T S T I M T D Y N P N Y C F A G K T S S I S D L K E V P R K N I T L I R G L G H G A F G E V Y E G Q V S G M P N D P S P L Q V A V K T L P E V C S E Q D E L D F L M E A L I I S K F N H Q N I V R C I G V S L Q S L P R F I L L E L M A G G D L K S F L R E T R P R P S Q P S S L A M L D L L H V A R D I A C G C Q Y L E E N H F I H R D I A A R N C L L T C P G P G R V A K I G D F G M A R D I Y R A S Y Y R K G G C A M L P V K W M P P E A F M E G I F T S K T D T W S F G V L L W E I F S L G Y M P Y P S K S N Q E V L E F V T S G G R M D P P K N C P G P V Y R I M T Q C W Q H Q P E D R P N F A I I L E R I E Y C T Q D P D V I N T A L P I E Y G P L V E E E E K V P V R P K D P E G V P P L L V S Q Q A K R E E E R S P A A P P P L P T T S S G K A A K K P T A A E I S V R V P R G P A V E G G H V N M A F S Q S N P P S E L H K V H G S R N K P T S L W N P T Y G S W F T E K P T K K N N P I A K K E P H D R G N L G L E G S C T V P P N V A T G R L P G A S L L L E P S S L T A N M K E V P L F R L R H F P C G N V N Y G Y Q Q Q G L P L E A A T A P G A G H Y E D T I L K S K N S M N Q P G P * A R S H T H F S S L G S L R P W R R E R Q W L L H K P E T K C H V L F C A N L F * S T T K K A V F * K C F R K V L S M G S S Y S F E R R K Y H K N E * * I Q G P D V V A * G F Y A C L L Y T S L C F F Q I V C A L L Q C S Q N * L L L C F I V G V I D V S L P C * C G H E P F E G R G N G N K G V I C N D *
[0124] In some embodiments, ALK antigen or immunogen amino acid sequence comprises an amino acid sequence that is at least 95%, at least 98%, at least 99%, or 100% identical to a sequence provided in Table 3, Table 4, or Table 5. In some embodiments, the ALK antigen or immunogen comprises an amino acid sequence that is at least 95%, at least 98%, at least 99%, or 100% identical to: RPRPSQPSSL. In some embodiments, the ALK antigen or immunogen comprises an amino acid sequence that is at least 95%, at least 98%, at least 99%, or 100% identical to: IVRCIGVSL. In some embodiments, the ALK antigen or immunogen comprises an amino acid sequence that is at least 95%, at least 98%, at least 99%, or 100% identical to: VPRKNITLI. In some embodiments, the ALK antigen or immunogen comprises an amino acid sequence that is at least 95%, at least 98%, at least 99%, or 100% identical to: TAAEVSVRV. In some embodiments, the ALK antigen or immunogen comprises an amino acid sequence that is at least 95%, at least 98%, at least 99%, or 100% identical to: AMLDLLHVA.
[0125] In some embodiments, the ALK antigen or immunogen is conjugated to an amphiphile or amphiphilic tail (see, e.g.,
[0126] In some embodiments, the ALK antigen or immunogen conjugated to an amphiphile or amphiphilic tail comprises an amino acid sequence from Table 3. In some embodiments, the ALK antigen or immunogen conjugated to an amphiphile or amphiphilic tail comprises an amino acid sequence from Table 4. In some embodiments, the ALK antigen or immunogen conjugated to an amphiphile or amphiphilic tail comprises an amino acid sequence from Table 5. In some embodiments, the ALK antigen or immunogen conjugated to an amphiphile or amphiphilic tail comprises flanking amino acid sequences. In some embodiments, the flanking amino acid sequences are on either side or on both sides of the ALK antigen or immunogen sequence. In some embodiments, the ALK antigen or immunogen conjugated to an amphiphile or amphiphilic tail comprises a central core amino acid sequence with flanking amino acid sequences on both sides of the core. In some embodiments, the core amino acid sequence is about 9 to 10 amino acids in length. In some embodiments, the flanking amino acid sequences are between 5 to 15 amino acids. In some embodiments, the ALK antigen or immunogen conjugated to an amphiphile or amphiphilic tail comprises an amino acid sequence that is about 9 to about 30 amino acids in length. In some embodiments, the ALK antigen or immunogen conjugated to an amphiphile or amphiphilic tail comprises an amino acid sequence from Table 3 with flanking amino acid sequences.
[0127] In some embodiments, the ALK antigen or immunogen is a polynucleotide sequence. In some embodiments, the ALK antigen or immunogen is a polynucleotide sequence that encodes a polypeptide or peptide antigen or fragment thereof as described herein. In some embodiments, the nucleic acid molecule encoding an ALK polypeptide or fragment thereof (antigen or antigen protein or immunogen) is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the full-length ALK nucleic acid sequence from Homo Sapiens provided below:
TABLE-US-00021 2 ggggcggcagcggtggtagcagctggtacctcccgccgcctctgttcggagggtcgcggg 61 62 gcaccgaggtgctttccggccgccctctggtcggccacccaaagccgcgggcgctgatga 121 122 tgggtgaggagggggcggcaagatttcgggcgcccctgccctgaacgccctcagctgctg 181 182 ccgccggggccgctccagtgcctgcgaactctgaggagccgaggcgccggtgagagcaag 241 242 gacgctgcaaacttgcgcagcgcgggggctgggattcacgcccagaagttcagcaggcag 301 302 acagtccgaagccttcccgcagcggagagatagcttgagggtgcgcaagacggcagcctc 361 362 cgccctcggttcccgcccagaccgggcagaagagcttggaggagccaaaaggaacgcaaa 421 422 aggcggccaggacagcgtgcagcagctgggagccgccgttctcagccttaaaagttgcag 481 482 agattggaggctgccccgagaggggacagaccccagctccgactgcggggggcaggagag 541 542 gacggtacccaactgccacctcccttcaaccatagtagttcctctgtaccgagcgcagcg 601 602 agctacagacgggggcgcggcactcggcgcggagagcgggaggctcaaggtcccagccag 661 662 tgagcccagtgtgcttgagtgtctctggactcgcccctgagcttccaggtctgtttcatt 721 722 tagactcctgctcgcctccgtgcagttgggggaaagcaagagacttgcgcgcacgcacag 781 782 tcctctggagatcaggtggaaggagccgctgggtaccaaggactgttcagagcctcttcc 841 842 catctcggggagagcgaagggtgaggctgggcccggagagcagtgtaaacggcctcctcc 901 902 ggcgggatgggagccatcgggctcctgtggctcctgccgctgctgctttccacggcagct 961 962 gtgggctccgggatggggaccggccagcgcgcgggctccccagctgcggggccgccgctg 1021 1022 cagccccgggagccactcagctactcgcgcctgcagaggaagagtctggcagttgacttc 1081 1082 gtggtgccctcgctcttccgtgtctacgcccgggacctactgctgccaccatcctcctcg 1141 1142 gagctgaaggctggcaggcccgaggcccgcggctcgctagctctggactgcgccccgctg 1201 1202 ctcaggttgctggggccggcgccgggggtctcctggaccgccggttcaccagccccggca 1261 1262 gaggcccggacgctgtccagggtgctgaagggcggctccgtgcgcaagctccggcgtgcc 1321 1322 aagcagttggtgctggagctgggcgaggaggcgatcttggagggttgcgtcgggcccccc 1381 1382 ggggaggcggctgtggggctgctccagttcaatctcagcgagctgttcagttggtggatt 1441 1442 cgccaaggcgaagggcgactgaggatccgcctgatgcccgagaagaaggcgtcggaagtg 1501 1502 ggcagagagggaaggctgtccgcggcaattcgcgcctcccagccccgccttctcttccag 1561 1562 atcttcgggactggtcatagctccttggaatcaccaacaaacatgccttctccttctcct 1621 1622 gattattttacatggaatctcacctggataatgaaagactccttccctttcctgtctcat 1681 1682 cgcagccgatatggtctggagtgcagctttgacttcccctgtgagctggagtattcccct 1741 1742 ccactgcatgacctcaggaaccagagctggtcctggcgccgcatcccctccgaggaggcc 1801 1802 tcccagatggacttgctggatgggcctggggcagagcgttctaaggagatgcccagaggc 1861 1862 