Patent Publication Number: US-2023139506-A1

Title: Heteroclitic cancer vaccines

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of priority of U.S. Provisional Patent Application No. 62/990,696 filed on Mar. 17, 2020, the content of which is hereby incorporated by reference in its entirety. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH 
     This invention was made with government support under CA008748 awarded by the National Institutes of Health. The government has certain rights in the invention. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 16, 2021, is named MSKCC_045_WO1_SL.txt and is 73,360 bytes in size. 
     INCORPORATION BY REFERENCE 
     For the purposes of only those jurisdictions that permit incorporation by reference, all of the references cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers&#39; instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Numbers in superscript or parentheses following text herein refer to the numbered references identified in the “Reference List” section of this patent application. 
     BACKGROUND 
     The majority of JAK2 mutant-negative myeloproliferative neoplasms (MPN) have a disease-initiating frameshift mutation in calreticulin (CALR) resulting in a common 44-amino acid novel C-terminal mutant fragment (CALR MUT ), representing an attractive potential source of neoantigens for cancer vaccines. However, prior studies examining CALR MUT  fragment immunogenicity found that T cells from CALR MUT  MPN patients had diminished immunoreactivity to CALR MUT -derived peptides compared to healthy individuals—even though the peptides were predicted to be immunogenic. Accordingly, there is a need in the art for new approaches to the development of CALR MUT  cancer vaccines. The present invention addresses this need. 
     SUMMARY OF THE INVENTION 
     Some of the main aspects of the present invention are summarized below. Additional aspects are described in the Detailed Description of the Invention, Examples, Figures (Drawings), Brief Description of the Figures, and Claims sections of this disclosure. The description in each section of this patent disclosure, regardless of any heading or sub-heading titles, is intended to be read in conjunction with all other sections. Furthermore, the various embodiments described in each section of this disclosure can be combined in various different ways, and all such combinations are intended to fall within the scope of the present invention. 
     The present invention is based, in part, on certain discoveries that are described in more detail in the “Examples” section of this patent application. In brief, we investigated two independent myeloproliferative neoplasm (MPN) patient cohorts and found that six MHC-I alleles predicted to efficiently bind to multiple CALR MUT -derived peptides are less frequently observed in CALR MUT  MPN patients. This strongly pointed to a higher risk of developing CALR MUT  MPN in patients lacking these MHC-I alleles and, at the same time, suggested to us that individuals with these MHC-I alleles could potentially control primordial CALR MUT -expressing tumors as part of the immunoediting process. In addition, this suggested to us that CALR MUT -positive MPN patients were unlikely to respond to cancer vaccines composed of the CALR MUT  fragment. Therefore, we analyzed the CALR MUT  fragment for peptides that could be modified into heteroclitic peptides and designed numerous heteroclitic CALR MUT  peptides to serve as more potent anti-CALR MUT  immunogens. We tested our heteroclitic CALR MUT  peptides in vitro using human peripheral blood mononuclear cells (PBMCs) from healthy donors unable to respond to CALR MUT  peptides, and found that the same T cells could be induced to release IFNγ when primed using the heteroclitic peptides that we designed. Then, to verify whether these heteroclitic CALR MUT  peptides could control the growth of CALR MUT  tumors in vivo, we performed tests in a pre-clinical mouse model. We showed that mice that were unable to mount an immune response against the original CALR MUT  fragment, had significantly delayed tumor growth when given a heteroclitic CALR MUT  peptide vaccine of the same specificity and that this was further enhanced by PD1 blockade. 
     Based on these studies, the present invention provides numerous heteroclitic CALR MUT  peptides that were specifically designed and selected to elicit an immune response to CALR MUT . The amino acid sequences and SEQ ID NOs of these peptides, as well as those of the parental non-heteroclitic CALR MUT  peptides from which they were derived, are provided in Table A and Table B in the Detailed Description section of this patent disclosure. The present invention also provides nucleic acid molecules encoding these peptides, and numerous related compositions and methods, as described further herein. 
     Accordingly, in some embodiments, the present invention provides an isolated heteroclitic CALR MUT  peptide derived from SEQ ID NO. 268, wherein the peptide comprises at least one point mutation as compared to SEQ ID NO. 268. In some such embodiments the heteroclitic CALR MUT  peptide is 9-12 amino acids in length. 
     In some embodiments, the present invention provides an isolated heteroclitic CALR MUT  peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 1-262. 
     Similarly, in some embodiments, the present invention provides an isolated heteroclitic CALR MUT  peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 1-46. 
     In some embodiments, the present invention provides an isolated heteroclitic CALR MUT  peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 1, 2, 4, 5, 6, 8 and 40. 
     In some embodiments, the present invention provides an isolated heteroclitic CALR MUT  peptide comprising the amino acid sequence of SEQ ID NO. 40. 
     In some embodiments, the present invention provides an isolated heteroclitic CALR MUT  peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 47-59. 
     In some embodiments, the present invention provides an isolated heteroclitic CALR MUT  peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 60-85. 
     In some embodiments, the present invention provides an isolated heteroclitic CALR MUT  peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 86-103. 
     In some embodiments, the present invention provides an isolated heteroclitic CALR MUT  peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 104-125. 
     In some embodiments, the present invention provides an isolated heteroclitic CALR MUT  peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 126-139. 
     In some embodiments, the present invention provides an isolated heteroclitic CALR MUT  peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 140-157. 
     In some embodiments, the present invention provides an isolated heteroclitic CALR MUT  peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 158-172. 
     In some embodiments, the present invention provides an isolated heteroclitic CALR MUT  peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 173-183. 
     In some embodiments, the present invention provides an isolated heteroclitic CALR MUT  peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 184-215. 
     In some embodiments, the present invention provides an isolated heteroclitic CALR MUT  peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 216-236 
     In some embodiments, the present invention provides an isolated heteroclitic CALR MUT  peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 237-262. 
     In some embodiments, the present invention provides an isolated peptide comprising a heteroclitic derivative of SEQ ID NO. 263 (CALR9p2). In some embodiments, such heteroclitic derivatives of CALR9p2 comprise a single point mutation selected from K6F, R1Y, R1F, K6I, K6L, K6V, K6M and T5F. Examples of such heteroclitic derivatives of CALR9p2 are those having the amino acid sequence of SEQ ID NO. 1 (K6F), SEQ ID NO. 2 (R1Y), SEQ ID NO. 3 (R1F), SEQ ID NO. 4 (K6I), SEQ ID NO. 5 (K6L), SEQ ID NO. 6 (K6V), SEQ ID NO. 8 (K6M), or SEQ ID NO. 40 (T5F). In some embodiments, such heteroclitic derivatives of CALR9p2 comprise two point-mutations selected from K6F, R1Y, R1F, K6I, K6L, K6V, K6M and T5F. 
     In some embodiments, the present invention provides an isolated peptide comprising a heteroclitic derivative of SEQ ID NO. 264. In some embodiments, such a heteroclitic derivative comprises a single point mutation. In some embodiments, such a heteroclitic derivative comprises two point mutations. 
     In some embodiments, the present invention provides an isolated peptide comprising a heteroclitic derivative of SEQ ID NO. 265. In some embodiments, such a heteroclitic derivative comprises a single point mutation. In some embodiments, such a heteroclitic derivative comprises two point mutations. 
     In some embodiments, the present invention provides an isolated peptide comprising a heteroclitic derivative of SEQ ID NO. 266. In some embodiments, such a heteroclitic derivative comprises a single point mutation. In some embodiments, such a heteroclitic derivative comprises two point mutations. 
     In some embodiments, the present invention provides an isolated peptide comprising a heteroclitic derivative of SEQ ID NO. 267. In some embodiments, such a heteroclitic derivative comprises a single point mutation. In some embodiments, such a heteroclitic derivative comprises two point mutations. 
     In some embodiments, the present invention provides an isolated peptide comprising a heteroclitic derivative of SEQ ID NO. 268. In some embodiments, such a heteroclitic derivative comprises a single point mutation. In some embodiments, such a heteroclitic derivative comprises two point mutations. 
     In some embodiments, the present invention provides an isolated peptide comprising a heteroclitic derivative of SEQ ID NO. 269. In some embodiments, such a heteroclitic derivative comprises a single point mutation. In some embodiments, such a heteroclitic derivative comprises two point mutations. 
     In some embodiments, the present invention provides an isolated peptide comprising a heteroclitic derivative of SEQ ID NO. 270. In some embodiments, such a heteroclitic derivative comprises a single point mutation. In some embodiments, such a heteroclitic derivative comprises two point mutations. 
     In some embodiments, the present invention provides an isolated peptide comprising a heteroclitic derivative of SEQ ID NO. 271. In some embodiments, such a heteroclitic derivative comprises a single point mutation. In some embodiments, such a heteroclitic derivative comprises two point mutations. 
     In some embodiments, the present invention provides an isolated peptide comprising a heteroclitic derivative of SEQ ID NO. 272. In some embodiments, such a heteroclitic derivative comprises a single point mutation. In some embodiments, such a heteroclitic derivative comprises two point mutations. 
     In some embodiments, the present invention provides an isolated peptide comprising a heteroclitic derivative of SEQ ID NO. 273. In some embodiments, such a heteroclitic derivative comprises a single point mutation. In some embodiments, such a heteroclitic derivative comprises two point mutations. 
     In some embodiments, the present invention provides an isolated peptide comprising a heteroclitic derivative of SEQ ID NO. 274. In some embodiments, such a heteroclitic derivative comprises a single point mutation. In some embodiments, such a heteroclitic derivative comprises two point mutations. 
     An analysis of the amino acid sequences of all of these heteroclitic CALR MUT  peptides, in comparison to the parental non-heteroclitic CALR MUT  peptides from which they were derived, enabled us to deduce certain common features and “consensus” amino acid sequences for our heteroclitic CALR MUT  peptides. 
     Accordingly, in some embodiments the present invention provides an isolated heteroclitic CALR MUT  peptide comprising the amino acid sequence: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO. 275) 
               
               
                   
                 X1X2M3X4X5X6X7X8X9  
               
            
           
         
       
         
         
           
             wherein, independently of each other, 
             X1 is selected from: R, Y, F, M, and W, 
             X2 is selected from: M, Y, P, S, T, A, E, R, Q, F, and W, 
             X4 is selected from: R, D, and E, 
             X5 is selected from: T, W, Y, H, K, R, and F, 
             X6 is selected from: K, F, I, L, V, M, W, Y, T, C, N, and S, 
             X7 is selected from: M, and W, 
             X8 is selected from: R, A, P, S, Y, and F, and 
             X9 is selected from: M, K, V, F, R, Y, W, and H, and 
             and wherein, the amino acid sequence comprises at least one point mutation as compared to CALR9p2 (SEQ ID NO. 263). 
           
         
       
    
     And in some embodiments the present invention provides an isolated heteroclitic CALR MUT  peptide comprising the amino acid sequence: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO. 276) 
               
               
                   
                 X1X2M3R4R5M6R7X8X9 
               
            
           
         
       
         
         
           
             wherein, independently of each other, 
             X1 is selected from: M, and R, 
             X2 is selected from: R, P, L, and M, 
             X8 is selected from: R, A, P, and S, and 
             X9 is selected from: T, L, M, I, V, F, and Y, and 
             wherein, the amino acid sequence comprises at least one point mutation as compared to CALRp8 (SEQ ID NO. 264). 
           
