Individualized vaccines for cancer

Abstract

The present invention relates to the provision of vaccines which are specific for a patient's tumor and are potentially useful for immunotherapy of the primary tumor as well as tumor metastases. In one aspect, the present invention relates to a method for providing an individualized cancer vaccine comprising the steps: (a) identifying cancer specific somatic mutations in a tumor specimen of a cancer patient to provide a cancer mutation signature of the patient; and (b) providing a vaccine featuring the cancer mutation signature obtained in step (a). In a further aspect, the present invention relates to vaccines which are obtainable by said method.

1 - 45 . (canceled) 
     
     
         46 . A method for preparing an individualized cancer vaccine comprising the steps:
 (a) identifying in a tumor specimen of a cancer patient cancer specific somatic mutations to provide a cancer mutation signature of the patient comprising cancer specific somatic mutations, wherein the cancer cells of the patient express antigens comprising said mutations and wherein said mutations are not found in non-cancerous cells of the patient; and   (b) preparing an RNA vaccine featuring the cancer mutation signature obtained in step (a), wherein the RNA vaccine comprises RNA encoding a recombinant polyepitopic polypeptide comprising neo-epitopes comprising said mutations fused together by peptide bonds or linkers.   
     
     
         47 . The method according to  claim 46 , wherein the step of identifying cancer specific somatic mutations comprises identifying the cancer mutation signature of the exome of one or more cancer cells. 
     
     
         48 . The method according to  46 , wherein the step of identifying cancer specific somatic mutations comprises single cell sequencing of one or more cancer cells. 
     
     
         49 . The method according to  claim 48 , wherein the cancer cells are circulating tumor cells. 
     
     
         50 . The method according to  claim 46 , wherein the step of identifying cancer specific somatic mutations involves using next generation sequencing (NGS). 
     
     
         51 . The method according to  claim 46 , wherein the step of identifying cancer specific somatic mutations comprises sequencing genomic DNA and/or RNA of the tumor specimen. 
     
     
         52 . The method according to  claim 46 , wherein the step of identifying cancer specific somatic mutations is replicated at least in duplicates. 
     
     
         53 . The method according to  claim 46 , comprising the further step of determining the usability of the identified mutations in epitopes for cancer vaccination, wherein determining the usability comprises one or more of the following:
 (i) assessing whether the identified mutations are located in known or predicted WIC presented epitopes,   (ii) in vitro and/or in silico testing whether the identified mutations are located in WIC presented epitopes, and   (iii) in vitro testing whether the identified mutations are able to stimulate T cells of the patient having the desired specificity.   
     
     
         54 . The method according to  claim 46 , wherein the polypeptide comprises 5 or more, 10 or more, or 20 or more neo-epitopes. 
     
     
         55 . The method according to  claim 46 , wherein the polypeptide comprises up to 30 neo-epitopes. 
     
     
         56 . The method according to  claim 46 , wherein the polypeptide further comprises epitopes not containing cancer specific somatic mutations which are expressed by cancer cells. 
     
     
         57 . The method according to  claim 46 , wherein the polypeptide comprises neo-epitopes that are based on primary basal mutations. 
     
     
         58 . The method according to  claim 46 , wherein the neo-epitopes are flanked by amino acid sequences flanking said neo-epitopes in the naturally occurring protein so as to form a vaccine sequence. 
     
     
         59 . The method according to  claim 46 , wherein the neo-epitopes are lined up head-to-tail. 
     
     
         60 . The method according to  claim 46 , wherein the neo-epitopes are spaced by linkers. 
     
     
         61 . The method according to  claim 46 , wherein the RNA further comprises an unmasked poly-A sequence. 
     
     
         62 . The method according to  claim 61 , wherein the unmasked poly-A sequence has a length of approximately 120 adenosine residues. 
     
     
         63 . The method according to  claim 46 , wherein the RNA further comprises a 3′ UTR. 
     
     
         64 . The method according to  claim 46 , wherein the recombinant polyepitopic polypeptide encoded by the RNA further comprises an MHC class I signal peptide fragment, a transmembrane domain or a cytosolic domain. 
     