tcctttctccttctcaacacctcagctgactccaagcacaccatcctgagtccgtggatg 1921 1922 aggagcagcagtgagcactgcacactggccgtctcggtgcacaggcacctgcagccctct 1981 1982 ggaaggtacattgcccagctgctgccccacaacgaggctgcaagagagatcctcctgatg 2041 2042 cccactccagggaagcatggttggacagtgctccagggaagaatcgggcgtccagacaac 2101 2102 ccatttcgagtggccctggaatacatctccagtggaaaccgcagcttgtctgcagtggac 2161 2162 ttctttgccctgaagaactgcagtgaaggaacatccccaggctccaagatggccctgcag 2221 2222 agctccttcacttgttggaatgggacagtcctccagcttgggcaggcctgtgacttccac 2281 2282 caggactgtgcccagggagaagatgagagccagatgtgccggaaactgcctgtgggtttt 2341 2342 tactgcaactttgaagatggcttctgtggctggacccaaggcacactgtcaccccacact 2401 2402 cctcaatggcaggtcaggaccctaaaggatgcccggttccaggaccaccaagaccatgct 2461 2462 ctattgctcagtaccactgatgtccccgcttctgaaagtgctacagtgaccagtgctacg 2521 2522 tttcctgcaccgatcaagagctctccatgtgagctccgaatgtcctggctcattcgtgga 2581 2582 gtcttgaggggaaacgtgtccttggtgctagtggagaacaaaaccgggaaggagcaaggc 2641 2642 aggatggtctggcatgtcgccgcctatgaaggcttgagcctgtggcagtggatggtgttg 2701 2702 cctctcctcgatgtgtctgacaggttctggctgcagatggtcgcatggtggggacaagga 2761 2762 tccagagccatcgtggcttttgacaatatctccatcagcctggactgctacctcaccatt 2821 2822 agcggagaggacaagatcctgcagaatacagcacccaaatcaagaaacctgtttgagaga 2881 2882 aacccaaacaaggagctgaaacccggggaaaattcaccaagacagacccccatctttgac 2941 2942 cctacagttcattggctgttcaccacatgtggggccagcgggccccatggccccacccag 3001 3002 gcacagtgcaacaacgcctaccagaactccaacctgagcgtggaggtggggagcgagggc 3061 3062 cccctgaaaggcatccagatctggaaggtgccagccaccgacacctacagcatctcgggc 3121 3122 tacggagctgctggcgggaaaggcgggaagaacaccatgatgcggtcccacggcgtgtct 3181 3182 gtgctgggcatcttcaacctggagaaggatgacatgctgtacatcctggttgggcagcag 3241 3242 ggagaggacgcctgccccagtacaaaccagttaatccagaaagtctgcattggagagaac 3301 3302 aatgtgatagaagaagaaatccgtgtgaacagaagcgtgcatgagtgggcaggaggcgga 3361 3362 ggaggagggggtggagccacctacgtatttaagatgaaggatggagtgccggtgcccctg 3421 3422 atcattgcagccggaggtggtggcagggcctacggggccaagacagacacgttccaccca 3481 3482 gagagactggagaataactcctcggttctagggctaaacggcaattccggagccgcaggt 3541 3542 ggtggaggtggctggaatgataacacttccttgctctgggccggaaaatctttgcaggag 3601 3602 ggtgccaccggaggacattcctgcccccaggccatgaagaagtgggggtgggagacaaga 3661 3662 gggggtttcggagggggtggaggggggtgctcctcaggtggaggaggcggaggatatata 3721 3722 ggcggcaatgcagcctcaaacaatgaccccgaaatggatggggaagatggggtttccttc 3781 3782 atcagtccactgggcatcctgtacaccccagctttaaaagtgatggaaggccacggggaa 3841 3842 gtgaatattaagcattatctaaactgcagtcactgtgaggtagacgaatgtcacatggac 3901 3902 cctgaaagccacaaggtcatctgcttctgtgaccacgggacggtgctggctgaggatggc 3961 3962 gtctcctgcattgtgtcacccaccccggagccacacctgccactctcgctgatcctctct 4021 4022 gtggtgacctctgccctcgtggccgccctggtcctggctttctccggcatcatgattgtg 4081 4082 taccgccggaagcaccaggagctgcaagccatgcagatggagctgcagagccctgagtac 4141 4142 aagctgagcaagctccgcacctcgaccateatgaccgactacaaccccaactactgcttt 4201 4202 gctggcaagacctcctccatcagtgacctgaaggaggtgccgcggaaaaacatcaccctc 4261 4262 attcggggtctgggccatggcgcctttggggaggtgtatgaaggccaggtgtccggaatg 4321 4322 cccaacgacccaagccccctgcaagtggctgtgaagacgctgcctgaagtgtgctctgaa 4381 4382 caggacgaactggatttcctcatggaagccctgatcatcagcaaattcaaccaccagaac 4441 4442 attgttcgctgcattggggtgagcctgcaatccctgccccggttcatcctgctggagctc 4501 4502 atggcggggggagacctcaagtccttcctccgagagacccgccctcgcccgagccagccc 4561 4562 tcctccctggccatgctggaccttctgcacgtggctcgggacattgcctgtggctgtcag 4621 4622 tatttggaggaaaaccacttcatccaccgagacattgctgccagaaactgcctcttgacc 4681 4682 tgtccaggccctggaagagtggccaagattggagacttcgggatggcccgagacatctac 4741 4742 agggcgagctactatagaaagggaggctgtgccatgctgccagttaagtggatgccccca 4801 4802 gaggccttcatggaaggaatattcacttctaaaacagacacatggtcctttggagtgctg 4861 4862 ctatgggaaatcttttctcttggatatatgccataccccagcaaaagcaaccaggaagtt 4921 4922 ctggagtttgtcaccagtggaggccggatggacccacccaagaactgccctgggcctgta 4981 4982 taccggataatgactcagtgctggcaacatcagcctgaagacaggcccaactttgccatc 5041 5042 attttggagaggattgaatactgcacccaggacccggatgtaatcaacaccgctttgccg 5101 5102 atagaatatggtccacttgtggaagaggaagagaaagtgcctgtgaggcccaaggaccct 5161 5162 gagggggttcctcctctcctggtctctcaacaggcaaaacgggaggaggagcgcagccca 5221 5222 gctgccccaccacctctgcctaccacctcctctggcaaggctgcaaagaaacccacagct 5281 5282 gcagagatctctgttcgagtccctagagggccggccgtggaagggggacacgtgaatatg 5341 5342 gcattctctcagtccaaccctccttcggagttgcacaaggtccacggatccagaaacaag 5401 5402 cccaccagcttgtggaacccaacgtacggctcctggtttacagagaaacccaccaaaaag 5461 5462 aataatcctatagcaaagaaggagccacacgacaggggtaacctggggctggagggaagc 5521 5522 tgtactgtcccacctaacgttgcaactgggagacttccgggggcctcactgctcctagag 5581 5582 ccctcttcgctgactgccaatatgaaggaggtacctctgttcaggctacgtcacttccct 5641 5642 tgtgggaatgtcaattacggctaccagcaacagggcttgcccttagaagccgctactgcc 5701 5702 cctggagctggtcattacgaggataccattctgaaaagcaagaatagcatgaaccagcct 5761 5762 gggccctgagctcggtcgcacactcacttctcttccttgggatccctaagaccgtggagg 5821 5822 agagagaggcaatggctccttcacaaaccagagaccaaatgtcacgttttgttttgtgcc 5881 5882 aacctattttgaagtaccaccaaaaaagctgtattttgaaaatgctttagaaaggttttg 5941 5942 agcatgggttcatcctattctttcgaaagaagaaaatatcataaaaatgagtgataaata 6001 6002 caaggcccagatgtggttgcataaggtttttatgcatgtttgttgtatacttccttatgc 6061 6062 ttctttcaaattgtgtgtgctctgcttcaatgtagtcagaattagctgcttctatgtttc 6121 6122 atagttggggtcatagatgtttccttgccttgttgatgtggacatgagccatttgagggg 6181 6182 agagggaacggaaataaaggagttatttgtaatgactaaaa 6222
[0128] In some embodiments, ALK polynucleotide sequences encode ALK antigen or immunogen amino acid sequences that are at least 95%, at least 98%, at least 99%, or 100% identical to the sequences provided in Table 3, Table 4, and Table 5. In some embodiments, the ALK polynucleotide sequence encodes the ALK antigen or immunogen amino acid sequence that is at least 95%, at least 98%, at least 99%, or 100% identical to: RPRPSQPSSL. In some embodiments, the ALK polynucleotide sequence encodes the ALK antigen or immunogen amino acid sequence that is at least 95%, at least 98%, at least 99%, or 100% identical to: IVRCIGVSL. In some embodiments, the ALK polynucleotide sequence encodes the ALK antigen or immunogen amino acid sequence that is at least 95%, at least 98%, at least 99%, or 100% identical to: VPRKNITLI. In some embodiments, the ALK polynucleotide sequence encodes the ALK antigen or immunogen amino acid sequence that is at least 95%, at least 98%, at least 99%, or 100% identical to: TAAEVSVRV. In some embodiments, the ALK polynucleotide sequence encodes the ALK antigen or immunogen amino acid sequence that is at least 95%, at least 98%, at least 99%, or 100% identical to: AMLDLLHVA.
[0129] In some embodiments, the amino acid sequence of the antigen or immunogen, e.g., the ALK protein, is reverse translated and optimized for expression in mammalian cells. As will be appreciated by a skilled practitioner in the art, optimization of the nucleic acid sequence includes optimization of the codons for expression of a sequence in mammalian cells and RNA optimization (such as RNA stability).