         
       
    
     In other embodiments the present invention provides an isolated heteroclitic CALR MUT  peptide comprising the amino acid sequence: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO. 277) 
               
               
                   
                 X1X2X3X4X5M6X7P8X9 
               
            
           
         
       
         
         
           
             wherein, independently of each other, 
             X1 is selected from: K, F, Y, and M, 
             X2 is selected from: M, and P, 
             X3 is selected from: R, F, M, I, W, Y, L, A, V, N, and S, 
             X4 is selected from: R, E, and D, 
             X5 is selected from: K, and F, 
             X7 is selected from: S, and W, and 
             X9 is selected from: A, Y, K, L, F, M, R, W, and V, and 
             wherein, the amino acid sequence comprises at least one point mutation as compared to CALR9p19 (SEQ ID NO. 265). 
           
         
       
    
     In yet further embodiments the present invention provides an isolated heteroclitic CALR MUT  peptide comprising the amino acid sequence: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO. 278) 
               
               
                   
                 R1X2X3C4R5X6A7C8X9 
               
            
           
         
       
         
         
           
             wherein, independently of each other, 
             X2 is selected from: T, P, E, Q, L, M, Y, and R, 
             X3 is selected from: S, K, and R, 
             X6 is selected from: E, F, H, R, W, and Y, and 
             X9 is selected from: L, K, W, R, and Y, and 
             wherein, the amino acid sequence comprises at least one point mutation as compared to CALR9p30 (SEQ ID NO. 266). 
           
         
       
    
     In other embodiments the present invention provides an isolated heteroclitic CALR MUT  peptide comprising the amino acid sequence: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO. 279) 
               
               
                   
                 X1X2X3M4R5M6R7X8X9 
               
            
           
         
       
         
         
           
             wherein, independently of each other, 
             X1 is selected from: R, D, E, F, H, and Y, 
             X2 is selected from: T, P, Q, and R, 
             X3 is selected from: K, M, F, Y, W, A, I, L, and V, 
             X8 is selected from: R, A, and P, and 
             X9 is selected from: M, R, K, W, and Y, and 
             wherein, the amino acid sequence comprises at least one point mutation as compared to CALR9p5 (SEQ ID NO. 267). 
           
         
       
    
     In some embodiments the present invention provides an isolated heteroclitic CALR MUT  peptide comprising the amino acid sequence: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO. 280) 
               
               
                   
                 X1X2X3R4R5T6R7R8K9X10 
               
            
           
         
       
         
         
           
             wherein, independently of each other, 
             X1 is selected from: R, F, Y, M, and W, 
             X2 is selected from: R, P, L, M, Q, S, T, Y, and E, 
             X3 is selected from: M, and P, and 
             X10 is selected from: M, and R, and 
             wherein, the amino acid sequence comprises at least one point mutation as compared to CALR10p11 (SEQ ID NO. 268). 
           
         
       
    
     In some embodiments the present invention provides an isolated heteroclitic CALR MUT  peptide comprising the amino acid sequence: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO. 281) 
               
               
                   
                 R1X2X3X4R5K6M7X8P9X10 
               
            
           
         
       
         
         
           
             wherein, independently of each other, 
             X2 is selected from: K, P, R, L, M, and E, 
             X3 is selected from: M, and P, 
             X4 is selected from: R, F, I, M, W, and Y, 
             X8 is selected from: S, and W, and 
             X10 is selected from: A, K, Y, F, R, M, and L, and 
             wherein, the amino acid sequence comprises at least one point mutation as compared to CALR10p18 (SEQ ID NO. 269). 
           
         
       
    
     In some embodiments the present invention provides an isolated heteroclitic CALR MUT  peptide comprising the amino acid sequence: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO. 282) 
               
               
                   
                 X1X2M3X4M5R6R7M8R9X10 
               
            
           
         
       
         
         
           
             wherein, independently of each other, 
             X1 is selected from: T, and R, 
             X2 is selected from: K, T, V, I, A, S, R, and M, 
             X4 is selected from: R, and Y, and 
             X10 is selected from: R, K, L, F, I, M, and V, and 
             wherein, the amino acid sequence comprises at least one point mutation as compared to CALR10p6 (SEQ ID NO. 270). 
           
         
       
    
     In some embodiments the present invention provides an isolated heteroclitic CALR MUT  peptide comprising the amino acid sequence: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO. 283) 
               
               
                   
                 R1X2X3X4X5R6X7X8X9R10K11M12 
               
            
           
         
       
         
         
           
             wherein, independently of each other, 
             X2 is selected from: M, and P, 
             X3 is selected from: R, M, and P, 
             X4 is selected from: R, and P, 
             X5 is selected from: M, and P, 
             X7 is selected from: R, and W, 
             X8 is selected from: T, and W, and 
             X9 is selected from: R, I, L, M, and Y, and 
             wherein, the amino acid sequence comprises at least one point mutation as compared to CALR12p9 (SEQ ID NO. 271). 
           
         
       
    
     In some embodiments the present invention provides an isolated heteroclitic CALR MUT  peptide comprising the amino acid sequence: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO. 284) 
               
               
                   
                 S1X2X3X4P5R6T7X8X9X10 
               
            
           
         
       
         
         
           
             wherein, independently of each other, 
             X2 is selected from: P, F, T, V, Y, I, S, A, M, L, Q, and W, 
             X3 is selected from: A, F, and M, 
             X4 is selected from: R, F, Y, and W, 
             X8 is selected from: S, W, and F, 
             X9 is selected from: C, I, L, M, V, Y, and F, and 
             X10 is selected from: R, L, F, I, M, V, A, K, and Y, and 
             wherein, the amino acid sequence comprises at least one point mutation as compared to CALR10p25 (SEQ ID NO. 272). 
           
         
       
    
     In some embodiments the present invention provides an isolated heteroclitic CALR MUT  peptide comprising the amino acid sequence: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO. 285) 
               
               
                   
                 X1T2K3M4R5M6R7X8M9X10 
               
            
           
         
       
         
         
           
             wherein, independently of each other, 
             X1 is selected from: R, M, E, F, H, N, and Y, 
             X8 is selected from: R, M, F, L, W, Y, I, and V, and 
             X10 is selected from: R, K, W, Y, F, M, I, L, and V, and 
             wherein, the amino acid sequence comprises at least one point mutation as compared to CALR10p5 (SEQ ID NO. 273). 
           
         
       
    
     In some embodiments the present invention provides an isolated heteroclitic CALR MUT  peptide comprising the amino acid sequence: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO. 286) 
               
               
                   
                 R1X2M3R4R5T6R7X8X9 
               
            
           
         
       
         
         
           
             wherein, independently of each other, 
             X2 is selected from: R, I, M, T, V, S, L, A, Q, F, W, Y, C, G, and N, 
             X8 is selected from: R, F, and Y, and 
             X9 is selected from: K, L, M, F, I, V, Y, W, A, C, and T, and 
             wherein, the amino acid sequence comprises at least one point mutation as compared to CALR9p11 (SEQ ID NO. 274). 
           
         
       