     
         65 . The method according to  claim 60 , wherein at least 50% of the amino acids of the linkers are glycine and/or serine residues. 
     
     
         66 . The method according to  claim 46 , wherein the RNA further comprises a 5′ cap. 
     
     
         67 . The method according to  claim 66 , wherein the 5′ cap is: 
       
         
           
           
               
               
           
         
       
       wherein R 1  and R 2  are independently hydroxy or methoxy, and W, X, and Y are independently oxygen, sulfur, selenium or BH 3 . 
     
     
         68 . The method according to  claim 67 , wherein R 1  and R 2  are hydroxy, and W, X, and Y are oxygen. 
     
     
         69 . The method according to  claim 67 , wherein one of R 1  and R 2  is hydroxy, the other one is methoxy, and W, X, and Y are oxygen. 
     
     
         70 . The method according to  claim 67 , wherein X is sulfur. 
     
     
         71 . The method according to  claim 70 , wherein W and Y are oxygen. 
     
     
         72 . The method according to  claim 66 , wherein the 5′ cap is m 2   7,2′-O Gpp s pG. 
     
     
         73 . The method according to  claim 72 , wherein m 2   7,2′-O Gpp s pG is in the Rp diastereoisomeric form. 
     
     
         74 . The method according to  claim 72 , wherein m 2   7,2′-O Gpp s pG is in the Sp diastereoisomeric form. 
     
     
         75 . An RNA vaccine which is obtained by the method according to  claim 46 , wherein the vaccine, when administered to the patient, provides a collection of MHC presented epitopes incorporating sequence changes based on the identified mutations. 
     
     
         76 . The RNA vaccine according to  claim 75 , wherein the MHC presented epitopes are MHC class II-presented epitopes that elicit a CD4+ helper T cell response against cells expressing antigens from which the MHC presented epitopes are derived and/or MHC class I-presented epitopes that elicit a CD8+ T cell response against cells expressing antigens from which the MHC presented epitopes are derived. 
     
     
         77 . An RNA vaccine comprising RNA encoding a recombinant polypeptide comprising neo-epitopes fused together by peptide bonds or linkers, said neo-epitopes resulting from cancer specific somatic mutations in a tumor specimen of a cancer patient. 
     
     
         78 . The RNA vaccine according to  claim 77 , wherein the polypeptide comprises 5 or more, 10 or more, or 20 or more neo-epitopes. 
     
     
         79 . The RNA vaccine according to  claim 77 , wherein the polypeptide comprises up to 30 neo-epitopes. 
     
     
         80 . The RNA vaccine according to  claim 77 , wherein the polypeptide further comprises epitopes not containing cancer specific somatic mutations which are expressed by cancer cells. 
     
     
         81 . The RNA vaccine according to  claim 77 , wherein the polypeptide comprises neo-epitopes that are based on primary basal mutations. 
     
     
         82 . The RNA vaccine according to  claim 77 , wherein the neo-epitopes are flanked by amino acid sequences flanking said neo-epitopes in the naturally occurring protein so as to form a vaccine sequence. 
     
     
         83 . The RNA vaccine according to  claim 77 , wherein the neo-epitopes are lined up head-to-tail. 
     
     
         84 . The RNA vaccine according to  claim 77 , wherein the neo-epitopes are spaced by linkers. 
     
     
         85 . The RNA vaccine according to  claim 77 , wherein the vaccine, when administered to the patient, provides a collection of MHC presented epitopes incorporating sequence changes based on the tumor specific mutations. 
     
     
         86 . The RNA vaccine according to  claim 85 , wherein the MHC presented epitopes are MHC class II-presented epitopes that elicit a CD4+ helper T cell response against cells expressing antigens from which the MHC presented epitopes are derived and/or MHC class I-presented epitopes that elicit a CD8+ T cell response against cells expressing antigens from which the MHC presented epitopes are derived. 
     
     
         87 . The RNA vaccine according to  claim 77 , wherein the RNA further comprises an unmasked poly-A sequence. 
     