[0130] In some embodiments, the ALK antigen or immunogen is isolated and/or purified. In some embodiments, the antigen or immunogen is formulated for administration to a subject in need. In some embodiments, the antigen or immunogen is administered to a subject in need thereof in an effective amount to elicit an immune response (e.g., a T-cell response) in the subject. In some embodiments, the immune response produces T-lymphocytes. In some embodiments, the immune response is prophylactic or therapeutic.
[0131] In some embodiments, fusion proteins comprising the ALK antigen polypeptides are described herein. In some embodiments, the ALK polypeptide can be fused to any heterologous amino acid sequence to form the fusion protein. By way of example, peptide components of ALK polypeptides may be generated independently and then fused together to produce an intact ALK polypeptide antigen, for use as an immunogen.
ALK Immunogenic Compositions and Vaccines
[0132] The ALK antigens or immunogens may be used in immunogenic compositions or vaccines to elicit an immune response, e.g., a T-cell response, against disease caused by oncogenic ALK gene fusions, rearrangements, duplications or mutations (e.g., ALK-positive cancers). In some embodiments, the immune response includes producing T-lymphocytes.
[0133] In particular embodiments, the ALK polypeptides of the immunogenic compositions or vaccines contain antigenic determinants that serve to elicit an immune response in a subject (e.g., the production of activated T-cells) that can treat and/or protect a subject against disease caused by oncogenic ALK gene fusions, rearrangements, duplications or mutations (e.g., ALK-positive cancers) and symptoms thereof.
[0134] In some embodiments, such immunogenic compositions or vaccines as described herein contain at least one ALK antigen or immunogen and are effective in treating, reducing, delaying, or preventing at least one disease caused by oncogenic ALK gene fusions, rearrangements, duplications or mutations (e.g., ALK-positive cancers). In some embodiments, such immunogenic compositions or vaccines as described herein contain two or more ALK antigens or immunogens and are effective in treating, reducing, or preventing at least one disease caused by oncogenic ALK gene fusions, rearrangements, duplications or mutations (e.g., ALK-positive cancers). In some embodiments, the two or more ALK antigens or immunogens comprise two or more amino acid sequences selected from the following: AMLDLLHVA; RPRPSQPSSL; IVRCIGVSL; VPRKNITLI; and/or TAAEVSVRV. In some embodiments, the two or more ALK antigens or immunogens comprise two or more amino acid sequences selected from Table 3, Table 4 and/or Table 5. In some embodiments, the immunogenic compositions or vaccines contain at least one ALK antigen or immunogen conjugated to an amphiphile or amphiphilic tail (see, e.g.
[0135] Because the ALK antigens or immunogens and the sequences thereof as described herein and used as immunogenic compositions or vaccines, elicit an immune response in an immunocompetent subject, they provide a superior vaccine against which an immune response (e.g., producing T-lymphocytes) is generated.
[0136] In some embodiments, an immunogenic composition or a vaccine is provided that elicits an immune response (e.g., producing T-lymphocytes) in a subject following introduction, administration, or delivery of the antigen or immunogen to the subject. The route of introduction, administration, or delivery is not limited and may include, for example, intravenous, subcutaneous, intramuscular, oral, or other routes. The immunogenic composition or vaccine may be therapeutic (e.g., administered to a subject following a symptom of disease caused by oncogenic ALK gene fusions, rearrangements, duplications or mutations (e.g., ALK-positive cancers)) or prophylactic (e.g., administered to a subject prior to the subject having or expressing a symptom of disease, or full-blown disease, caused by oncogenic ALK gene fusions, rearrangements, duplications or mutations (e.g., ALK-positive cancers)).
Vectors
[0137] Vectors containing a nucleotide sequence encoding an isolated ALK polypeptide or peptide antigen are provided. In some embodiments, the vectors comprise a nucleotide sequence encoding the full-length ALK polypeptide or peptide antigen that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a Homo Sapiens full-length ALK polypeptide as provided herein. In some embodiments, the vectors comprise a nucleotide sequence encoding the ALK polypeptide or peptide antigen that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a polynucleotide encoding at least one ALK polypeptide sequence provided in Table 3, Table 4 or Table 5. In some embodiments, the vector further includes a promoter operably linked to the nucleotide sequence encoding the ALK polypeptide. In a particular embodiment, the promoter is a cytomegalovirus (CMV) promoter. In some embodiments, the nucleotide sequence of the vector is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the polynucleotide sequence provided below. In some embodiments, the nucleotide sequence of the vector is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the Homo Sapiens full-length ALK nucleic acid sequence provided below.
TABLE-US-00022 2 ggggcggcagcggtggtagcagctggtacctcccgccgcctctgttcggagggtcgcggg 61 62 gcaccgaggtgctttccggccgccctctggtcggccacccaaagccgcgggcgctgatga 121 122 tgggtgaggagggggcggcaagatttcgggcgcccctgccctgaacgccctcagctgctg 181 182 ccgccggggccgctccagtgcctgcgaactctgaggagccgaggcgccggtgagagcaag 241 242 gacgctgcaaacttgcgcagcgcgggggctgggattcacgcccagaagttcagcaggcag 301 302 acagtccgaagccttcccgcagcggagagatagcttgagggtgcgcaagacggcagcctc 361 362 cgccctcggttcccgcccagaccgggcagaagagcttggaggagccaaaaggaacgcaaa 421 422 aggcggccaggacagcgtgcagcagctgggagccgccgttctcagccttaaaagttgcag 481 482 agattggaggctgccccgagaggggacagaccccagctccgactgcggggggcaggagag 541 542 gacggtacccaactgccacctcccttcaaccatagtagttcctctgtaccgagcgcagcg 601 602 agctacagacgggggcgcggcactcggcgcggagagcgggaggctcaaggtcccagccag 661 662 tgagcccagtgtgcttgagtgtctctggactcgcccctgagcttccaggtctgtttcatt 721 722 tagactcctgctcgcctccgtgcagttgggggaaagcaagagacttgcgcgcacgcacag 781 782 tcctctggagatcaggtggaaggagccgctgggtaccaaggactgttcagagcctcttcc 841 842 catctcggggagagcgaagggtgaggctgggcccggagagcagtgtaaacggcctcctcc 901 902 ggcgggatgggagccatcgggctcctgtggctcctgccgctgctgctttccacggcagct 961 962 gtgggctccgggatggggaccggccagcgcgcgggctccccagctgcggggccgccgctg 1021 1022 cagccccgggagccactcagctactcgcgcctgcagaggaagagtctggcagttgacttc 1081 1082 gtggtgccctcgctcttccgtgtctacgcccgggacctactgctgccaccatcctcctcg 1141 1142 gagctgaaggctggcaggcccgaggcccgcggctcgctagctctggactgcgccccgctg 1201 1202 ctcaggttgctggggccggcgccgggggtctcctggaccgccggttcaccagccccggca 1261 1262 gaggcccggacgctgtccagggtgctgaagggcggctccgtgcgcaagctccggcgtgcc 1321 1322 aagcagttggtgctggagctgggcgaggaggcgatcttggagggttgcgtcgggcccccc 1381 1382 ggggaggcggctgtggggctgctccagttcaatctcagcgagctgttcagttggtggatt 1441 1442 cgccaaggcgaagggcgactgaggatccgcctgatgcccgagaagaaggcgtcggaagtg 