    
     The present invention also provides nucleic acid molecules that encode the heteroclitic CALR MUT  peptides described above and elsewhere herein. For example, in some of such embodiments the present invention provides a nucleic acid molecule comprising a nucleic acid sequence that encodes a heteroclitic CALR MUT  peptide as described above and/or elsewhere herein. In some of such embodiments the nucleic acid molecule comprises both a nucleic acid sequence encoding a heteroclitic CALR MUT  peptide and a nucleic acid sequence encoding a signal peptide, wherein the nucleic acid sequence encoding the heteroclitic CALR MUT  peptide is downstream of the nucleic acid sequence encoding the signal peptide. In some embodiments the nucleic acid molecule is a DNA molecule. In some embodiments the nucleic acid molecule comprises a promoter that is operably linked to the nucleic acid sequence encoding the heteroclitic CALR MUT  peptide. In some embodiments the nucleic acid molecule is an RNA molecule. In some embodiments the nucleic acid molecule is an mRNA molecule. 
     The present invention also provides vectors that comprise nucleic acid molecules that encode the heteroclitic CALR MUT  peptides described above and elsewhere herein. In some such embodiments the vectors are viral vectors. In some such embodiments the vectors are selected from the group consisting of: adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, alphavirus vectors, and vaccinia virus vectors. 
     The present invention also provides cells that comprise nucleic acid molecules that encode the heteroclitic CALR MUT  peptides described above and elsewhere herein. 
     In addition to the various heteroclitic CALR MUT  peptides, nucleic acid molecules and vectors described above and elsewhere herein, the present invention also provides various compositions comprising such peptides, nucleic acid molecules, or vectors. In some embodiments such compositions comprise one or more carriers suitable for administration to mammalian subjects. In some embodiments such compositions comprise a delivery vehicle, such as a nanoparticle, a lipid nanoparticle, a liposome, a lipid, a lipid encapsulation system, a polymer or a polymersome. In some such embodiments such compositions comprise an adjuvant. 
     The present invention also provides various methods of treatment. For example, in some embodiments the present invention provides methods of treating JAK2 mutant-negative myeloproliferative neoplasms (MPNs) in subjects in need thereof, such methods involving administering to such subjects an effective amount of a heteroclitic CALR MUT  peptide, nucleic acid molecule, vector or composition as described herein. In some such embodiments the subject has a JAK2 V617F  mutant-negative MPN. In some embodiments such methods involve administering one dose of a heteroclitic CALR MUT  peptide, nucleic acid molecule, vector or composition to the subject. In some embodiments such methods involve administering two or more doses of a heteroclitic CALR MUT  peptide, nucleic acid molecule, vector or composition to the subject. For example, in some embodiments such treatment methods involve administering a priming dose and one or more booster doses of the heteroclitic CALR MUT  peptide, nucleic acid molecule, vector or composition to the subject. In some embodiments such methods also comprise administering an effective amount of an immune checkpoint inhibitor to the subject. Suitable immune checkpoint inhibitors include PD-1, PD-L1, PD-L2 and CTLA-4 inhibitors. In some embodiments the immune checkpoint inhibitor is an anti-PD1 antibody. In some embodiments the treatment methods result in one or more of: (a) an immune response to the JAK2 mutant-negative MPN, (b) a CD8+ T cell response to the JAK2 mutant-negative MPN, (c) an anti-CALR MUT  immune response, (d) an anti-CALR MUT  CD8+ T cell response, and (e) enhanced sensitivity of the to the JAK2 mutant-negative MPN to immune checkpoint blockade. 
     While some of the main embodiments of the present invention are summarized above, additional aspects and additional details are provided and described in the Brief Description of the Figures, Detailed Description of the Invention, Examples, Claims, and Figures sections of this patent application. Furthermore, it should be understood that variations and combinations of each of the embodiments described herein are contemplated and are intended to fall within the scope of the present invention. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG.  1 A-F . MHC-I alleles with predicted binding to CALR MUT -derived peptides are less frequent in CALR MUT  MPNs. A) Principal component analysis of MHC-I frequencies from CALR MUT  MPN patients, JAK2 V617F  MPN patients, and US Caucasian population B) Comparison of MHC-I allele frequencies from each group compared to each other in NEUS cohort and C) Danish cohort. Frequencies are expressed as percentages. MHC-I alleles that are over-represented in CALR MUT  patients are in darker gray (for both the NEUS cohort and Danish cohort), while MHC-I alleles that are under-represented in CALRMUT patients are in mid-gray (for the NEUS cohort and the Danish cohort). For NEUS cohort, only MHC-I alleles differentially expressed between CALR MUT  MPN patients compared to both JAK2 V617F  MPN patients and US Caucasian population were considered. D) Heatmap of predicted binding to each CALR MUT -derived peptides to each MHC-I allele from B (left panel). MHC-I alleles that are under-represented or over-represented in CALR MUT  MPN patients in both cohorts are noted with white or black circles, respectively. Actual MHC-I allele frequencies CALR MUT  and JAK2 V617F  MPN patient populations are also noted (right panel). E) Cohort breakdown of CALR MUT  MPN MHC-I allele frequencies of individual institution consisting of the NEUS cohort for the six less frequent MHC-I alleles. F) IFNγ ELISpot of PBMCs from healthy donors that had either at least one (black circles) or zero (white circles) under-represented MHC-I allele expanded with a peptide pool derived from the entire CALR MUT  fragment and restimulated after 10 days with either the irrelevant peptide (MOG) or the same CALR MUT  fragment peptide pool. 
         FIG.  2 A-F . MHC-I bias is selective against CALR MUT  fragment. A) Schema depicting calculation of Patient:Peptide Score (PPS). In brief, single peptides derived from longer sequences were applied to netMHCpan v3 to predict class-I HLA binding against all of six any given patient&#39;s class-I HLAs. In each patient-peptide combination, the strongest affinity was registered as the PPS. B) The mean PPS of individual peptides derived from indicated protein sequence (9-10mers) from CALR MUT  and JAK2 V617F  MPN patients and pseudo-population created from expected frequencies of US Caucasian population (NEUS only). Protein sequences are the 44-amino acid CALR MUT  sequence (CALR MUT-44aa ), the wild-type CALR sequence upstream of CALR MUT-44aa  (CALR 1-361 ) and the irrelevant foreign antigen neuraminidase (NA) from influenza. Also showing peptides broadly subdivided from predicted possible binding (&lt;10 4  nM) and predicted non-binding (&gt;10 4  nM) peptides. C) The difference in mean PPS of each group from  FIG.  2 B . The Student t test was performed to calculate significance. D) Top ten best predicted mean PPS of CALR MUT  MPN patients. Peptide sequences are labeled below and shorthand codes (CALR-length-p-start position) are identified above. E) Breakdown of HLA-I allele frequencies in CALR MUT  MPN patients from NEUS cohort or F) Danish cohort, versus the predicted binding affinity to the top peptide CALR9p2. 
         FIG.  3 A-E . Human CD8 +  T cells activated with heteroclitic CALR9p2 peptides cross-react with CALR9p2 peptide. A) Predicted mean PPS in CALR MUT  MPN patients of all single amino-acid substitution of CALR9p2. B) Predicted affinity of all single amino-acid substitution of CALR9p2 to HLA-A*02:01. Seven heteroclitic peptides were chosen for further testing and are identified here only by their amino-acid substitution in the CALR9p2 peptide. C) Binding affinity of CALR9p2 and indicated heteroclitic peptide to the most common MHC-I in the CALR MUT  MPN patient cohorts. Shadowed area indicates predicted binding affinity range of 5000-500 nM. D) Percent IFNγ + CD8 +  T cells after primary in vitro stimulation of PBMCs with CALR9p2 or CALR9p2 heteroclitic peptides followed by secondary restimulation with either control (MOG) peptide, CALR9p2 or initial CALR9p2 heteroclitic peptide. E) Summary of responding donor PBMCs to CALR9p2 or CALR9p2 heteroclitic peptides. 
         FIG.  4 A-F . CALRMUT sequence is not immunogenic in C57BL/6J mice. A) The predicted binding affinity of CALRMUT-derived peptides (8-10-mers shown) against all available murine MHC-I alleles. The strongest binding peptide CALR9p2 is identified in the figures (see dark gray bar and arrow). B) H-2Kb stabilization assay using TAP-deficient RMA/S cells was performed for CALR9p2 in the presence and absence of serum. The chicken ovalbumin (OVA)-derived peptide SIINFEKL was used as a positive control. C) Timeline of DNA immunization schedule and CD8+ T cell collection for the experiment in D. D) IFNγ ELISpot depicting secondary reactivity of CD8+ T cells isolated from draining lymph nodes of mice DNA immunized with full-length CALRWT, CALRMUT, and OVA. E) Timeline of peptide immunization and CD8+ T cell collection for the experiment in F. F) IFNγ ELISpot depicting secondary reactivity of CD8+ T cells isolated from draining lymph nodes of mice peptide immunized with adjuvant and DMSO, CALR9p2 or SIINFEKL. Data shown represent results from one repeat of experiments performed at least three times. 
         FIG.  5 A-M . Heteroclitic CALR9p2 peptide vaccine elicits cross-reactive CD8 +  T cell response against CALR9p2 and controls tumor growth. A) Predicted binding affinity to H-2K b  of all single amino-acid substitution variants of CALR9p2. Top predicted peptide CALR9p2(T5F) is shown. B) Cartoon of the expected effect of T5F substitution in CALR9p2 peptide conformational binding into H-2K b . Known dominant anchor sites and minor anchor sites are depicted in red and blue, respectively C) MHC-I stabilization assay using TAP-deficient RMA/S cells was performed for CALR9p2 and CALR9p2(T5F) in absence of serum. SIINFEKL was used as a positive control. Representative results from one repeat of an experiment performed at least three times. D) IFNγ ELISpot depicting secondary reactivity of CD8 +  T cells isolated from draining lymph nodes of mice peptide immunized with adjuvant and DMSO, CALR9p2 or CALR9p2(T5F). Representative results from one repeat of an experiment performed at least three times. The Student&#39;s t test was performed to calculate significance. E) Killing assay of peptide-pulsed B16 cells by CD8 +  T cells isolated from peptide-immunized mice. Representative results from one repeat of an experiment performed at least three times. The Student&#39;s t test was performed to calculate significance. F) Timeline of peptide immunization and tumor implantation for prophylactic vaccine G) RMV/s pER-CALR9p2  tumor growth over time following prophylactic peptide immunization for individual tumors or H) averaged up until the second mouse reaching the endpoint. I) Survival of mice following the prophylactic vaccine. J) Timeline of peptide immunization and tumor implantation for therapeutic vaccine and in combination with anti-PD1 therapy. K) Tumor growth over time following therapeutic vaccine for individual tumors or L) averaged up until the second mouse reaching the endpoint. M) Survival of mice following the therapeutic vaccine, with or without anti-PD1 therapy. Data from tumor growth experiments represent results from one repeat of experiments performed twice. Significance for tumor growth experiments was calculated by performing a Student&#39;s t test on the area under the curve of each tumor. Significance for survival was calculated by performing a log-rank test. 
         FIG.  6 A-C . MHC-II alleles skewing in CALR MUT  MPN patients compared to JAK2 V617F  MPN patients from NEUS cohort. A) Principal component analysis of HLA-II allele frequencies from CALR MUT  MPN patients, JAK2 V617F  MPN patients and US Caucasian population. B) Volcano plot of Barnard&#39;s unconditional test or chi square test P value versus difference in MHC-II allele frequencies. Dotted line represents a P value of 0.05. C) Comparison of HLA-I allele frequencies from each group compared to each other in NEUS cohort. HLA-I alleles with greater (depicted as mid-gray dots) or lesser (depicted as dark-gray dots) are shown here. only HLA-I alleles differentially expressed between CALR MUT  MPN patients compared to both JAK2 V617F  MPN patients and US Caucasian population were considered. 
         FIG.  7 A-B . Contingency analysis of MHC-I allele frequencies in NEUS and Danish cohorts. A) Volcano plot of Barnard&#39;s unconditional test or chi square test P value versus difference in MHC-I allele frequencies in NEUS cohort and B) Danish cohort. Dotted line represents a P value of 0.05. 
         FIG.  8   . MHC-I alleles with predicted binding to CALR MUT -derived peptides are less frequent in CALR MUT  MPNs. Heatmap of predicted binding to each CALR MUT -derived 10mer peptide to each differentially expressed MHC-I allele. Upper boxes represent MHC-I alleles that are more frequent and lower boxes represent MHC-I alleles that are less frequent—in CALR MUT  MPN patients. 
         FIG.  9   . MHC-II alleles with predicted binding to CALR MUT -derived peptides are more frequent in CALR MUT  MPNs. Heatmap of predicted binding to each CALR MUT -derived 15mer peptide to each differentially expressed MHC-II allele. Upper boxes represent MHC-I alleles that are more frequent and lower boxes represent MHC-I alleles that are less frequent—in CALR MUT  MPN patients. 
         FIG.  10   . CALR9p2 heteroclitic peptides increase HLA-A*02:01 stabilization compared to CALR9p2. MHC-I stabilization assay using human TAP-deficient T2 cells was performed for CALR9p2 and CALR9p2 heteroclitic peptides. The MART1-A2 peptide was used as a positive control. 
         FIG.  11   . Control secondary stimulatory conditions of rapid T cell assay of human healthy donor PBMCs. Graphed results of secondary stimulation MOG, CEFT and PMA+Ionomycin (PMA), of human healthy donors that had received the initial stimulation of DMSO, CEFT, CALR9p2 (9p2) and all pool heteroclitic peptides (hetPool). In some cases, not all conditions could be tested. 
         FIG.  12   . CALR9p2 heteroclitic peptides can mount cross-reactive response to CALR9p2 through HLA-A*02:01. PBMCs from two healthy donors were activated in vitro with CALR9p2 heteroclitic peptides and final restimulation was provided by peptide-pulsed HLA-A*02:01-transduced K562 cells. Reactivity was assessed by intracellular staining for IFNγ and TNFα by flow cytometry. PBMC from two other healthy donors showed no reactivity (not shown). 
         FIG.  13 A-C . CALRMUT does not inhibit antigen processing and presentation. A) Representative flow cytometry image of B16F10 cells presentation of SIINFEKL bound to H-2Kb following co-transfection with OVA and different CALR constructs. B) Quantification of the percentage of B16F10 cells presenting SIINFEKL H-2Kb and C) total expression of H-2Kb. Data shown represent results from one repeat of experiments performed at least three times. A Student&#39;s t test was used to determine significance. 
         FIG.  14 A-F . Full-length CALRMUT encoding CALR9p2(T5F) elicits activated antigen-specific CD8+ T cells. A) Depiction and Sanger sequencing validation of site-directed mutagenesis introducing CALR9p2(T5F) variant in the pING-CALRMUT DNA vaccine sequence. Only relevant section of DNA construct is depicted. B) IFNγ ELISpot following co-culture of CD8+ T cells from dLNs of pING or pING-CALRMUT-CALR9p2(T5F)-immunized mice with peptide-pulsed T cell-depleted splenocytes. C) Representative image of CALR9p2(T5F)-tetramer staining of live CD8+ T cells from spleen. D) Quantification of CALR9p2(T5F)-tetramer-positive live CD8+ T cells from C). E) Representative image of CD44, Tim3 and Pd1 levels on CALR9p2(T5F)-tetramer-negative and -positive live CD8+ T cells from spleen of pING-CALRMUT-CALR9p2(T5F)-immunized mice. F). Quantification of CD44HITim3HI and CD44HIPd1HI, as well as geometric MFI of Tim3 and Pd1 of CALR9p2(T5F)-tetramer-negative and -positive live CD8+ T cells from pING-CALRMUT- CALR9p2(T5F) -immunized mice from E). Showing one of two representative experiment. Statistical significance was calculated using the Student t test. 
         FIG.  15 A-F . Demonstration that CALR9p2(T5F)-specific CD8 +  T cells also recognize CALR9p2 following in vitro secondary restimulation. A) Representative image of CALR9p2(T5F)-tetramer staining on live CD8 +  T cells from dLNs of d7 peptide-immunized mice. Samples shown are from secondary control restimulation of DMSO-pulsed splenocytes. B) Quantification of live CD8 +  T cells CALR9p2(T5F)-tetramer staining from A). C) Representative image of IFNγ and TNFα intracellular staining following secondary restimulation with peptide-pulsed splenocytes or PMA/Ionomycin of all, CALR9p2(T5F)-tetramer negative, or CALR9p2(T5F)-tetramer positive live CD8 +  T cells from CALR9p2(T5F) peptide-immunized mice. D) Quantification of live of IFNγ or TNFα positivity in CALR9p2(T5F)-tetramer negative or positive CD8 +  T cells from C). E-F) Quantification of E) Pd1 and F) Tim3 surface levels by flow cytometry of CALR9p2(T5F)-tetramer negative or positive of live CD8 +  T cells from CALR9p2(T5F)-immunized mice restimulated with splenocytes-pulsed with indicated peptides or PMA/Ionomycin. To control for possible staining artifacts, also showing results from background stained CALR9p2(T5F)-tetramer negative or positive live CD8 +  T cells from DMSO-immunized mice. Experiment shown is representative of experiment performed twice. Statistical significance was calculated using the Student t test. 
         FIG.  16   . CALR9p2 cross-reactivity of CD8 +  T cells primed by CALR9p2(T5F) diminishes over time and is not maintained by subsequent CALR9p2 boosts. IFNγ ELISpot depicting secondary restimulation of CD8 +  T cells isolated from draining lymph nodes of hock peptide-immunized at different timepoints. Boost #1 and Boost #2 occur at days 7 and 14, respectively. 
     