     
         88 . The RNA vaccine according to  claim 87 , wherein the unmasked poly-A sequence has a length of approximately 120 adenosine residues. 
     
     
         89 . The RNA vaccine according to  claim 77 , wherein the RNA further comprises a 3′ UTR. 
     
     
         90 . The RNA vaccine according to  claim 77 , wherein the polyepitopic polypeptide encoded by the RNA further comprises an MHC class I signal peptide fragment, a transmembrane domain, or a cytosolic domain. 
     
     
         91 . The RNA vaccine according to  claim 84 , wherein at least 50% of the amino acids of the linkers are glycine and/or serine residues. 
     
     
         92 . The RNA vaccine according to  claim 77 , wherein the RNA further comprises a 5′ cap. 
     
     
         93 . The RNA vaccine according to  claim 92 , wherein the 5′ cap is: 
       
         
           
           
               
               
           
         
       
       wherein R 1  and R 2  are independently hydroxy or methoxy, and W, X, and Y are independently oxygen, sulfur, selenium or BH 3 . 
     
     
         94 . The RNA vaccine according to  claim 93 , wherein R 1  and R 2  are hydroxy, and W, X, and Y are oxygen. 
     
     
         95 . The RNA vaccine according to  claim 93 , wherein one of R 1  and R 2  is hydroxy, the other one is methoxy, and W, X, and Y are oxygen. 
     
     
         96 . The RNA vaccine according to  claim 93 , wherein X is sulfur. 
     
     
         97 . The RNA vaccine according to  claim 96 , wherein W and Y are oxygen. 
     
     
         98 . The RNA vaccine according to  claim 92 , wherein the 5′ cap is m 2   7,2′-O Gpp s pG. 
     
     
         99 . The RNA vaccine according to  claim 98 , wherein m 2   7,2′-O Gpp s pG is in the Rp diastereoisomeric form. 
     
     
         100 . The RNA vaccine according to  claim 98 , wherein m 2   7,2′-O Gpp s pG is in the Sp diastereoisomeric form. 
     
     
         101 . A method for preparing an individualized cancer vaccine comprising the steps:
 (a) identifying cancer specific somatic mutations in a tumor specimen of a cancer patient to provide a cancer mutation signature of the cancer patient comprising cancer specific somatic mutations, comprising
 (aa) obtaining nucleic acid sequence information by sequencing genomic DNA and/or RNA of the tumor specimen of the cancer patient, 
 (bb) obtaining reference nucleic acid sequence information by sequencing DNA and/or RNA of normal non-cancerous cells, and 
 (cc) comparing the nucleic acid sequence information from the tumor specimen obtained in step (aa) with the reference nucleic acid sequence information obtained in step (bb); and 
   (b) preparing an RNA vaccine featuring the cancer mutation signature obtained in step (a), wherein the RNA vaccine comprises RNA encoding a recombinant polyepitopic polypeptide comprising neo-epitopes fused together by peptide bonds or linkers.   
     
     
         102 . The method according to  claim 101 , wherein the step of identifying cancer specific somatic mutations comprises identifying the cancer mutation signature of the exome of one or more cancer cells. 
     
     
         103 . The method according to  claim 101 , wherein the step of identifying cancer specific somatic mutations comprises single cell sequencing of one or more cancer cells. 
     
     
         104 . The method according to  claim 103 , wherein the cancer cells are circulating tumor cells. 
     
     
         105 . The method according to  claim 101 , wherein the step of identifying cancer specific somatic mutations involves using next generation sequencing (NGS). 
     
     
         106 . The method according to  claim 101 , wherein the normal non-cancerous cells are obtained from the cancer patient. 
     
     
         107 . The method according to  claim 101 , wherein the reference nucleic acid sequence information is obtained from genomic DNA obtained from peripheral blood mononuclear cells (PBMCs). 
     