1501 1502 ggcagagagggaaggctgtccgcggcaattcgcgcctcccagccccgccttctcttccag 1561 1562 atcttcgggactggtcatagctccttggaatcaccaacaaacatgccttctccttctcct 1621 1622 gattattttacatggaatctcacctggataatgaaagactccttccctttcctgtctcat 1681 1682 cgcagccgatatggtctggagtgcagctttgacttcccctgtgagctggagtattcccct 1741 1742 ccactgcatgacctcaggaaccagagctggtcctggcgccgcatcccctccgaggaggcc 1801 1802 tcccagatggacttgctggatgggcctggggcagagcgttctaaggagatgcccagaggc 1861 1862 tcctttctccttctcaacacctcagctgactccaagcacaccatcctgagtccgtggatg 1921 1922 aggagcagcagtgagcactgcacactggccgtctcggtgcacaggcacctgcagccctct 1981 1982 ggaaggtacattgcccagctgctgccccacaacgaggctgcaagagagatcctcctgatg 2041 2042 cccactccagggaagcatggttggacagtgctccagggaagaatcgggcgtccagacaac 2101 2102 ccatttcgagtggccctggaatacatctccagtggaaaccgcagcttgtctgcagtggac 2161 2162 ttctttgccctgaagaactgcagtgaaggaacatccccaggctccaagatggccctgcag 2221 2222 agctccttcacttgttggaatgggacagtcctccagcttgggcaggcctgtgacttccac 2281 2282 caggactgtgcccagggagaagatgagagccagatgtgccggaaactgcctgtgggtttt 2341 2342 tactgcaactttgaagatggcttctgtggctggacccaaggcacactgtcaccccacact 2401 2402 cctcaatggcaggtcaggaccctaaaggatgcccggttccaggaccaccaagaccatgct 2461 2462 ctattgctcagtaccactgatgtccccgcttctgaaagtgctacagtgaccagtgctacg 2521 2522 tttcctgcaccgatcaagagctctccatgtgagctccgaatgtcctggctcattcgtgga 2581 2582 gtcttgaggggaaacgtgtccttggtgctagtggagaacaaaaccgggaaggagcaaggc 2641 2642 aggatggtctggcatgtcgccgcctatgaaggcttgagcctgtggcagtggatggtgttg 2701 2702 cctctcctcgatgtgtctgacaggttctggctgcagatggtcgcatggtggggacaagga 2761 2762 tccagagccatcgtggcttttgacaatatctccatcagcctggactgctacctcaccatt 2821 2822 agcggagaggacaagatcctgcagaatacagcacccaaatcaagaaacctgtttgagaga 2881 2882 aacccaaacaaggagctgaaacccggggaaaattcaccaagacagacccccatctttgac 2941 2942 cctacagttcattggctgttcaccacatgtggggccagcgggccccatggccccacccag 3001 3002 gcacagtgcaacaacgcctaccagaactccaacctgagcgtggaggtggggagcgagggc 3061 3062 cccctgaaaggcatccagatctggaaggtgccagccaccgacacctacagcatctcgggc 3121 3122 tacggagctgctggcgggaaaggcgggaagaacaccatgatgcggtcccacggcgtgtct 3181 3182 gtgctgggcatcttcaacctggagaaggatgacatgctgtacatcctggttgggcagcag 3241 3242 ggagaggacgcctgccccagtacaaaccagttaatccagaaagtctgcattggagagaac 3301 3302 aatgtgatagaagaagaaatccgtgtgaacagaagcgtgcatgagtgggcaggaggcgga 3361 3362 ggaggagggggtggagccacctacgtatttaagatgaaggatggagtgccggtgcccctg 3421 3422 atcattgcagccggaggtggtggcagggcctacggggccaagacagacacgttccaccca 3481 3482 gagagactggagaataactcctcggttctagggctaaacggcaattccggagccgcaggt 3541 3542 ggtggaggtggctggaatgataacacttccttgctctgggccggaaaatctttgcaggag 3601 3602 ggtgccaccggaggacattcctgcccccaggccatgaagaagtgggggtgggagacaaga 3661 3662 gggggtttcggagggggtggaggggggtgctcctcaggtggaggaggcggaggatatata 3721 3722 ggcggcaatgcagcctcaaacaatgaccccgaaatggatggggaagatggggtttccttc 3781 3782 atcagtccactgggcatcctgtacaccccagctttaaaagtgatggaaggccacggggaa 3841 3842 gtgaatattaagcattatctaaactgcagtcactgtgaggtagacgaatgtcacatggac 3901 3902 cctgaaagccacaaggtcatctgcttctgtgaccacgggacggtgctggctgaggatggc 3961 3962 gtctcctgcattgtgtcacccaccccggagccacacctgccactctcgctgatcctctct 4021 4022 gtggtgacctctgccctcgtggccgccctggtcctggctttctccggcatcatgattgtg 4081 4082 taccgccggaagcaccaggagctgcaagccatgcagatggagctgcagagccctgagtac 4141 4142 aagctgagcaagctccgcacctcgaccateatgaccgactacaaccccaactactgcttt 4201 4202 gctggcaagacctcctccatcagtgacctgaaggaggtgccgcggaaaaacatcaccctc 4261 4262 attcggggtctgggccatggcgcctttggggaggtgtatgaaggccaggtgtccggaatg 4321 4322 cccaacgacccaagccccctgcaagtggctgtgaagacgctgcctgaagtgtgctctgaa 4381 4382 caggacgaactggatttcctcatggaagccctgatcatcagcaaattcaaccaccagaac 4441 4442 attgttcgctgcattggggtgagcctgcaatccctgccccggttcatcctgctggagctc 4501 4502 atggcggggggagacctcaagtccttcctccgagagacccgccctcgcccgagccagccc 4561 4562 tcctccctggccatgctggaccttctgcacgtggctcgggacattgcctgtggctgtcag 4621 4622 tatttggaggaaaaccacttcatccaccgagacattgctgccagaaactgcctcttgacc 4681 4682 tgtccaggccctggaagagtggccaagattggagacttcgggatggcccgagacatctac 4741 4742 agggcgagctactatagaaagggaggctgtgccatgctgccagttaagtggatgccccca 4801 4802 gaggccttcatggaaggaatattcacttctaaaacagacacatggtcctttggagtgctg 4861 4862 ctatgggaaatcttttctcttggatatatgccataccccagcaaaagcaaccaggaagtt 4921 4922 ctggagtttgtcaccagtggaggccggatggacccacccaagaactgccctgggcctgta 4981 4982 taccggataatgactcagtgctggcaacatcagcctgaagacaggcccaactttgccatc 5041 5042 attttggagaggattgaatactgcacccaggacccggatgtaatcaacaccgctttgccg 5101 5102 atagaatatggtccacttgtggaagaggaagagaaagtgcctgtgaggcccaaggaccct 5161 5162 gagggggttcctcctctcctggtctctcaacaggcaaaacgggaggaggagcgcagccca 5221 5222 gctgccccaccacctctgcctaccacctcctctggcaaggctgcaaagaaacccacagct 5281 5282 gcagagatctctgttcgagtccctagagggccggccgtggaagggggacacgtgaatatg 5341 5342 gcattctctcagtccaaccctccttcggagttgcacaaggtccacggatccagaaacaag 5401 5402 cccaccagcttgtggaacccaacgtacggctcctggtttacagagaaacccaccaaaaag 5461 5462 aataatcctatagcaaagaaggagccacacgacaggggtaacctggggctggagggaagc 5521 5522 tgtactgtcccacctaacgttgcaactgggagacttccgggggcctcactgctcctagag 5581 5582 ccctcttcgctgactgccaatatgaaggaggtacctctgttcaggctacgtcacttccct 5641 5642 tgtgggaatgtcaattacggctaccagcaacagggcttgcccttagaagccgctactgcc 5701 5702 cctggagctggtcattacgaggataccattctgaaaagcaagaatagcatgaaccagcct 5761 5762 gggccctgagctcggtcgcacactcacttctcttccttgggatccctaagaccgtggagg 5821 5822 agagagaggcaatggctccttcacaaaccagagaccaaatgtcacgttttgttttgtgcc 5881 5882 aacctattttgaagtaccaccaaaaaagctgtattttgaaaatgctttagaaaggttttg 5941 5942 agcatgggttcatcctattctttcgaaagaagaaaatatcataaaaatgagtgataaata 6001 6002 caaggcccagatgtggttgcataaggtttttatgcatgtttgttgtatacttccttatgc 6061 6062 ttctttcaaattgtgtgtgctctgcttcaatgtagtcagaattagctgcttctatgtttc 6121 6122 atagttggggtcatagatgtttccttgccttgttgatgtggacatgagccatttgagggg 6181 6182 agagggaacggaaataaaggagttatttgtaatgactaaaa 6222
[0138] The vectors used to express an ALK antigen as described herein may be any suitable expression vector known and used in the art. In some embodiments, the vector is a prokaryotic or eukaryotic vector. In some embodiments, the vector is an expression vector, such as a eukaryotic (e.g., mammalian) expression vector. In another embodiment, the vector is a plasmid (prokaryotic or bacterial) vector. In another embodiment, the vector is a viral vector.
[0139] Provided are isolated, non-naturally occurring polypeptide antigens, e.g., ALK polypeptide antigens, produced by transfecting a host cell with an expression vector as known and used in the art under conditions sufficient to allow for expression of the polypeptide, e.g., an ALK polypeptide, in the cell. Isolated cells containing the vectors are also provided.
[0140] Also provided is an ALK polypeptide, as described herein, produced by transfecting a host cell with a vector containing a polynucleotide encoding the ALK polypeptide. Also provided in some embodiments is an ALK polypeptide, as described herein, produced by transfecting a host cell with a vector encoding the ALK polypeptide under conditions sufficient to allow for expression of the ALK protein. Collections of plasmids (vectors) are also contemplated. In certain embodiments, the collection of plasmids includes plasmid encoding an ALK protein as described herein.