    
    
     DETAILED DESCRIPTION 
     The sub-headings provided below, and throughout this patent disclosure, are not intended to denote limitations of the various aspects or embodiments of the invention, which are to be understood by reference to the specification as a whole. For example, this Detailed Description is intended to read in conjunction with, and to expand upon, the description provided in the Summary of the Invention section of this application. 
     Definitions &amp; Abbreviations 
     As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably. 
     Furthermore, “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone). 
     Units, prefixes, and symbols are denoted in their Systéme International de Unites (SI) accepted form. 
     Numeric ranges provided herein are inclusive of the numbers defining the range. Where a numeric term is preceded by “about,” the term includes the stated number and values ±10% of the stated number. 
     Numbers in parentheses or superscript following text in this patent disclosure refer to the numbered references provided in the “Reference List” section at the end of this patent disclosure. 
     Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of” are included. 
     As used herein the abbreviation CALR refers to calreticulin. 
     As used herein the term CALR MUT  refers to the 44-amino acid C-terminal fragment of CALR having the amino acid sequence: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO. 288) 
               
               
                   
                 RRMMRTKMRMRRMRRTRRKMRRKMSPA 
               
               
                   
                   
               
               
                   
                 RPRTSCREACLQGWTEA  
               
            
           
         
       
     
     that is generated in response to various mutations (including several known, specific frameshift mutations) in calreticulin, and also refers to any calreticulin mutation (including frameshift mutation) that results in the generation of this 44-amino acid C-terminal fragment. 
     As used herein the terms “CALR′ peptide” and “native CALR MUT  peptide” and “parental CALR MUT  peptide” refer a peptide comprising some portion of the 44-amino acid C-terminal mutant fragment of CALR (CALR MUT ), i.e., SEQ ID NO. 288. The CALR MUT  peptides described herein are typically about 8-13, e.g., 9-12 amino acids long, but can be longer or shorter. 
     As used herein the abbreviation CTLA-4 refers to cytotoxic T-lymphocyte-associated protein 
     The terms “heteroclitic,” “heteroclitic peptide” and “heteroclitic CALR MUT  peptide” are used herein consistent with the normal meaning of the term “heteroclitic” in the field of the invention, and, as used herein, refer to a mutated version of a peptide that has superior properties as compared to its non-mutant counterpart. The non-mutant counterparts of the hetereoclitic peptides described herein are sometimes referred to herein as “native” peptides or “parental” peptides or “non-heteroclitic peptides” or “CALR MUT  peptides” or “native CALR MUT  peptides” or “parental CALR MUT  peptides.” The heteroclitic peptides provided herein have at least one amino acid point mutation as compared to the native peptides from which they are derived, and were designed and/or selected to have one or more of the following superior properties: (a) superior immunogenicity as compared to their native counterparts, (b) superior HLA binding (e.g. affinity) as compared to their native counterparts, (c) an HLA-I binding affinity of &lt;500 nm, (d) an HLA-I binding affinity of &lt;100 nm, (d) being a superior T cell receptor (TCR) epitope as compared to their native counterparts, (e) superior (e.g., increased) TCR agonist activity as compared to their native counterparts, (f) superior induction of T cell responses as compared to their native counterparts, and (g) induction of superior (e.g. increased) antigen-specific (i.e. CALRMUT-specific) antitumor immunity as compared to their native counterparts. The term “heteroclitic” may also be further understood with reference to: Gold et al., (2003) “ A Single Heteroclitic Epitope Determines Cancer Immunity After Xenogeneic DNA Immunization Against a Tumor Differentiation Antigen ,” J. Immunol May 15, 2003, 170 (10) 5188-5194; 13. Solinger et al. (1979), “ Lymphocyte response to cytochrome c.; Demonstration of a T - cell heteroclitic proliferative response and identification of a topographic antigenic determinant on pigeon cytochrome c whose immune recognition requires two complementing major histocompatibility complex - linked immune response genes ,” J. Exp. Med. 150:830; Wang et al. (1999), “ The stimulation of low - affinity, nontolerized clones by heteroclitic antigen analogues causes the breaking of tolerance established to an immunodominant T cell epitope .” J. Exp. Med. 190:983; Dyall et al., (1998). “ Heteroclitic immunization induces tumor immunity ,” J. Exp. Med. 188:1553; Slansky et al., (2000). “ Enhanced antigen - specific antitumor immunity with altered peptide ligands that stabilize the MHC - peptide - TCR complex. Immunity,”  13:529; Bakker et al., (1997), “ Analogues of CTL epitopes with improved MHC class - I binding capacity elicit anti - melanoma CTL recognizing the wildtype epitope .” Int. J. Cancer 70.:302; Parkhurst et al., (1996), “ Improved induction of melanoma - reactive CTL with peptides from the melanoma antigen gp 100  modified at HLA - A* 0201- binding residues .” J. Immunol. 157:2539; and Valmori et al., (1998), “ Enhanced generation of specific tumor - reactive CTL in vitro by selected Melan - A/MART -1  immunodominant peptide analogues ,” J. Immunol. 160:1750. 
     The abbreviation “HLA” refers to human leukocyte antigen. 
     In each of the embodiments that involve peptides and/or nucleic acid molecules, the peptides and/or nucleic acid molecules can optionally be in “isolated” form. An “isolated” peptide or nucleic acid molecule is not within a living subject or cell and is typically in a form not found in nature. In some embodiments an isolated peptide or nucleic acid molecule may have been purified to a degree that it is not in a form in which it is found in nature. In some embodiments, a peptide or nucleic acid molecule that is isolated is substantially pure. In some embodiments, a protein or nucleic acid molecule that is isolated has a purity of greater than 75%, or greater than 80%, or greater than 90%, or greater than 95%. 
     The terms “identical” or “percent identity” in the context of a comparison between two peptides refer to amino acid sequences that are the same (identical) or have a specified percentage of amino acid residues that are the same (percent identity), when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity of two peptides can be determined using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid sequences and determine identity and/or percentage identity. 
     As used herein the abbreviation “MPN” refers to myeloproliferative neoplasms. 
     As used herein the abbreviation “PD-1” refers to Programmed Death 1, which is also known as Programmed Death Protein 1 or Programmed Cell Death Protein 1. 
     As used herein the abbreviation PD-L1 refers to Programmed Cell Death Ligand 1—which is a ligand for PD-1. 
     As used herein the abbreviation PD-L2 refers to Programmed Cell Death Ligand 2. 
     Various other terms are defined elsewhere in this patent disclosure, where used. Furthermore, terms that are not specifically defined herein may be more fully understood in the context in which the terms are used and/or by reference to the specification in its entirety. Where no explicit definition is provided all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which this invention pertains. 
     Heteroclitic CALR MUT  Peptides 
     In certain embodiments the present invention provides heteroclitic CALR MUT  peptides, including those for which amino acid sequences are provided in the below Tables A and B. 
     Tables A and B also provide the amino acid sequences of the parental CALR MUT  peptides from which the various heteroclitic CALR MUT  peptides were derived. 
     
       
         
           
               
             
               
                 TABLE A 
               
             
            
               
                   
               
               
                 Peptide Sequences 
               
            
           
           
               
               
               
               
               
            
               
                 Hetero- 
                   
                   
                   
                   
               
               
                 clitic 
                 Hetero- 
                 Parental 
                 Parental 
                   
               
               
                 peptide 
                 clitic 
                 peptide 
                 peptide 
                 Parental 
               
               
                 aa 
                 peptide 
                 aa 
                 SEQ ID 
                 peptide 
               
               
                 sequence 
                 SEQ ID NO. 
                 sequence 
                 NO. 
                 Name 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 RMMRTFMRM 
                 1 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 YMMRTKMRM 
                 2 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 FMMRTKMRM 
                 3 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRTIMRM 
                 4 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRTLMRM 
                 5 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRTVMRM 
                 6 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRWKMRM 
                 7 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRTMMRM 
                 8 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRTKMRK 
                 9 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMDTKMRM 
                 10 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMETKMRM 
                 11 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRTKMRV 
                 12 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRTKMRF 
                 13 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RYMRTKMRM 
                 14 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRTWMRM 
                 15 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRTKMRR 
                 16 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRTKMRY 
                 17 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRTKMAM 
                 18 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RPMRTKMRM 
                 19 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRTKMRW 
                 20 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRTKMPM 
                 21 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRTKMSM 
                 22 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRTKMRH 
                 23 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RSMRTKMRM 
                 24 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RTMRTKMRM 
                 25 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 MMMRTKMRM 
                 26 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRYKMRM 
                 27 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 WMMRTKMRM 
                 28 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRHKMRM 
                 29 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRKKMRM 
                 30 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRRKMRM 
                 31 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RAMRTKMRM 
                 32 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRTYMRM 
                 33 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRTKMYM 
                 34 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 REMRTKMRM 
                 35 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RRMRTKMRM 
                 36 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RQMRTKMRM 
                 37 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RFMRTKMRM 
                 38 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RWMRTKMRM 
                 39 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRFKMRM 
                 40 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRTTMRM 
                 41 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRTCMRM 
                 42 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRTNMRM 
                 43 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRTSMRM 
                 44 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRTKWRM 
                 45 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 RMMRTKMFM 
                 46 
                 RMMRTKMRM 
                 263 
                 CALR9p2 
               
               
                   
               
               
                 MRMRRMRRL 
                 47 
                 MRMRRMRRT 
                 264 
                 CALR9p8 
               
               
                   
               
               
                 MRMRRMRRM 
                 48 
                 MRMRRMRRT 
                 264 
                 CALR9p8 
               
               
                   
               
               
                 MRMRRMRRI 
                 49 
                 MRMRRMRRT 
                 264 
                 CALR9p8 
               
               
                   
               
               
                 MRMRRMRRV 
                 50 
                 MRMRRMRRT 
                 264 
                 CALR9p8 
               
               
                   
               
               
                 MRMRRMRRF 
                 51 
                 MRMRRMRRT 
                 264 
                 CALR9p8 
               
               
                   
               
               
                 MRMRRMRRY 
                 52 
                 MRMRRMRRT 
                 264 
                 CALR9p8 
               
               
                   
               
               
                 MPMRRMRRT 
                 53 
                 MRMRRMRRT 
                 264 
                 CALR9p8 
               
               
                   
               
               
                 MLMRRMRRT 
                 54 
                 MRMRRMRRT 
                 264 
                 CALR9p8 
               
               
                   
               
               
                 MMMRRMRRT 
                 55 
                 MRMRRMRRT 
                 264 
                 CALR9p8 
               
               
                   
               
               
                 MRMRRMRAT 
                 56 
                 MRMRRMRRT 
                 264 
                 CALR9p8 
               
               
                   
               
               