     
         108 . The method according to  claim 107 , wherein the tumor specimen is from a primary tumor, and wherein the step of identifying cancer specific somatic mutations in the tumor specimen further comprises the steps:
 (dd) preparing a phylogenetic tree of cancer specific somatic mutations, wherein the reference nucleic acid sequence information obtained in step (bb) is used to root the tree,   (ee) reproducing ancestral sequences, wherein the ancestral sequences are sequences of nodes near the root of the phylogenetic tree containing primary basal mutations, wherein the primary basal mutations are the earliest mutations predicted to exist in the primary tumor; and   (ff) selecting the primary basal mutations from the ancestral sequences identified in step (ee).   
     
     
         109 . The method according to  claim 101 , wherein the step of identifying cancer specific somatic mutations is replicated at least in duplicates. 
     
     
         110 . The method according to  claim 101 , comprising the further step of determining the usability of the identified mutations in epitopes for cancer vaccination, wherein determining the usability comprises one or more of the following:
 (i) assessing whether the identified mutations are located in known or predicted WIC presented epitopes,   (ii) in vitro and/or in silico testing whether the identified mutations are located in WIC presented epitopes, and   (iii) in vitro testing whether the identified mutations are able to stimulate T cells of the patient having the desired specificity.   
     
     
         111 . The method according to  claim 101 , wherein the polypeptide comprises 5 or more, 10 or more, or 20 or more neo-epitopes. 
     
     
         112 . The method according to  claim 101 , wherein the polypeptide comprises up to 30 neo-epitopes. 
     
     
         113 . The method according to  claim 101 , wherein the polypeptide further comprises epitopes not containing cancer specific somatic mutations which are expressed by cancer cells. 
     
     
         114 . The method according to  claim 101 , wherein the polypeptide comprises neo-epitopes that are based on primary basal mutations. 
     
     
         115 . The method according to  claim 101 , wherein the neo-epitopes are flanked by amino acid sequences flanking said neo-epitopes in the naturally occurring protein so as to form a vaccine sequence. 
     
     
         116 . The method according to  claim 101 , wherein the neo-epitopes are lined up head-to-tail and/or are spaced by linkers. 
     
     
         117 . The method according to  claim 101 , wherein the RNA further comprises an unmasked poly-A sequence. 
     
     
         118 . The method according to  claim 117 , wherein the unmasked poly-A sequence has a length of approximately 120 adenosine residues. 
     
     
         119 . The method according to  claim 101 , wherein the RNA further comprises a 3′ UTR. 
     
     
         120 . The method according to  claim 101 , wherein the polyepitopic polypeptide encoded by the RNA further comprises an MHC class I signal peptide fragment, a transmembrane domain or a cytosolic domain. 
     
     
         121 . The method according to  claim 116 , wherein at least 50% of the amino acids of the linkers are glycine and/or serine residues. 
     
     
         122 . The method according to  claim 101 , wherein the RNA further comprises a 5′ cap. 
     
     
         123 . The method according to  claim 122 , wherein the 5′ cap is: 
       
         
           
           
               
               
           
         
         wherein R 1  and R 2  are independently hydroxy or methoxy, and W, X, and Y are independently oxygen, sulfur, selenium or BH 3 . 
       
     
     
         124 . The method according to  claim 123 , wherein R 1  and R 2  are hydroxy, and W, X, and Y are oxygen. 
     
     
         125 . The method according to  claim 123 , wherein one of R 1  and R 2  is hydroxy, the other one is methoxy, and W, X, and Y are oxygen. 
     
     
         126 . The method according to  claim 123 , wherein X is sulfur. 
     
     
         127 . The method according to  claim 126 , wherein W and Y are oxygen. 
     
     
         128 . The method according to  claim 122 , wherein the 5′ cap is m 2   7,2′-O Gpp s pG. 
     
     
         129 . The method according to  claim 128 , wherein m 2   7,2′-O Gpp s pG is in the Rp diastereoisomeric form. 
     
     
         130 . The method according to  claim 128 , wherein m 2   7,2′-O Gpp s pG is in the Sp diastereoisomeric form. 
     
     
         131 . An RNA vaccine which is obtained by the method according to  claim 101 , wherein the vaccine, when administered to the patient, provides a collection of MHC presented epitopes incorporating sequence changes based on the identified mutations. 
     