Compositions and Pharmaceutical Compositions for Administration
[0141] Compositions comprising at least one ALK protein, or a polynucleotide encoding at least one ALK protein, as described herein are provided. In some embodiments, the compositions further comprise a pharmaceutically acceptable carrier, diluent, excipient, or vehicle. In some embodiments, an adjuvant (a pharmacological or immunological agent that modifies or boosts an immune response, e.g., to produce more antibodies that are longer-lasting) is also employed. For example, without limitation, the adjuvant can be an inorganic compound, such as alum, aluminum hydroxide, or aluminum phosphate; mineral or paraffin oil; squalene; detergents such as Quil A; plant saponins; Freund's complete or incomplete adjuvant, a biological adjuvant (e.g., cytokines such as IL-1, IL-2, or IL-12); bacterial products such as killed Bordetella pertussis, or toxoids; or immunostimulatory oligonucleotides (such as CpG oligonucleotides). In some embodiments, the adjuvant is conjugated to an amphiphile as previously described (H. Liu et al., Structure-based programming of lymph-node targeting in molecular vaccines. Nature 507, 5199522 (2014)). In some embodiments, the amphiphile is N-hydroxy succinimidyl ester-end-functionalized poly(ethylene glycol)-lipid (NHS-PEG2KDa-DSPE)
[0142] Compositions and preparations (e.g., physiologically or pharmaceutically acceptable compositions) containing ALK polypeptides or polynucleotides for parenteral administration include, without limitation, sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Nonlimiting examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and canola oil, and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include, for example, fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present in such compositions and preparations, such as, for example, antimicrobials, antioxidants, chelating agents, colorants, stabilizers, inert gases and the like.
[0143] Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base-addition salt, formed by reaction with inorganic acids, such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids, such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, tri-alkyl and aryl amines and substituted ethanolamines.
[0144] Provided herein are pharmaceutical compositions which include a therapeutically effective amount of an isolated ALK polypeptide or polynucleotide antigen, alone, or in combination with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carrier and composition can be sterile, and the formulation suits the mode of administration. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid or aqueous solution, suspension, emulsion, dispersion, tablet, pill, capsule, powder, or sustained release formulation. A liquid or aqueous composition can be lyophilized and reconstituted with a solution or buffer prior to use. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. Any of the commonly known pharmaceutical carriers, such as sterile saline solution or sesame oil, can be used. The medium can also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, buffers, preservatives and the like. Other media that can be used in the compositions and administration methods as described are normal saline and sesame oil.
Methods of Treatment, Administration and Delivery
[0145] Methods of treating a disease, or symptoms thereof, caused by the oncogenic ALK gene fusions, rearrangements, duplications or mutations (e.g., ALK-positive cancers) are provided. The methods comprise administering a therapeutically effective amount of an antigen, immunogen, immunogenic composition, or vaccine, as described herein, or a pharmaceutical composition comprising the immunogen or a vaccine, as described herein, to a subject (e.g., a mammal), in particular, a human subject. The invention provides methods of treating a subject suffering from, or at risk of, or susceptible to disease, or a symptom thereof, or delaying the progression of a disease caused by oncogenic ALK gene fusions, rearrangements, duplications or mutations (e.g., ALK-positive cancers). In some embodiments, the method includes administering to the subject (e.g., a mammalian subject), an amount or a therapeutic amount of an immunogenic composition or a vaccine comprising at least one ALK antigen polypeptide, sufficient to treat the disease, delay the growth of, or treat the symptoms thereof, caused by the oncogenic ALK gene under conditions in which the disease and/or the symptoms thereof are treated.
[0146] In some embodiments, the methods herein include administering to the subject (including a human subject identified as in need of such treatment) an effective amount of an isolated, ALK antigen or immunogen polypeptide, or an immunogenic composition or vaccine, or a pharmaceutical composition thereof, as described herein to produce such effect. The treatment methods are suitably administered to subjects, particularly humans, suffering from, are susceptible to, or at risk of having a disease, or symptoms thereof, caused by oncogenic ALK gene fusions, rearrangements, duplications or mutations, namely, ALK-positive cancers. Nonlimiting examples of ALK-positive cancers include non-small cell lung cancer (NSCLC), anaplastic large cell lymphoma (ALCL), neuroblastoma, B-cell lymphoma, thyroid cancer, colon cancer, breast cancer, inflammatory myofibroblastic tumors (IMT), renal carcinoma, esophageal cancer, melanoma, or a combination thereof.
[0147] Identifying a subject in need of such treatment can be based on the judgment of the subject or of a health care professional and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method). Briefly, the determination of those subjects who are in need of treatment or who are “at risk” or “susceptible” can be made by any objective or subjective determination by a diagnostic test (e.g., blood sample, biopsy, genetic test, enzyme or protein marker assay), marker analysis, family history, and the like, including an opinion of the subject or a health care provider. In some embodiments, the subject in need of treatment can be identified by measuring ALK specific autoantibodies and ALK-specific T-cell responses in a patient sample (e.g., blood sample) or by assessing infiltrating immune cell subsets from a tumor core biopsy from a subject (see Example 3). The ALK antigens and immunogens, such as ALK polypeptides, immunogenic compositions and vaccines as described herein, may also be used in the treatment of any other disorders in which disease caused by oncogenic ALK gene fusions, rearrangements, duplications or mutations may be implicated. A subject undergoing treatment can be a non-human mammal, such as a veterinary subject, or a human subject (also referred to as a “patient”).
[0148] In addition, prophylactic methods of preventing or protecting against a disease, or symptoms thereof, caused by oncogenic ALK gene fusions, rearrangements, duplications or mutations are provided. Such methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an ALK immunogenic composition or vaccine as described herein to a subject (e.g., a mammal, such as a human), in particular, prior to development or onset of a disease, such as ALK-positive tumors or cancers.
[0149] In another embodiment, a method of monitoring the progress of a disease caused by oncogenic ALK gene fusions, rearrangements, duplications or mutations (e.g., ALK-positive cancers), or monitoring treatment of the disease is provided. The method includes a diagnostic measurement (e.g., CT scan, screening assay or detection assay) in a subject suffering from or susceptible to disease or symptoms thereof associated with oncogenic ALK gene fusions, rearrangements, duplications or mutations (e.g., ALK-positive cancers), in which the subject has been administered an amount (e.g., a therapeutic amount) of an isolated ALK protein, as described herein, or an immunogenic composition or vaccine as described herein, sufficient to treat the disease or symptoms thereof. The diagnostic measurement in the method can be compared to samples from healthy, normal controls; in a pre-disease sample of the subject; or in other afflicted/diseased patients to establish the treated subject's disease status. For monitoring, a second diagnostic measurement may be obtained from the subject at a time point later than the determination of the first diagnostic measurement, and the two measurements can be compared to monitor the course of disease or the efficacy of the therapy/treatment. In certain embodiments, a pre-treatment measurement in the subject (e.g., in a sample or biopsy obtained from the subject or CT scan) is determined prior to beginning treatment as described; this measurement can then be compared to a measurement in the subject after the treatment commences and/or during the course of treatment to determine the efficacy of (monitor the efficacy of) the disease treatment. In some embodiments, efficacy of the disease treatment can be performed with antibody marker analysis and/or interferon-gamma (IFN-γ) ELISPOT assays.
[0150] The isolated ALK antigen polypeptide, or compositions thereof, can be administered to a subject by any of the routes normally used for introducing a recombinant protein or composition containing the recombinant protein into a subject. Routes and methods of administration include, without limitation, intradermal, intramuscular, intraperitoneal, intrathecal, parenteral, such as intravenous (IV) or subcutaneous (SC), vaginal, rectal, intranasal, inhalation, intraocular, intracranial, or oral. Parenteral administration, such as subcutaneous, intravenous or intramuscular administration, is generally achieved by injection (immunization). Injectables can be prepared in conventional forms and formulations, either as liquid solutions or suspensions, solid forms (e.g., lyophilized forms) suitable for solution or suspension in liquid prior to injection, or as emulsions. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets. Administration can be systemic or local.
[0151] The isolated ALK polypeptides or compositions thereof, can be administered in any suitable manner, such as with pharmaceutically acceptable carriers, diluents, or excipients as described supra. Pharmaceutically acceptable carriers are determined in part by the particular immunogen or composition being administered, as well as by the particular method used to administer the composition. Accordingly, a pharmaceutical composition comprising the isolated ALK antigen polypeptides or compositions thereof, can be prepared using a wide variety of suitable and physiologically and pharmaceutically acceptable formulations.
[0152] Further provided is a method of eliciting or generating an immune response in a subject with a disease caused by oncogenic ALK gene fusions, rearrangements, duplications or mutations (e.g., ALK-positive cancers) by administering to the subject an isolated ALK protein antigen or immunogen, or immunogenic composition or vaccine thereof, as described herein. In some embodiments, the ALK protein can be administered using any suitable route of administration, such as, for example, by intramuscular injection. In some embodiments, the ALK protein is administered as a composition comprising a pharmaceutically acceptable carrier. In some embodiments, the composition comprises an adjuvant selected from, for example, alum, Freund's complete or incomplete adjuvant, a biological adjuvant or immunostimulatory oligonucleotides (such as CpG oligonucleotides). In some embodiments, the adjuvant is conjugated to an amphiphile. In other embodiments, the composition may be administered in combination with one or more therapeutic agents or molecules.
[0153] Also provided is a method of immunizing a subject against disease or the symptoms thereof caused by oncogenic ALK gene fusions, rearrangements, duplications or mutations (e.g., ALK-positive cancers), in which the method involves administering to the subject an isolated ALK protein as described herein, or administering an immunogenic composition or vaccine thereof. In some embodiments of the method, the composition further comprises a pharmaceutically acceptable carrier, diluent, excipient, and/or an adjuvant. For example, the adjuvant can be alum, Freund's complete or incomplete adjuvant, a biological adjuvant or immunostimulatory oligonucleotides (such as CpG oligonucleotides). In some embodiments, the adjuvant is conjugated to an amphiphile. In some embodiments, the ALK peptides (or compositions thereof) are administered intramuscularly.