                 MRMRRMRPT 
                 57 
                 MRMRRMRRT 
                 264 
                 CALR9p8 
               
               
                   
               
               
                 MRMRRMRST 
                 58 
                 MRMRRMRRT 
                 264 
                 CALR9p8 
               
               
                   
               
               
                 RRMRRMRRT 
                 59 
                 MRMRRMRRT 
                 264 
                 CALR9p8 
               
               
                   
               
               
                 KMRRKMWPA 
                 60 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 KMFRKMSPA 
                 61 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 KMMRKMSPA 
                 62 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 KMIRKMSPA 
                 63 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 KMWRKMSPA 
                 64 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 KMYRKMSPA 
                 65 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 KMLRKMSPA 
                 66 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 KMRRKMSPY 
                 67 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 KMRRKMSPK 
                 68 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 KPRRKMSPA 
                 69 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 KMRRKMSPL 
                 70 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 KMRRKMSPF 
                 71 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 KMRRKMSPM 
                 72 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 FMRRKMSPA 
                 73 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 KMARKMSPA 
                 74 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 KMVRKMSPA 
                 75 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 KMRRKMSPR 
                 76 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 KMRRKMSPW 
                 77 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 YMRRKMSPA 
                 78 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 KMREKMSPA 
                 79 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 KMRDKMSPA 
                 80 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 MMRRKMSPA 
                 81 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 KMNRKMSPA 
                 82 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 KMSRKMSPA 
                 83 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 KMRRFMSPA 
                 84 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 KMRRKMSPV 
                 85 
                 KMRRKMSPA 
                 265 
                 CALR9p19 
               
               
                   
               
               
                 RPSCREACL 
                 86 
                 RTSCREACL 
                 266 
                 CALR9p30 
               
               
                   
               
               
                 RTSCREACK 
                 87 
                 RTSCREACL 
                 266 
                 CALR9p30 
               
               
                   
               
               
                 RTSCREACW 
                 88 
                 RTSCREACL 
                 266 
                 CALR9p30 
               
               
                   
               
               
                 RESCREACL 
                 89 
                 RTSCREACL 
                 266 
                 CALR9p30 
               
               
                   
               
               
                 RTKCREACL 
                 90 
                 RTSCREACL 
                 266 
                 CALR9p30 
               
               
                   
               
               
                 RTRCREACL 
                 91 
                 RTSCREACL 
                 266 
                 CALR9p30 
               
               
                   
               
               
                 RTSCRFACL 
                 92 
                 RTSCREACL 
                 266 
                 CALR9p30 
               
               
                   
               
               
                 RTSCRHACL 
                 93 
                 RTSCREACL 
                 266 
                 CALR9p30 
               
               
                   
               
               
                 RTSCRRACL 
                 94 
                 RTSCREACL 
                 266 
                 CALR9p30 
               
               
                   
               
               
                 RTSCRWACL 
                 95 
                 RTSCREACL 
                 266 
                 CALR9p30 
               
               
                   
               
               
                 RTSCRYACL 
                 96 
                 RTSCREACL 
                 266 
                 CALR9p30 
               
               
                   
               
               
                 RTSCREACR 
                 97 
                 RTSCREACL 
                 266 
                 CALR9p30 
               
               
                   
               
               
                 RQSCREACL 
                 98 
                 RTSCREACL 
                 266 
                 CALR9p30 
               
               
                   
               
               
                 RLSCREACL 
                 99 
                 RTSCREACL 
                 266 
                 CALR9p30 
               
               
                   
               
               
                 RMSCREACL 
                 100 
                 RTSCREACL 
                 266 
                 CALR9p30 
               
               
                   
               
               
                 RTSCREACY 
                 101 
                 RTSCREACL 
                 266 
                 CALR9p30 
               
               
                   
               
               
                 RYSCREACL 
                 102 
                 RTSCREACL 
                 266 
                 CALR9p30 
               
               
                   
               
               
                 RRSCREACL 
                 103 
                 RTSCREACL 
                 266 
                 CALR9p30 
               
               
                   
               
               
                 RPKMRMRRM 
                 104 
                 RTKMRMRRM 
                 267 
                 CALR9p5 
               
               
                   
               
               
                 RTKMRMRAM 
                 105 
                 RTKMRMRRM 
                 267 
                 CALR9p5 
               
               
                   
               
               
                 RTKMRMRPM 
                 106 
                 RTKMRMRRM 
                 267 
                 CALR9p5 
               
               
                   
               
               
                 RTMMRMRRM 
                 107 
                 RTKMRMRRM 
                 267 
                 CALR9p5 
               
               
                   
               
               
                 RTFMRMRRM 
                 108 
                 RTKMRMRRM 
                 267 
                 CALR9p5 
               
               
                   
               
               
                 RTYMRMRRM 
                 109 
                 RTKMRMRRM 
                 267 
                 CALR9p5 
               
               
                   
               
               
                 RTKMRMRRR 
                 110 
                 RTKMRMRRM 
                 267 
                 CALR9p5 
               
               
                   
               
               
                 RTKMRMRRK 
                 111 
                 RTKMRMRRM 
                 267 
                 CALR9p5 
               
               
                   
               
               
                 RTKMRMRRW 
                 112 
                 RTKMRMRRM 
                 267 
                 CALR9p5 
               
               
                   
               
               
                 RTWMRMRRM 
                 113 
                 RTKMRMRRM 
                 267 
                 CALR9p5 
               
               
                   
               
               
                 RTKMRMRRY 
                 114 
                 RTKMRMRRM 
                 267 
                 CALR9p5 
               
               
                   
               
               
                 RQKMRMRRM 
                 115 
                 RTKMRMRRM 
                 267 
                 CALR9p5 
               
               
                   
               
               
                 RRKMRMRRM 
                 116 
                 RTKMRMRRM 
                 267 
                 CALR9p5 
               
               
                   
               
               
                 RTAMRMRRM 
                 117 
                 RTKMRMRRM 
                 267 
                 CALR9p5 
               
               
                   
               
               
                 RTIMRMRRM 
                 118 
                 RTKMRMRRM 
                 267 
                 CALR9p5 
               
               
                   
               
               
                 RTLMRMRRM 
                 119 
                 RTKMRMRRM 
                 267 
                 CALR9p5 
               
               
                   
               
               
                 RTVMRMRRM 
                 120 
                 RTKMRMRRM 
                 267 
                 CALR9p5 
               
               
                   
               
               
                 DTKMRMRRM 
                 121 
                 RTKMRMRRM 
                 267 
                 CALR9p5 
               
               
                   
               
               
                 ETKMRMRRM 
                 122 
                 RTKMRMRRM 
                 267 
                 CALR9p5 
               
               
                   
               
               
                 FTKMRMRRM 
                 123 
                 RTKMRMRRM 
                 267 
                 CALR9p5 
               
               
                   
               
               
                 HTKMRMRRM 
                 124 
                 RTKMRMRRM 
                 267 
                 CALR9p5 
               
               
                   
               
               
                 YTKMRMRRM 
                 125 
                 RTKMRMRRM 
                 267 
                 CALR9p5 
               
               
                   
               
               
                 FRMRRTRRKM 
                 126 
                 RRMRRTRRKM 
                 268 
                 CALR10p11 
               
               
                   
               
               
                 YRMRRTRRKM 
                 127 
                 RRMRRTRRKM 
                 268 
                 CALR10p11 
               
               
                   
               
               
                 RPMRRTRRKM 
                 128 
                 RRMRRTRRKM 
                 268 
                 CALR10p11 
               
               
                   
               
               
                 RRPRRTRRKM 
                 129 
                 RRMRRTRRKM 
                 268 
                 CALR10p11 
               
               
                   
               
               
                 MRMRRTRRKM 
                 130 
                 RRMRRTRRKM 
                 268 
                 CALR10p11 
               
               
                   
               
               
                 WRMRRTRRKM 
                 131 
                 RRMRRTRRKM 
                 268 
                 CALR10p11 
               
               
                   
               
               
                 RLMRRTRRKM 
                 132 
                 RRMRRTRRKM 
                 268 
                 CALR10p11 
               
               
                   
               
               
                 RMMRRTRRKM 
                 133 
                 RRMRRTRRKM 
                 268 
                 CALR10p11 
               
               
                   
               
               
                 RRMRRTRRKR 
                 134 
                 RRMRRTRRKM 
                 268 
                 CALR10p11 
               
               
                   
               
               
                 RQMRRTRRKM 
                 135 
                 RRMRRTRRKM 
                 268 
                 CALR10p11 
               
               
                   
               
               
                 RSMRRTRRKM 
                 136 
                 RRMRRTRRKM 
                 268 
                 CALR10p11 
               
               
                   
               
               
                 RTMRRTRRKM 
                 137 
                 RRMRRTRRKM 
                 268 
                 CALR10p11 
               
               
                   
               
               
                 RYMRRTRRKM 
                 138 
                 RRMRRTRRKM 
                 268 
                 CALR10p11 
               
               
                   
               
               
                 REMRRTRRKM 
                 139 
                 RRMRRTRRKM 
                 268 
                 CALR10p11 
               
               
                   
               
               
                 RKMRRKMSPK 
                 140 
                 RKMRRKMSPA 
                 269 
                 CALR10p18 
               
               
                   
               
               
                 RPMRRKMSPA 
                 141 
                 RKMRRKMSPA 
                 269 
                 CALR10p18 
               
               
                   
               
               
                 RKPRRKMSPA 
                 142 
                 RKMRRKMSPA 
                 269 
                 CALR10p18 
               
               
                   
               
               
                 RKMRRKMSPY 
                 143 
                 RKMRRKMSPA 
                 269 
                 CALR10p18 
               
               
                   
               
               
                 RKMRRKMSPF 
                 144 
                 RKMRRKMSPA 
                 269 
                 CALR10p18 
               
               
                   
               
               
                 RKMRRKMSPR 
                 145 
                 RKMRRKMSPA 
                 269 
                 CALR10p18 
               
               
                   
               
               
                 RKMRRKMSPM 
                 146 
                 RKMRRKMSPA 
                 269 
                 CALR10p18 
               
               
                   
               
               
                 RRMRRKMSPA 
                 147 
                 RKMRRKMSPA 
                 269 
                 CALR10p18 
               
               
                   
               
               
                 RLMRRKMSPA 
                 148 
                 RKMRRKMSPA 
                 269 
                 CALR10p18 
               
               
                   
               
               
                 RMMRRKMSPA 
                 149 
                 RKMRRKMSPA 
                 269 
                 CALR10p18 
               
               
                   
               
               
                 RKMFRKMSPA 
                 150 
                 RKMRRKMSPA 
                 269 
                 CALR10p18 
               
               
                   
               
               
                 RKMIRKMSPA 
                 151 
                 RKMRRKMSPA 
                 269 
                 CALR10p18 
               
               
                   
               
               
                 RKMMRKMSPA 
                 152 
                 RKMRRKMSPA 
                 269 
                 CALR10p18 
               
               
                   
               
               
                 RKMWRKMSPA 
                 153 
                 RKMRRKMSPA 
                 269 
                 CALR10p18 
               
               
                   
               
               
                 RKMYRKMSPA 
                 154 
                 RKMRRKMSPA 
                 269 
                 CALR10p18 
               
               
                   
               
               
                 RKMRRKMWPA 
                 155 
                 RKMRRKMSPA 
                 269 
                 CALR10p18 
               
               
                   
               
               
                 REMRRKMSPA 
                 156 
                 RKMRRKMSPA 
                 269 
                 CALR10p18 
               
               
                   
               
               
                 RKMRRKMSPL 
                 157 
                 RKMRRKMSPA 
                 269 
                 CALR10p18 
               
               
                   
               
               