     
         132 . The RNA vaccine according to  claim 131 , wherein the MHC presented epitopes are MHC class II-presented epitopes that elicit a CD4+ helper T cell response against cells expressing antigens from which the MHC presented epitopes are derived and/or MHC class I-presented epitopes that elicit a CD8+ T cell response against cells expressing antigens from which the MHC presented epitopes are derived. 
     
     
         133 . Use of the individualized cancer vaccine prepared by the method according to  claim 101  for treating the cancer of said patient. 
     
     
         134 . Use of the RNA vaccine according to  claim 131  for treating the cancer of said patient. 
     
     
         135 . Use of the individualized cancer vaccine prepared by the method according to  claim 46  for treating the cancer of said patient. 
     
     
         136 . Use of the RNA vaccine according to  claim 75  for treating the cancer of said patient. 
     
     
         137 . A method of treating cancer, the method comprising a step of:
 administering to cancer patients an RNA vaccine, wherein:   
       each patient receives an individualized vaccine comprising an RNA that encodes a recombinant polyepitopic polypeptide comprising a plurality of neoepitopes expressed by that patient's tumor, the neoepitopes being linked to one another in the polyeptopic polypeptide by linker sequences. 
     
     
         138 . A method of producing a vaccine specific for an individual patient's tumor, the method comprising steps of:
 (a) synthesizing an RNA that encodes a recombinant polyepitopic polypeptide comprising a plurality of neoeptiopes spaced apart by linker sequences, wherein each of the neoepitopes:
 i. is expressed by the individual patient's tumor; 
 ii. includes a somatic mutation relative to non-tumor sequence in the individual patient; 
 iii. satisfies WIC presentation prediction. 
   
     
     
         139 . The method of  claim 138 , wherein detection of RNA encoding the neoepitope in a tumor sample from the individual patient determines that the neoeptitope is expressed in the individual patient's tumor. 
     
     
         140 . The method of  claim 138 , wherein the plurality of neoepitopes comprises neoepitopes from a plurality of different antigens. 
     
     
         141 . The method of  claim 138 , further comprising a step of administering the synthesized RNA to the individual patient. 
     
     
         142 . An individualized cancer vaccine comprising an RNA encoding a recombinant polyepitopic polypeptide, wherein said polypeptide comprises two or more immunogenic neo-epitopes expressed by tumor cells in a particular patient, the vaccine produced by a method comprising steps of:
 (a) obtaining nucleic acid sequence information from a sample comprising tumor cells from a patient;   (b) obtaining nucleic acid sequence information from a sample comprising non-tumor cells from the same patient;   (c) comparing the tumor cell sequence information with the non-tumor-cell sequence information so that somatic mutations present in the tumor cell sequence information are identified;   (d) classifying as immunogenic neo-epitopes at least two of the identified somatic mutations, wherein each immunogenic neo-epitope is characterized in that it:
 i. occurs in a transcript; 
 ii. occurs in a protein-coding region; 
 iii. introduces a change in amino acid sequence; 
 iv. is predicted to exhibit MHC binding; and 
   (e) producing an RNA encoding a recombinant polyepitopic polypeptide, wherein said polypeptide comprises two or more immunogenic neo-epitopes identified in steps (a)-(d) and the neo-epitopes are fused together by peptide bonds or linkers.   
     
     
         143 . The individualized vaccine of  claim 142 , wherein the method further comprises a step of formulating the RNA with one or more pharmaceutically acceptable components. 
     
     
         144 . The individualized vaccine of  claim 143 , wherein the one or more pharmaceutically acceptable components are or comprise one or more lipids. 
     
     
         145 . An individualized cancer vaccine comprising an RNA encoding a recombinant polyepitopic polypeptide, wherein said polypeptide comprises two or more immunogenic neo-epitopes expressed by tumor cells in a particular patient, wherein each immunogenic neo-epitope is characterized in that it:
 i. occurs in a transcript;   ii. occurs in a protein-coding region;   iii. introduces a change in amino acid sequence; and   iv. is predicted to exhibit MHC binding.