[0154] An advantage of the immunogens and immunogenic compositions comprising ALK antigens described herein is that an immune response is elicited against not only the ALK-expressing tumor or cell line from which the antigen was derived, but also against one or more, or all, ALK-positive cancers, e.g., non-small cell lung cancer (NSCLC), anaplastic large cell lymphoma (ALCL), neuroblastoma, B-cell lymphoma, thyroid cancer, colon cancer, breast cancer, inflammatory myofibroblastic tumors (IMT), renal carcinoma, esophageal cancer, melanoma, or a combination thereof. In some embodiments, the immunogens and immunogenic compositions described herein elicit immune responses against NSCLC. In some embodiments, the immunogens and immunogenic compositions described herein elicit immune responses against ALCL. Thus, the ALK immunogens are more cost effective to produce, and beneficially elicit an immune response, thus, obviating a need to make and administer a poly- or multivalent immunogenic composition or vaccine.
[0155] Administration of the isolated ALK antigen polypeptides or compositions thereof, can be accomplished by single or multiple doses. In some embodiments, the doses can be administered according to the vaccination schedule described in Example 3 and
Adjuvants and Combination Therapies
[0156] The ALK immunogens or immunogenic compositions or vaccines containing an ALK-specific peptide antigen can be administered alone or in combination with other therapeutic agents to enhance antigenicity or immunogenicity, i.e., to increase an immune response, such as the elicitation of specific or neutralizing antibodies, in a subject. For example, the ALK-specific peptide can be administered with an adjuvant, such as alum, Freund's incomplete adjuvant, Freund's complete adjuvant, biological adjuvant, or immunostimulatory oligonucleotides (such as CpG oligonucleotides). The adjuvant may be conjugated to an amphiphile as previously described (H. Liu et al., Structure-based programming of lymph-node targeting in molecular vaccines. Nature 507, 5199522 (2014)). In some embodiments, the amphiphile conjugated to the adjuvant is N-hydroxy succinimidyl ester-end-functionalized poly(ethylene glycol)-lipid (NHS-PEG2KDa-DSPE).
[0157] One or more cytokines, such as interleukin-1 (IL-2), interleukin-6 (IL-6), interleukin-12 (IL-12), the protein memory T-cell attractant “Regulated on Activation, Normal T Expressed and Secreted” (RANTES), granulocyte-macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-α), or interferon-gamma (IFN-γ); one or more growth factors, such as GM-CSF or granulocyte-colony stimulation factor (G-CSF); one or more molecules such as the TNF ligand superfamily member 4 ligand (OX40L) or the type 2 transmembrane glycoprotein receptor belonging to the TNF superfamily (4-1BBL), or combinations of these molecules, can be used as biological adjuvants, if desired or warranted (see, e.g., Salgaller et al., 1998, J. Surg. Oncol. 68(2):122-38; Lotze et al., 2000, Cancer J. Sci. Am. 6(Suppl 1):S61-6; Cao et al., 1998, Stem Cells 16(Suppl 1):251-60; Kuiper et al., 2000, Adv. Exp. Med. Biol. 465:381-90). These molecules can be administered systemically (or locally) to a subject.
[0158] Several ways of inducing cellular responses, both in vitro and in vivo, are known and practiced in the art. Lipids have been identified as agents capable of assisting in priming cytotoxic lymphocytes (CTL) in vivo against various antigens. For example, palmitic acid residues can be attached to the alpha and epsilon amino groups of a lysine residue and then linked (for example, via one or more linking residues, such as glycine, glycine-glycine, serine, serine-serine, or the like) to an immunogenic peptide (U.S. Pat. No. 5,662,907). The lipidated peptide can then be injected directly in a micellar form, incorporated in a liposome, or emulsified in an adjuvant. As another example, E. coli lipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine can be used to prime tumor-specific CTL when covalently attached to an appropriate peptide (see, e.g., Deres et al., 1989, Nature 342:561). Moreover, the induction of neutralizing antibodies can also be primed with the same molecule conjugated to a peptide which displays an appropriate epitope, and two compositions can be combined to elicit both humoral and cell-mediated responses where such a combination is deemed desirable.
[0159] The ALK-specific peptides can also be administered as a combination therapy with one or more other therapeutic agents, such as ALK inhibitors, tyrosine kinase inhibitors (TKIs), and/or immune checkpoint inhibitors. Non-limiting examples of ALK inhibitors include lorlatinib (Lobrena®). Non-limiting examples of checkpoint inhibitors include programmed cell death protein 1 (PD-1) inhibitors, programmed death-ligand 1 (PD-L1), and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) inhibitors. Nonlimiting examples of PD-1 inhibitors include pembrolizumab (Keytruda®) and nivolumab (Opdivo®). Nonlimiting examples of CTLA-4 inhibitors include ipilimumab (Yervoy®). Non-limiting examples of TKI inhibitors include crizotinib, ceritinib, alectinib, brigatinib, and lorlatinib.
[0160] In some embodiments, one or more ALK inhibitors, immune checkpoint inhibitors, and/or TKI inhibitors is administered simultaneously or sequentially with ALK-specific peptide antigens, immunogens, or immunogenic compositions or vaccines containing an ALK-specific peptide antigen or immunogen. In some embodiments, the ALK-specific peptide antigen, immunogen, or immunogenic composition or vaccine containing an ALK-specific peptide antigen or immunogen is administered with a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody. In some embodiments, the ALK-specific peptide antigen, immunogen, or immunogenic composition or vaccine containing an ALK-specific peptide antigen or immunogen is administered with a PD-1 inhibitor according to the schedule described in Example 3 and/or
[0161] In some embodiments, the ALK-specific peptide antigen, immunogen, or immunogenic composition or vaccine containing an ALK-specific peptide antigen or immunogen is administered with a TKI inhibitor. In some embodiments, the ALK-specific peptide antigen, immunogen, or immunogenic composition or vaccine containing an ALK-specific peptide antigen or immunogen is administered with an ALK inhibitor. In some embodiments, the ALK-specific peptide antigen, immunogen, or immunogenic composition or vaccine containing an ALK-specific peptide antigen or immunogen is administered with lorlatinib.
[0162] In some embodiments, the ALK-specific peptide antigen, immunogen, or immunogenic composition or vaccine containing an ALK-specific peptide antigen or immunogen is administered with a PD-1 inhibitor in combination with an ALK inhibitor. In some embodiments, the ALK-specific peptide antigen, immunogen, or immunogenic composition or vaccine containing an ALK-specific peptide antigen or immunogen is administered with an anti-PD-1 antibody in combination with lorlatinib. In some embodiments, the ALK-specific peptide antigen, immunogen, or immunogenic composition or vaccine containing an ALK-specific peptide antigen or immunogen is administered with an anti-PD-1 antibody in combination with lorlatinib according to the schedule described in Example 4 and/or
[0163] While treatment methods may involve the administration of a vaccine containing a ALK immunogenic protein as described herein, one skilled in the art will appreciate that the ALK protein itself, as a component of a pharmaceutically acceptable composition or as a fusion protein, can be administered to a subject in need thereof to elicit an immune response against an ALK-positive cancer in the subject.
Kits
[0164] Also provided are kits containing the ALK antigen or immunogen as described, or an immunogenic composition, or a vaccine, or a pharmaceutically acceptable composition containing the antigen or immunogen and a pharmaceutically acceptable carrier, diluent, or excipient, for administering to a subject, for example. The antigen or immunogen may be in the form of an ALK protein (polypeptide) or a polynucleotide (a polynucleotide encoding an ALK polypeptide), as described herein. Kits containing one or more of the plasmids, or a collection of plasmids as described herein, are also provided. As will be appreciated by the skilled practitioner in the art, such a kit may contain one or more containers that house the antigen, immunogen, vaccine, or composition, carriers, diluents or excipients, as necessary, and instructions for use.
[0165] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Useful techniques for particular embodiments will be discussed in the sections that follow.
[0166] The following examples are put forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
EXAMPLES
[0167] The following examples are provided to illustrate certain particular features and/or embodiments. The examples should not be construed to limit the disclosure to the particular features or embodiments described.
Example 1. Identification of ALK-Specific Peptides for an ALK-Positive Cancer Vaccine
[0168] The direct identification of ALK peptides that are effectively presented on the surface of ALK-expressing cancer cells in the context of the most frequent patient haplotypes will provide a powerful resource to design the most effective vaccine to elicit ALK-specific T-cell responses.