                 TKMRMRRMRK 
                 158 
                 TKMRMRRMRR 
                 270 
                 CALR10p6 
               
               
                   
               
               
                 TKMYMRRMRR 
                 159 
                 TKMRMRRMRR 
                 270 
                 CALR10p6 
               
               
                   
               
               
                 TTMRMRRMRR 
                 160 
                 TKMRMRRMRR 
                 270 
                 CALR10p6 
               
               
                   
               
               
                 TVMRMRRMRR 
                 161 
                 TKMRMRRMRR 
                 270 
                 CALR10p6 
               
               
                   
               
               
                 TIMRMRRMRR 
                 162 
                 TKMRMRRMRR 
                 270 
                 CALR10p6 
               
               
                   
               
               
                 TKMRMRRMRL 
                 163 
                 TKMRMRRMRR 
                 270 
                 CALR10p6 
               
               
                   
               
               
                 TKMRMRRMRF 
                 164 
                 TKMRMRRMRR 
                 270 
                 CALR10p6 
               
               
                   
               
               
                 TKMRMRRMRI 
                 165 
                 TKMRMRRMRR 
                 270 
                 CALR10p6 
               
               
                   
               
               
                 TKMRMRRMRM 
                 166 
                 TKMRMRRMRR 
                 270 
                 CALR10p6 
               
               
                   
               
               
                 TKMRMRRMRV 
                 167 
                 TKMRMRRMRR 
                 270 
                 CALR10p6 
               
               
                   
               
               
                 TAMRMRRMRR 
                 168 
                 TKMRMRRMRR 
                 270 
                 CALR10p6 
               
               
                   
               
               
                 TSMRMRRMRR 
                 169 
                 TKMRMRRMRR 
                 270 
                 CALR10p6 
               
               
                   
               
               
                 RKMRMRRMRR 
                 170 
                 TKMRMRRMRR 
                 270 
                 CALR10p6 
               
               
                   
               
               
                 TRMRMRRMRR 
                 171 
                 TKMRMRRMRR 
                 270 
                 CALR10p6 
               
               
                   
               
               
                 TMMRMRRMRR 
                 172 
                 TKMRMRRMRR 
                 270 
                 CALR10p6 
               
               
                   
               
               
                 RPRRMRRTRRKM 
                 173 
                 RMRRMRRTRRKM 
                 271 
                 CALR12p9 
               
               
                   
               
               
                 RMRRMRRTIRKM 
                 174 
                 RMRRMRRTRRKM 
                 271 
                 CALR12p9 
               
               
                   
               
               
                 RMRRMRRTLRKM 
                 175 
                 RMRRMRRTRRKM 
                 271 
                 CALR12p9 
               
               
                   
               
               
                 RMRRMRRTMRKM 
                 176 
                 RMRRMRRTRRKM 
                 271 
                 CALR12p9 
               
               
                   
               
               
                 RMRRPRRTRRKM 
                 177 
                 RMRRMRRTRRKM 
                 271 
                 CALR12p9 
               
               
                   
               
               
                 RMMRMRRTRRKM 
                 178 
                 RMRRMRRTRRKM 
                 271 
                 CALR12p9 
               
               
                   
               
               
                 RMRRMRWTRRKM 
                 179 
                 RMRRMRRTRRKM 
                 271 
                 CALR12p9 
               
               
                   
               
               
                 RMRRMRRWRRKM 
                 180 
                 RMRRMRRTRRKM 
                 271 
                 CALR12p9 
               
               
                   
               
               
                 RMRRMRRTYRKM 
                 181 
                 RMRRMRRTRRKM 
                 271 
                 CALR12p9 
               
               
                   
               
               
                 RMPRMRRTRRKM 
                 182 
                 RMRRMRRTRRKM 
                 271 
                 CALR12p9 
               
               
                   
               
               
                 RMRPMRRTRRKM 
                 183 
                 RMRRMRRTRRKM 
                 271 
                 CALR12p9 
               
               
                   
               
               
                 SPARPRTSCL 
                 184 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SPARPRTSCF 
                 185 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SPARPRTSCI 
                 186 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SPARPRTSCM 
                 187 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SPARPRTSCV 
                 188 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SPARPRTSCA 
                 189 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SPARPRTSIR 
                 190 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SPARPRTSLR 
                 191 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SPARPRTSMR 
                 192 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SPARPRTSVR 
                 193 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SFARPRTSCR 
                 194 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 STARPRTSCR 
                 195 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SVARPRTSCR 
                 196 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SYARPRTSCR 
                 197 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SPARPRTSYR 
                 198 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SPARPRTWCR 
                 199 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SPARPRTSFR 
                 200 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SIARPRTSCR 
                 201 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SSARPRTSCR 
                 202 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SPAFPRTSCR 
                 203 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SAARPRTSCR 
                 204 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SPAYPRTSCR 
                 205 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SPARPRTSCK 
                 206 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SMARPRTSCR 
                 207 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SPFRPRTSCR 
                 208 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SPMRPRTSCR 
                 209 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SLARPRTSCR 
                 210 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SQARPRTSCR 
                 211 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SWARPRTSCR 
                 212 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SPAWPRTSCR 
                 213 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SPARPRTFCR 
                 214 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 SPARPRTSCY 
                 215 
                 SPARPRTSCR 
                 272 
                 CALR10p25 
               
               
                   
               
               
                 RTKMRMRMMR 
                 216 
                 RTKMRMRRMR 
                 273 
                 CALR10p5 
               
               
                   
               
               
                 RTKMRMRRMK 
                 217 
                 RTKMRMRRMR 
                 273 
                 CALR10p5 
               
               
                   
               
               
                 RTKMRMRFMR 
                 218 
                 RTKMRMRRMR 
                 273 
                 CALR10p5 
               
               
                   
               
               
                 RTKMRMRLMR 
                 219 
                 RTKMRMRRMR 
                 273 
                 CALR10p5 
               
               
                   
               
               
                 RTKMRMRWMR 
                 220 
                 RTKMRMRRMR 
                 273 
                 CALR10p5 
               
               
                   
               
               
                 RTKMRMRYMR 
                 221 
                 RTKMRMRRMR 
                 273 
                 CALR10p5 
               
               
                   
               
               
                 RTKMRMRIMR 
                 222 
                 RTKMRMRRMR 
                 273 
                 CALR10p5 
               
               
                   
               
               
                 RTKMRMRVMR 
                 223 
                 RTKMRMRRMR 
                 273 
                 CALR10p5 
               
               
                   
               
               
                 MTKMRMRRMR 
                 224 
                 RTKMRMRRMR 
                 273 
                 CALR10p5 
               
               
                   
               
               
                 ETKMRMRRMR 
                 225 
                 RTKMRMRRMR 
                 273 
                 CALR10p5 
               
               
                   
               
               
                 FTKMRMRRMR 
                 226 
                 RTKMRMRRMR 
                 273 
                 CALR10p5 
               
               
                   
               
               
                 HTKMRMRRMR 
                 227 
                 RTKMRMRRMR 
                 273 
                 CALR10p5 
               
               
                   
               
               
                 NTKMRMRRMR 
                 228 
                 RTKMRMRRMR 
                 273 
                 CALR10p5 
               
               
                   
               
               
                 YTKMRMRRMR 
                 229 
                 RTKMRMRRMR 
                 273 
                 CALR10p5 
               
               
                   
               
               
                 RTKMRMRRMW 
                 230 
                 RTKMRMRRMR 
                 273 
                 CALR10p5 
               
               
                   
               
               
                 RTKMRMRRMY 
                 231 
                 RTKMRMRRMR 
                 273 
                 CALR10p5 
               
               
                   
               
               
                 RTKMRMRRMF 
                 232 
                 RTKMRMRRMR 
                 273 
                 CALR10p5 
               
               
                   
               
               
                 RTKMRMRRMM 
                 233 
                 RTKMRMRRMR 
                 273 
                 CALR10p5 
               
               
                   
               
               
                 RTKMRMRRM1 
                 234 
                 RTKMRMRRMR 
                 273 
                 CALR10p5 
               
               
                   
               
               
                 RTKMRMRRML 
                 235 
                 RTKMRMRRMR 
                 273 
                 CALR10p5 
               
               
                   
               
               
                 RTKMRMRRMV 
                 236 
                 RTKMRMRRMR 
                 273 
                 CALR10p5 
               
               
                   
               
               
                 RRMRRTRRL 
                 237 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RRMRRTRRM 
                 238 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RRMRRTRRF 
                 239 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RIMRRTRRK 
                 240 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RMMRRTRRK 
                 241 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RTMRRTRRK 
                 242 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RVMRRTRRK 
                 243 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RSMRRTRRK 
                 244 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RLMRRTRRK 
                 245 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RAMRRTRRK 
                 246 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RQMRRTRRK 
                 247 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RFMRRTRRK 
                 248 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RWMRRTRRK 
                 249 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RYMRRTRRK 
                 250 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RCMRRTRRK 
                 251 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RGMRRTRRK 
                 252 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RNMRRTRRK 
                 253 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RRMRRTRFK 
                 254 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RRMRRTRYK 
                 255 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RRMRRTRRI 
                 256 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RRMRRTRRV 
                 257 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RRMRRTRRY 
                 258 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RRMRRTRRW 
                 259 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RRMRRTRRA 
                 260 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RRMRRTRRC 
                 261 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
               
                 RRMRRTRRT 
                 262 
                 RRMRRTRRK 
                 274 
                 CALR9p11 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE B 
               
             
            
               
                   
               
               
                 Consensus Sequences 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Heteroclitic 
                   
                 Parental 
                 Parental 
               
               
                 Heteroclitic peptide 
                 peptide 
                 Parental peptide 
                 peptide 
                 peptide 
               
               
                 consensus aa sequence 
                 SEQ ID NO. 
                 aa sequence 
                 name 
                 SEQ ID NO. 
               
               
                   
               
               
                 X1X2M3X4X5X6X7X8X9 (SEQ ID NO. 275) 
                 275 
                 RMMRTKMRM 
                 CALR9p2 
                 263 
               
               
                 wherein: 
                   
                   
                   
                   
               
               
                 X1 is: R, Y, F, M, or W 
                   
                   
                   
                   
               
               
                 X2 is: M, Y, P, S, T, A, E, R, Q, F, or W 
                   
                   
                   
                   
               
               
                 X4 is: R, D, or E 
                   
                   
                   
                   
               
               
                 X5 is: T, W, Y, H, K, R, or F 
                   
                   
                   
                   
               
               
                 X6 is: K, F, I, L, V, M, W, Y, T, C, N, or S 
                   
                   
                   
                   
               
               
                 X7 is: M, or W 
                   
                   
                   
                   
               
               
                 X8 is: R, A, P, S, Y, or F 
                   
                   
                   
                   
               
               
                 X9 is: M, K, V, F, R, Y, W, or H 
                   
                   
                   
                   
               
               
                   
               
               
                 X1X2M3R4R5M6R7X8X9 (SEQ ID NO. 276) 
                 276 
                 MRMRRMRRT 
                 CALR9p8 
                 264 
               
               
                 Wherein: 
                   
                   
                   
                   
               
               
                 X1 is: M, or R 
                   
                   
                   
                   
               
               
                 X2 is: R, P, L, or M 
                   
                   
                   
                   
               
               
                 X8 is: R, A, P, or S 
                   
                   
                   
                   
               
               
                 X9 is: T, L, M, I, V, F, or Y 
                   
                   
                   
                   
               
               
                   
               
               
                 X1X2X3X4X5M6X7P8X9 (SEQ ID NO. 277) 
                 277 
                 KMRRKMSPA 
                 CALR9p19 
                 265 
               
               
                 Wherein: 
                   
                   
                   
                   
               
               
                 X1 is: K, F, Y, or M 
                   
                   
                   