Example 1.1 Identification of ALK-Specific Peptides in Different HLA-Haplotypes
[0169] The B721.221 human lymphoblastic cell line does not express endogenous HLA class I (A, B and C) due to gamma-ray-induced mutations in the HLA complex. (Shimizu Y, DeMars R. Production of human cells expressing individual transferred HLA-A, -B, -C genes using an HLA-A, -B, -C null human cell line. J. Immunol. 1989; 142(9):3320-3328). Fifty (50) cell lines each expressing a single HLA class I allele (monoallelic cell lines) were generated by transducing B721.221 cells with retroviruses encoding HLA class I genes. This tool allows the unequivocal assignation of peptides identified by mass spectrometry to a specific HLA class I allele. The HLA-peptide complexes were immunoprecipitated with an anti-HLA A/B/C antibody (clone W6/32, Santa Cruz) and the peptides released were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Using retroviral transduction, the EML4-ALK variant 1, the most frequent EML4-ALK fusion protein (Lin J J, et al. Impact of EML4-ALK Variant on Resistance Mechanisms and Clinical Outcomes in ALK-Positive Lung Cancer. J. Clin. Oncol. 2018: JCO2017762294), was expressed into different HLA-monoallelic B721.221 cell lines (
[0170] Using the most sensitive targeted mass spectrometry, two (2) peptides predicted by the algorithms from the immune epitope database (IEDB), AMLDLLHVA and SLAMLDLLHV, were isotope-labelled (heavy standard) and searched in HLA*02:01 elute from B721.221/A*02:01 cells. Only the 9-mer, AMLDLLHVA, ALK-specific peptide binding the HLA A*02:01 allele was identified (
TABLE-US-00023 TABLE 1 Percentage of different HLA class I alleles among 100 ALK-positive lung cancer patients. HLA A HLA B HLA C Allele Frequency Allele Frequency Allele Frequency *02:01 43% *07:02 18% *07:01 27% *01:01 26% *08:01 15% *04:01 26% *24:02 24% *35:01 12% *06:02 24% *03:01 22% *18:01 10% *07:02 22% *11:01 12% *44:02 10% *03:04 15%
Example 1.2 Identification of ALK-Specific Peptides in Different Cell Lines
[0171] The low levels of EML4-ALK expression reached by transducing B721.221 cells make it difficult to identify ALK-specific peptides normally expressed on the surface of tumor cells. In order to avoid this technical challenge, different ALK-positive anaplastic large cell lymphoma (ALCL) cell lines encoding frequent HLA-alleles were selected (Karpas-299, DEL, and SR-786). ALCL cell lines express high levels of the NPM-ALK fusion protein, a fusion protein containing the exactly same ALK portion as the EML4-ALK fusion protein. The HLA genotype and HLA expression on the selected cell lines were confirmed before proceeding with mass spectrometry experiments (Table 2). The two HLA-A, -B and -C alleles were determined by Next Generation sequencing (NGS). Expression levels of HLA were determined by flow cytometry with a pan-HLA antibody. Positive or negative antibody staining for anti-HLA-A*02, which recognizes all the allelic variants of HLA-A*02, and anti-DR, which recognizes all the HLA class II DR (HLA-DR) subclasses, was determined by flow cytometry.
TABLE-US-00024 TABLE 2 Characterization of ALK-positive ALCL cell lines. Next Generation Sequencing Antibody Staining HLA A(1) A(2) B(1) B(2) C(1) C(2) Pan-HLA A02 DR ALCL KARPAS *03:01 *11:01 *07:02 *35:01 *04:01 *07:02 (+++) No Yes Cell DEL *26:01 *02:01 *14:01 *27:05 *08:02 *05:01 (++++) Yes Yes Lines SR786 *02:01 *68:02 *55:01 *57:01 *03:03 *06:02 (+++) Yes No
[0172] Since the HLA A*02:01 allele can be immunoprecipitated by a specific antibody (BB7.2 clone), the peptide, AMLDLLHVA, previously identified in B721.221/HLA A*02:01 cells, was isotope-labeled (heavy standard) and its presence was confirmed by targeted mass spectrometry in the HLA A*02:01 elute of DEL cells (
[0173] This data showed the physiological processing and presentation of four (4) different ALK-peptides on the groove of two frequent HLA class I haplotypes, HLA A*02:01 and HLA B*07:02, by cancer cells (Table 3). The peptides described in Table 3 belong to the intracytoplasmic portion of ALK; therefore, are comprised in all the possible forms of ALK aberrant expression, such as overexpression (neuroblastoma) or the EML4-ALK (NSCLC) and NPM-ALK (ALCL) translocations. Based on the frequency of these HLA class I alleles among individuals with ALK-positive NSCLC, a therapeutic vaccine against these peptides would be applicable to about 54% of patients.
[0174] In addition, the 9-mer peptide, TAAEVSVRV, was identified in two independent experiments using pan-HLA elute from SR-786 cells (
TABLE-US-00025 TABLE 3 Peptides discovered by mass spectrometry. Sequence Predicted Allele AMLDLLHVA HLA A*02:01 RPRPSQPSSL HLA B*07:02 IVRCIGVSL HLA B*07:02 VPRKNITLI HLA B*07:02 TAAEVSVRV HLA A*68:02
Example 1.3 Identified ALK-Specific Peptides Induce Immune Response In Vitro
[0175] The ability of the identified peptides to induce an immune response was assessed in vitro. Cryopreserved PBMCs from ALK-positive NSCLC patients or PBMCs from healthy donors were incubated in vitro with autologous antigen presenting cells (APC) as previously described (Cai A, et al. Mutated BCR-ABL generates immunogenic T-cell epitopes in Chronic myelogenous leukemia (CML) patients (Rajasagi et al., Clin. Cancer Res. 2012; 18(20):5761-5772; Rajasagi M, et al. Blood. 2014; 124(3):453-462). 10×10.sup.6 PBMCs were incubated in a 24-well plate with autologous dendritic cells (DCs) pulsed with 10 μM of the corresponding peptide (ratio 100:1) in a media supplemented with 10% human AB serum, rhIL7 (long/ml) and rhIL2 (20 IU/ml). After 6 days, CD8.sup.+ T-cells were isolated from the bulk population by magnetic beads (StemCell Technologies) and incubated with irradiated B721.221-HLA monoallele pulsed with 10 μM of the corresponding peptide (ratio 10:1). Cells were re-stimulated on days 7, 14, 21 and 28 by adding rhIL7 (long/ml) and rhIL2 (20 IU/ml) on days 8, 15, 22 and 29. The specificity of the T-cells was evaluated one week after the last stimulation. T-cells were incubated with irradiated B721.221/corresponding HLA/peptide-pulsed or B721.221/corresponding HLA/EML4-ALK transduced (ratio 1:1) cells in an IFNγ-ELISPOT plate (Mabtech). As an alternative approach, T-cells were stained by dextramers that were synthesized based on the identified peptide in a complex with the corresponding HLA allele (Immunex).
[0176] The capability of these peptides in generating T-cells responses was also assessed. HLA-A*02:01 transgenic mice (C57BL/6-Mcph1.sup.Tg(HLA-A2.1)Enge/J) were vaccinated with the AMLDLLHVA peptide together with Freund's complete adjuvant. Three (3) weeks after vaccination, splenocytes from the vaccinated mice were challenged with AMLDLLHVA, OVA-peptide (negative control), and media (negative control) in an interferon-gamma (IFN-γ) ELISPOT assay (
[0177] For the ALK-specific HLA B*07:02 binding peptides, IVRCIGVSL and RPRPSQPSSL, a significant response was not detected in the direct IFN-γ-ELISPOT performed with PBMCs from four (4) ALK-positive NSCLC patients. Thus, peptide-specific CD8.sup.+ T-cell expansion with IVRCIGVSL and RPRPSQPSSL was evaluated in ALK-positive NSCLC HLA B*07:02 patient PBMCs according to the schematic in
[0178] When performing an IFN-γ-ELISPOT using the IVRCIGVSL-expanded CD8+ T-cells, about four (4) times more spot forming units in wells challenged with IVRCIGVSL-pulsed B-cells was detected compared to those wells challenged with B-cells pulsed with control peptides (
Example 2. ALK Amphiphile Peptides
[0179] Without wishing to be bound by theory, in vitro and in vivo data suggest that amphiphile-peptide (amph-peptide) vaccines function by binding to endogenous albumin present in interstitial fluid following injection, with subsequent albumin-mediated transport to lymph nodes for efficient capture by dendritic cells (DCs) (H. Liu et al., Structure-based programming of lymph-node targeting in molecular vaccines. Nature 507, 5199522 (2014)). A schematic of this amph-peptide vaccine “albumin hitchhiking” concept is shown in
Example 2.1 ALK Peptide Selection for Amphiphile-Peptide Conjugation
[0180] In silico prediction of peptide-MHC binding is considered a reliable strategy to rank the probability of shared or neoantigens to be effectively presented on human HLA subtypes (E. F. Fritsch et al., HLA-binding properties of tumor neoepitopes in humans. Cancer Immunol Res 2, 5229529 (2014)). To determine whether conjugation with an amphiphilic tail can be applied to an ALK-specific peptide vaccine, in silico prediction and ELISpot-based screening techniques were used to identify immunogenic ALK peptides that induce strong immune responses in mouse models.