                   
               
               
                 X2 is: M, or P 
                   
                   
                   
                   
               
               
                 X3 is: R, F, M, I, W, Y, L, A, V, N, or S 
                   
                   
                   
                   
               
               
                 X4 is: R, E, or D 
                   
                   
                   
                   
               
               
                 X5 is: K, or F 
                   
                   
                   
                   
               
               
                 X7 is: S, or W 
                   
                   
                   
                   
               
               
                 X9 is: A, Y, K, L, F, M, R, W, or V 
                   
                   
                   
                   
               
               
                   
               
               
                 R1X2X3C4R5X6A7C8X9 (SEQ ID NO. 278) 
                 278 
                 RTSCREACL 
                 CALR9p30 
                 266 
               
               
                 Wherein: 
                   
                   
                   
                   
               
               
                 X2 is: T, P, E, Q, L, M, Y, or R 
                   
                   
                   
                   
               
               
                 X3 is: S, K, or R 
                   
                   
                   
                   
               
               
                 X6 is: E, F, H, R, W, or Y 
                   
                   
                   
                   
               
               
                 X9 is: L, K, W, R, or Y 
                   
                   
                   
                   
               
               
                   
               
               
                 X1X2X3M4R5M6R7X8X9 (SEQ ID NO. 279) 
                 279 
                 RTKMRMRRM 
                 CALR9p5 
                 267 
               
               
                 Wherein: 
                   
                   
                   
                   
               
               
                 X1 is: R, D, E, F, H, or Y 
                   
                   
                   
                   
               
               
                 X2 is: T, P, Q, or R 
                   
                   
                   
                   
               
               
                 X3 is: K, M, F, Y, W, A, I, L, or V 
                   
                   
                   
                   
               
               
                 X8 is: R, A, or P 
                   
                   
                   
                   
               
               
                 X9 is: M, R, K, W, or Y 
                   
                   
                   
                   
               
               
                   
               
               
                 X1X2X3R4R5T6R7R8K9X10(SEQ ID NO. 280) 
                 280 
                 RRMRRTRRKM 
                 CALR10p11 
                 268 
               
               
                 Wherein: 
                   
                   
                   
                   
               
               
                 X1 is: R, F, Y, M, or W 
                   
                   
                   
                   
               
               
                 X2 is: R, P, L, M, Q, S, T, Y, or E 
                   
                   
                   
                   
               
               
                 X3 is: M, or P 
                   
                   
                   
                   
               
               
                 X10 is: M, or R 
                   
                   
                   
                   
               
               
                   
               
               
                 R1X2X3X4R5K6M7X8P9X10 (SEQ ID NO. 281) 
                 281 
                 RKMRRKMSPA 
                 CALR10p18 
                 269 
               
               
                 Wherein: 
                   
                   
                   
                   
               
               
                 X2 is: K, P, R, L, M, or E 
                   
                   
                   
                   
               
               
                 X3 is: M, or P 
                   
                   
                   
                   
               
               
                 X4 is: R, F, I, M, W, or Y 
                   
                   
                   
                   
               
               
                 X8 is: S, or W 
                   
                   
                   
                   
               
               
                 X10 is: A, K, Y, F, R, M, or L 
                   
                   
                   
                   
               
               
                   
               
               
                 X1X2M3X4M5R6R7M8R9X10 (SEQ ID NO. 282) 
                 282 
                 TKMRMRRMRR 
                 CALR10p6 
                 270 
               
               
                 Wherein: 
                   
                   
                   
                   
               
               
                 X1 is: T, or R 
                   
                   
                   
                   
               
               
                 X2 is: K, T, V, I, A, S, R, or M 
                   
                   
                   
                   
               
               
                 X4 is: R, or Y 
                   
                   
                   
                   
               
               
                 X10 is: R, K, L, F, I, M, or V 
                   
                   
                   
                   
               
               
                   
               
               
                 R1X2X3X4X5R6X7X8X9R10K11M12 (SEQ ID NO. 
                 283 
                 RMRRMRRTRRKM 
                 CALR12p9 
                 271 
               
               
                 283) 
                   
                   
                   
                   
               
               
                 Wherein: 
                   
                   
                   
                   
               
               
                 X2 is: M, or P 
                   
                   
                   
                   
               
               
                 X3 is: R, M, or P 
                   
                   
                   
                   
               
               
                 X4 is: R, or P 
                   
                   
                   
                   
               
               
                 X5 is: M, or P 
                   
                   
                   
                   
               
               
                 X7 is: R, or W 
                   
                   
                   
                   
               
               
                 X8 is: T, or W 
                   
                   
                   
                   
               
               
                 X9 is: R, I, L, M, or Y 
                   
                   
                   
                   
               
               
                   
               
               
                 S1X2X3X4P5R6T7X8X9X10 (SEQ ID NO. 284) 
                 284 
                 SPARPRTSCR 
                 CALR10p25 
                 272 
               
               
                 Wherein: 
                   
                   
                   
                   
               
               
                 X2 is: P, F, T, V, Y, I, S, A, M, L, Q, or W 
                   
                   
                   
                   
               
               
                 X3 is: A, F, or M 
                   
                   
                   
                   
               
               
                 X4 is: R, F, Y, or W 
                   
                   
                   
                   
               
               
                 X8 is: S, W, or F 
                   
                   
                   
                   
               
               
                 X9 is: C, I, L, M, V, Y, or F 
                   
                   
                   
                   
               
               
                 X10 is: R, L, F, I, M, V, A, K, or Y 
                   
                   
                   
                   
               
               
                   
               
               
                 X1T2K3M4R5M6R7X8M9X10 (SEQ ID NO. 285) 
                 285 
                 RTKMRMRRMR 
                 CALR10p5 
                 273 
               
               
                 Wherein: 
                   
                   
                   
                   
               
               
                 X1 is: R, M, E, F, H, N, or Y 
                   
                   
                   
                   
               
               
                 X8 is: R, M, F, L, W, Y, I, or V 
                   
                   
                   
                   
               
               
                 X10 is: R, K, W, Y, F, M, I, L, or V 
                   
                   
                   
                   
               
               
                   
               
               
                 R1X2M3R4R5T6R7X8X9 (SEQ ID NO. 286) 
                 286 
                 RRMRRTRRK 
                 CALR9p11 
                 274 
               
               
                 Wherein: 
                   
                   
                   
                   
               
               
                 X2 is: R, I, M, T, V, S, L, A, 
                   
                   
                   
                   
               
               
                 Q, F, W, Y, C, G, or N 
                   
                   
                   
                   
               
               
                 X8 is: R, F, or Y 
                   
                   
                   
                   
               