[0181] Out of twenty-eight (28) patients analyzed for HLA haplotypes, thirteen (13) patients were HLA A*02:01, eight (8) patients were HLA B*07:02, and eight (8) patients were HLA C*07:02. NetMHCpan and IEDB algorithms were used to predict which ALK peptides were most likely to be presented on HLA A*02:01, HLA B*07:02, and HLA C*07:02. The predicted ALK peptides for these three HLA haplotypes are shown in Table 4.
TABLE-US-00026 TABLE 4 Selection of ALK peptides for synthesis of an ALK vaccine Percentile NetMHCpan nM IEDB Rank HLA A*02:01 9 mer 9 mer VLLWEIFSL 5 VLLWEIFSL 0.3 AMLDLLHVA 8 FLMEALIIS 0.5 KTDTWSFGV 11 AMLDLLHVA 0.5 FLMEALIIS 16 KTDTWSFGV 1.1 GMARDIYRA 77 GMARDIYRA 1.2 10 mer 10 mer SLAMLDLLHV 16 SLAMLDLLHV 0.4 FLMEALIISK 35 FLMEALIISK 0.55 SLPRFILLEL 53 SLPRFILLEL 1.45 LLLEPSSLTA 71 GVLLWEIFSL 1.55 GVLLWEIFSL 93 LLLEPSSLTA 1.6 HLA B*07:02 9 mer 9 mer KPTKKNNPI 14 RPSQPSSLA 0.3 RPSQPSSLA 16 LPRFILLEL 0.5 LPRFILLEL 18 VPRKNITLI 0.5 IVRCIGVSL 20 KPTKKNNPI 1.1 VPRKNITLI 27 RPRPSQPSS 1.2 10 mer 10 mer RPRPSQPSSL 7 RPRPSQPSSL 0.1 RPSQPSSLAM 14 RPSQPSSLAM 0.25 YPSKSNQEVL 55 LPRFILLELM 0.3 LPRFILLELM 60 YPSKSNQEVL 0.65 SPLQVAVKTL 85 SPLQVAVKTL 0.85 HLA C*07:02 9 mer 9 mer YYRKGGCAM 62 VYEGQVSGM 0.7 YRKGGCAML 194 YRKGGCAML 0.75 VYRRKHQEL 225 VYRRKHQEL 0.85 KWMPPEAFM 257 VRVPRGPAV 1.2 GRLPGASLL 316 YYRKGGCAM 1.35 10 mer 10 mer YYRKGGCAML 64 YYRKGGCAML 0.2 SYYRKGGCAM 145 SYYRKGGCAM 0.5 ERSPAAPPPL 429 RRKHQELQAM 1.2 RRKHQELQAM 443 ERSAPAAPPPL 1.2 GRLPGASLLL 495 SFGVLLWEIF 1.3
[0182] Twenty-three (23) synthetic long peptides (SLP) were synthesized, each approximately thirty (30) amino acids in length (Table 5). The SLPs were synthesized to overlap with one another and span the entire region of ALK, which is translocated in human cancers. The peptides were synthesized by standard solid-phase GMP synthesis. Each peptide was purified by HPLC, and pooled and coupled through amino terminal residues with N-hydroxy succinimidyl ester-end-functionalized poly(ethylene glycol)-lipid (NHS-PEG2KDa-DSPE), forming the amphiphile-peptide conjugate, followed by size exclusion chromatography (SEC) purification.
TABLE-US-00027 TABLE 5 ALK synthetic long peptides (SLPs) 1 VYRRKHQELQAMQMELQSPEYKLSKLRTSTIMTDYN 2 KLRTSTIMTDYNPNYCFAGKTSSISDLKEVPRKNIT 3 SDLKEVPRKNITLIRGLGHGAFGEVYEGQVSGMPND 4 VYEGQVSGMPNDPSPLQVAVKTLPEVCSEQDELDFL 5 EVCSEQDELDFLMEALIISKFNHQNIVRCIGVSLQS 6 NIVRCIGVSLQSLPRFILLELMAGGDLKSFLRETRP 7 GDLKSFLRETRPRPSQPSSLAMLDLLHVARDIACGC 8 LLHVARDIACGCQYLEENHFIHRDIAARNCLLTCPG 9 IAARNCLLTCPGPGRVAKIGDFGMARDIYRASYYRK 10 ARDIYRASYYRKGGCAMLPVKWMPPEAFMEGIFTSK 11 PEAFMEGIFTSKTDTWSFGVLLWEIFSLGYMPYPSK 12 IFSLGYMPYPSKSNQEVLEFVTSGGRMDPPKNCPGP 13 GRMDPPKNCPGPVYRIMTQCWQHQPEDRPNFAIILE 14 PEDRPNFAIILERIEYCTQDPDVINTALPIEYGPLV 15 NTALPIEYGPLVEEEEKVPVRPKDPEGVPPLLVSQQ 16 PEGVPPLLVSQQAKREEERSPAAPPPLPTTSSGKAA 17 PPLPTTSSGKAAKKPTAAEISVRVPRGPAVEGGHVN 18 PRGPAVEGGHVNMAFSQSNPPSELHKVHGSRNKPTS 19 HKVHGSRNKPTSLWNPTYGSWFTEKPTKKNNPIAKK 20 KPTKKNNPIAKKEPHDRGNLGLEGSCTVPPNVATGR 21 SCTVPPNVATGRLPGASLLLEPSSLTANMKEVPLFR 22 LTANMKEVPLFRLRHFPCGNVNYGYQQQGLPLEAAT 23 GYQQQGLPLEAATAPGAGHYEDTILKSKNSMNQPGP
Example 2.2 ALK Amph-Peptides Elicit Protective T-Cell Responses
[0183] To determine whether, ALK amph-peptides elicited a T-cell response, Balb/c mice (Charles River Laboratories) were immunized with a series of different ALK amph-peptides and then assayed for ex vivo antigen-specific cytokine production. ALK amph-peptides may be generated as provided in H. Liu et al., Structure-based programming of lymph-node targeting in molecular vaccines. Nature 507, 5199522 (2014). The ALK amph-peptides assayed include: VYRRKHQEL (ALK Peptide 1); PGPGRVAKI (ALK Peptide 9 short); PGPGRVAKIGDFGMARDIY (ALK Peptide 9 long); and CGNVNYGYQQQGLPLEAAT (ALK Peptide 22/23).
Example 3. ALK Vaccination Schedule for NSCLC Patients Harboring ALK Rearrangements
[0184] In selecting patients with advanced ALK-positive NSCLC for administration of an ALK vaccine, patients were required to have progressive disease and at least one prior ALK tyrosine kinase inhibitor treatment or treatment with multiple ALK tyrosine kinase inhibitors. A washout period of three (3) weeks for standard chemotherapy or five (5) half-lives for oral tyrosine kinase inhibitors was required prior to vaccine administration. At baseline, all patients were required to undergo both blood collection to measure ALK specific autoantibodies and ALK-specific T-cell responses as well as a tumor core biopsy in order to assess for infiltrating immune cell subsets. The first portion of this study consisted of peptide administration alone. Six (6) patients (Cohort 1A) received “naked” ALK peptides, and six (6) patients (Cohort 1B) received amphiphilic-conjugated peptides. Twenty (20) peptides were synthesized and pools of five (5) peptides and the adjuvant poly-ICLC, which is a synthetic complex of carboxymethylcellulose, polyinosinic-polycytidylic acid, and poly-L-lysine double-stranded RNA, were injected into four (4) different anatomic sites that drain to separate lymph node basins in order to reduce potential antigenic competition between peptides binding to the same HLA molecule. ALK vaccine was administered on days 1, 8, 15, 29, 43, and 57 with a booster vaccine at 16 and 24 weeks according to the schedule shown in
Example 4. Efficacy of ALK-Peptide Vaccine in Combination with ALK Inhibitor Therapy and Immune Checkpoint Therapy
[0185] To test the efficacy of an ALK-peptide vaccine in combination with ALK inhibitor therapy and immune checkpoint therapy, a non-small cell lung cancer (NSCLC) mouse model was utilized. To perform the experiments in immunocompetent mice, EML4-ALK rearranged NSCLC cell line syngeneic to the BALB/c mouse strain was generated. These cells were further edited by CRISPR/Cas9 technology to introduce in the ALK gene a CD8.sup.+ epitope specific for BALB/c mice. These NSCLC cell lines colonize the lung when injected into the tail vein and generate lung tumors. Once tumors formed in the lungs, mice were treated with either: i) the ALK-specific inhibitor, lorlatinib, for 15 days (n=9); ii) with a combination of lorlatinib and the check-point inhibitor, PD-1, (n=9); iii) with lorlatinib and the ALK vaccine (n=9); or iv) a combination of lorlatinib, anti-PD1 and ALK vaccine (n=8) (
OTHER EMBODIMENTS
[0186] From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
[0187] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of some embodiments herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
[0188] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.