               
                 X9 is: K, L, M, F, I, V, Y, W, A, C, or T 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the heteroclitic CALR MUT  peptides described herein have one or more the following superior properties: (a) superior immunogenicity as compared to their native counterparts, (b) superior HLA binding (e.g. affinity) as compared to their native counterparts, (c) an HLA-I binding affinity of &lt;500 nm, (d) an HLA-I binding affinity of &lt;100 nm, (d) being a superior T cell receptor (TCR) epitope as compared to their native counterparts, (e) superior (e.g., increased) TCR agonist activity as compared to their native counterparts, (f) superior induction of CD8+ T cell responses as compared to their native counterparts, (g) induction of superior (e.g. increased) antigen-specific (i.e. CALRMUT-specific) antitumor immunity as compared to their native counterparts. 
     In certain embodiments the present invention also provides variants of the heteroclitic CALR MUT  peptides described herein. In some embodiments such variants comprise 1 or 2 or 3 or more amino acid point mutations as compared to any of SEQ ID Nos 1-262, or have an amino acid sequence that is at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to any of SEQ ID Nos 1-262, provided that such variants are heteroclitic, and/or exhibit one or more the superior properties described above. 
     In some embodiments a heteroclitic CALR MUT  peptide as described herein is 8, or 9, or 10, or 11, or 12, or 13 amino acids in length. In some embodiments a heteroclitic CALR MUT  peptide as described herein is 8-13 amino acids in length. In some embodiments a heteroclitic CALR MUT  peptide as described herein is 9-12 amino acids in length. 
     In some embodiments a heteroclitic CALR MUT  peptide as described herein comprises one amino acid point mutation as compared to the parental peptide from which it is derived. In some embodiments a heteroclitic CALR MUT  peptide as described herein comprises two amino acid point mutations as compared to the parental peptide from which it is derived. In some embodiments, where the heteroclitic CALR MUT  peptides comprise two amino acid point mutations, those mutations can be a combination of any two of the single amino acid point mutations described herein (e.g. the single point mutations present in SEQ ID Nos. 1-262). 
     Nucleic Acid Molecules 
     In some embodiments the present invention provides nucleic acid molecules that encode the heteroclitic CALR MUT  peptides described herein. In some embodiments the nucleic acid molecules are DNA. In some embodiments the nucleic acid molecules are RNA. In some embodiments the nucleic acid molecules are mRNA. All such nucleic acid molecules can comprise naturally occurring nucleotides or synthetic and/or chemically modified nucleotides—such as those that are modified to increase their stability or otherwise improve their suitability for administration to subjects. 
     Vectors 
     In some embodiments the present invention provides “vectors” that comprise nucleic acid molecules that encode the heteroclitic CALR MUT  peptides described herein. 
     The term “vector,” as used herein, means a construct suitable for delivery of a nucleic acid molecule to a cell. Examples of vectors include, but are not limited to, viruses, viral-derived vectors, naked DNA or RNA vectors, plasmid vectors, cosmid vectors, phage vectors, and the like. In some embodiments a vector may be an “expression vector” that is capable of delivering a nucleic acid molecule to a cell and that also contains elements required for expression of the nucleic acid molecule in the cell. 
     In some embodiments the vectors are viral vectors. Examples of suitable viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, alphavirus vectors, and vaccinia virus vectors. 
     Compositions 
     The present invention provides various compositions comprising the heteroclitic CALR MUT  peptides, nucleic acid molecules, or vectors described herein. 
     In some embodiments the compositions described herein comprise one or more additional components suitable for administration to a subject and/or useful in formulating a composition for delivery to a subject, including, but not limited to, diluents, buffers, carriers, stabilizers, dispersing agents, suspending agents, thickening agents, excipients, preservatives, and the like. 
     In some embodiments, the compositions described herein also comprise an adjuvant. Adjuvants are well known in the art and any suitable adjuvant can be used. Examples of adjuvants that can be used in the compositions and methods of the present invention include, but are not limited to: inorganic or organic adjuvants, oil-based adjuvants, virosomes, liposomes, lipopolysaccharide (LPS), monophosphoryl lipid A (MPL), saponin, saponin QS-21, CpG oligonucleotides, molecular cages for antigens (such as immune-stimulating complexes (“ISCOMS”)), Ag-modified saponin/cholesterol micelles that form stable cage-like structures that are transported to the draining lymph nodes), components of bacterial cell walls, nucleic acids (such as double-stranded RNA (dsRNA), single-stranded DNA (ssDNA), and unmethylated CpG dinucleotide-containing DNA), alum, aluminum phosphate, aluminum hydroxide, squalene, Freund&#39;s Complete Adjuvant, Freund&#39;s Incomplete Adjuvant, and the like. 
     Delivery Vehicles 
     In some embodiments the compositions of the present invention comprise a delivery vehicle. The term “delivery vehicle,” as used herein, refers to a substance useful for the delivery of either a nucleic acid molecule or a peptide to a cell. Examples of delivery vehicles that can be used in conjunction with the present invention include, but are not limited to, nanoparticles, lipid nanoparticles, liposomes, lipids, lipid encapsulation systems, polymers, and polymersomes. 
     Methods of Treatment 
     The present invention provides various methods of treatment. For example, in some embodiments the present invention provides methods of treating JAK2 mutant-negative myeloproliferative neoplasms (MPNs) in subjects in need thereof, such methods comprising administering to a subject an effective amount of a heteroclitic CALR MUT  peptide, nucleic acid molecule, vector or composition as described herein. In some embodiments such methods involve administering one dose of a heteroclitic CALR MUT  peptide, nucleic acid molecule, vector or composition to the subject. In some embodiments such methods involve administering two or more doses of a heteroclitic CALR MUT  peptide, nucleic acid molecule, vector or composition to the subject. For example, in some embodiments such treatment methods involve administering a priming dose and one or more booster doses of the heteroclitic CALR MUT  peptide, nucleic acid molecule, vector or composition to the subject. In some embodiments such methods also comprise administering an effective amount of an immune checkpoint inhibitor to the subject. Suitable immune checkpoint inhibitors include PD-1, PD-L1, PD-L2 and CTLA-4 inhibitors. In some embodiments the immune checkpoint inhibitor is an anti-PD1 antibody. 
     As used herein, the terms “treat,” “treating,” and “treatment” refer achieving, and/or administering an agent or agents (e.g., a heteroclitic CALR MUT  peptide, nucleic acid molecule, vector or composition as described herein) to a subject to achieve, to a detectable degree, an improvement in one or more clinically relevant parameters in a subject (e.g., a subject with a JAK2 mutant-negative MPN), or in a cancer/tumor (e.g. a JAK2 mutant-negative MPN), or in tumor cells (e.g., JAK2 mutant-negative JAK2 mutant-negative MPN tumor cells). Such clinically relevant parameters include, but are not limited to, reducing the rate of growth of a tumor (or tumor cells), halting the growth of a tumor (or of tumor cells), causing regression of a tumor (or of tumor cells), reducing the size of a tumor (for example as measured in terms of tumor volume or tumor mass), reducing the grade of a tumor, eliminating a tumor (or tumor cells), preventing, delaying, or slowing recurrence (rebound) of a cancer/tumor, improving symptoms associated with a cancer/tumor, improving survival from a cancer/tumor, inhibiting or reducing spreading of a cancer/tumor (e.g., metastases), and the like. Importantly, in the context of the present invention, such clinically relevant parameters also include (a) an immune response to a tumor or tumor cells, (b) a CD8+ T cell response to a tumor or tumor cells, (c) an anti-CALR MUT  immune response, (d) an anti-CALR MUT  CD8+ T cell response, and (e) enhanced sensitivity of a tumor or tumor cells to immune checkpoint blockade. All of the above are desirable biological outcomes of the present methods. In some embodiments, the improvement in the one or more clinically relevant parameters is assessed in comparison to a suitable baseline or suitable control. For example, in some embodiments the improvement in the one or more clinically relevant parameters is assessed in comparison to the level/extent of that clinically relevant parameter in the same subject prior to that subject being treated with a heteroclitic CALR MUT  peptide, nucleic acid molecule, vector or composition as described herein. Similarly, in some embodiments the improvement in the one or more clinically relevant parameters is assessed in comparison to the level/extent of that clinically relevant parameter in a suitable control subject or group of control subjects not treated with a heteroclitic CALR MUT  peptide, nucleic acid molecule, vector or composition as described herein (e.g., in a subject or group or group of subjects treated with a placebo). In some embodiments the improvement in the one or more clinically relevant parameters is a statistically significant improvement. 
     In some embodiments the present methods and compositions can be used to treat any JAK2 mutant-negative MPN in a subject in need thereof. 
     In some embodiments the subject has a tumor that is resistant to treatment using other methodologies and/or compositions. As used herein, the terms “resistant” and “resistance” are used consistent with their normal usage in the art and consistent with the understanding of the term by physicians who treat cancer. For example, consistent with its usual meaning in the art, a tumor or a subject may be considered “resistant” to a certain treatment method or treatment with a certain agent (or combination of agents), if, despite using that method or administering that agent (or combination of agents), a subject&#39;s tumor (or tumor cells) grows, and/or progresses, and/or spreads, and/or metastasizes, and/or recurs. In some instances, a tumor may initially be sensitive to treatment with a certain method or agent (or combination of agents), but later became resistant to such treatment. 
     As used herein the term “subject” encompasses all mammalian species, including, but not limited to, humans, non-human primates, dogs, cats, rodents (such as rats, mice and guinea pigs), cows, pigs, sheep, goats, horses, and the like—including all mammalian animal species used in animal husbandry, as well as animals kept as pets and in zoos, etc. In preferred embodiments the subjects are human. 
     In some embodiments the subject has a JAK2 mutant-negative MPN. In some such embodiments the subject has a JAK2 V617F  mutant-negative MPN. In some embodiments the subject has a JAK2 mutant-negative MPN that has recurred following a prior treatment with other compositions or methods, including, but not limited to, chemotherapy, radiation therapy, or surgical resection, or any combination thereof. In some embodiments the subject has a JAK2 mutant-negative MPN that has not previously been treated. 
     As used herein the term “effective amount” refers to an amount of an active agent (e.g., a heteroclitic CALR MUT  peptide, nucleic acid molecule, vector or composition) as described herein that is sufficient to achieve, or contribute towards achieving, one or more desirable clinical outcomes, such as those described in the “treatment” description above. An appropriate “effective amount” in any individual case may be determined using standard techniques known in the art, such as dose escalation studies, and may be determined taking into account such factors as the desired route of administration (e.g., systemic vs. intratumoral), desired frequency of dosing, and patient characteristics such as a subject&#39;s age, sex, body weight, etc. Furthermore, an “effective amount” may be determined in the context of any co-administration method to be used. One of skill in the art can readily perform such dosing studies (whether using single agents or combinations of agents) to determine appropriate doses to use, for example using assays such as those described in the Examples section of this patent application—which involve administration of the agents described herein to subjects (such as animal subjects routinely used in the pharmaceutical sciences for performing dosing studies). For example, in some embodiments an “effective amount” an active agent (e.g., a heteroclitic CALR MUT  peptide, nucleic acid molecule, vector or composition) as described herein may be calculated based on studies in humans or other mammals carried out to determine efficacy of the active agent. 
     In some embodiments one or more of the active agents (e.g., a heteroclitic CALR MUT  peptide, nucleic acid molecule, vector or composition) described herein is used at approximately its maximum tolerated dose, for example as determined in phase I clinical trials and/or in dose escalation studies. In some embodiments one or more of the active agents is used at about 90% of its maximum tolerated dose. In some embodiments one or more of the active agents is used at about 80% of its maximum tolerated dose. In some embodiments one or more of the active agents is used at about 70% of its maximum tolerated dose. In some embodiments one or more of the active agents is used at about 60% of its maximum tolerated dose. In some embodiments one or more of the active agents is used at about 50% of its maximum tolerated dose. In some embodiments one or more of the active agents is used at about 50% of its maximum tolerated dose. In some embodiments one or more of the active agents is used at about 40% of its maximum tolerated dose. In some embodiments one or more of the active agents is used at about 30% of its maximum tolerated dose. 
     In carrying out the treatment methods described herein, any suitable method or route of administration can be used to deliver the active agents described herein. In some embodiments systemic administration may be employed, for example, oral or intravenous administration, or any other suitable method or route of systemic administration known in the art. In some embodiments intratumoral delivery may be employed. For example, the active agents described herein may be administered either systemically or locally by injection, by infusion through a catheter, using an implantable drug delivery device, or by any other means known in the art. One of skill in the art will be able to select the appropriate delivery method or route depending on the situation, for example depending on whether active agents or cells are being administered, and in the case of active agents, depending on the nature of the active agent (e.g., its stability, half-life, etc.). 
     In certain embodiments the compositions and methods of treatment provided herein may be employed together with other compositions and treatment methods known to be useful for tumor therapy, including, but not limited to, surgical methods (e.g., for tumor resection), radiation therapy methods, treatment with chemotherapeutic agents, treatment with antiangiogenic agents, treatment with tyrosine kinase inhibitors or treatment with immune checkpoint inhibitors. Similarly, in certain embodiments the methods of treatment provided herein may be employed together with procedures used to monitor disease status/progression, such as biopsy methods and diagnostic methods (e.g., MRI methods or other imaging methods). 
     For example, in some embodiments the methods described herein may be performed prior to performing surgical resection of a tumor, for example in order to shrink a tumor prior to surgical resection. In other embodiments the methods described herein may be performed both before and after performing surgical resection of a tumor. 
     In some embodiments the treatment methods described herein may be employed in conjunction with performing a diagnostic test to determine if the subject has a tumor that that is likely to be responsive to therapy. For example, in some embodiments, the treatment methods provided herein comprise performing a diagnostic test to determine if the subject has a JAK2 mutant-negative MPN. Typically, such a test will be performed prior to administering one or more of the active agents (e.g., heteroclitic CALR MUT  peptides, nucleic acid molecules, vectors or compositions) described herein. 
     The invention is further described in the following non-limiting Examples. 
     EXAMPLES 
     Numbers in parentheses in these Examples refer to the numbered references in the Reference List that follows this Examples section. 
     Example 1 
     Overview 
     The majority of JAK2 V617F -negative myeloproliferative neoplasms (MPN) have disease-initiating frameshift mutations in calreticulin (CALR) resulting in a common novel C-terminal mutant fragment (CALRMUT), representing an attractive source of neoantigens for cancer vaccines. However, studies have shown that CALRMUT-specific T cells are rare in CALRMUT MPN patients. The underlying reasons for this phenomenon are unknown. In this study, we examined class-I major histocompatibility complex (MHC-I) allele frequency in CALRMUT MPN patients from two independent cohorts and observed that MHC-I alleles that present CALRMUT neoepitopes with high affinity are under-represented in CALRMUT MPN patients. We believe that this is due to an increased chance of immune-mediated tumor rejection by individuals expressing one of these MHC-I alleles such that the disease never clinically manifests. As a consequence of this MHC-I allele restriction, we hypothesized that CALRMUT MPN patients might not efficiently respond to cancer vaccines composed of the CALRMUT fragment, but might do so when immunized with a properly modified CALRMUT heteroclitic peptide vaccine approach. We found that heteroclitic CALRMUT peptides specifically designed for CALRMUT MPN patient MHC-I alleles efficiently elicited a cross-reactive CD8+ T cell response in human PBMC samples otherwise unable to respond to the matched weakly immunogenic CALRMUT native peptides. We also modeled this effect in mice and observed that C57BL/6J mice, which are unable to mount an immune response to the human CALRMUT fragment, can mount a cross-reactive CD8+ T cell response against a CALRMUT-derived peptide upon heteroclitic peptide immunization and this was further amplified by combining the heteroclitic peptide vaccine with blockade of the immune checkpoint molecule PD-1. Together, our data demonstrate the therapeutic potential of heteroclitic peptide-based cancer vaccines in CALRMUT MPN patients. 
     INTRODUCTION 
     Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) are myeloid blood cancers arising from hematopoietic stem cells (1, 2) and are characterized by hyperactivated JAK-STAT signaling (3). The majority of JAK2 V617F -negative MPN tumors have an insertion or deletion (INDEL) mutation in the C-terminal region of calreticulin (CALR) creating a +1 base-pair frameshift (4, 5). While multiple unique INDELs are found, nearly all generate a 44 amino acid common peptide, although a few rare cases generate a shorter 36 amino acid fragment (4, 5). Mutant CALR (CALRMUT) develops a pathogenic binding interaction with the extracellular portion of the thrombopoietin receptor (MPL), inducing ligand-independent constitutive JAK-STAT signaling pathways activation and oncogenesis (6-8). Consequently, the oncogenic CALRMUT fragment is an attractive source of mutational frameshift neoantigens for cancer vaccines in CALRMUT-positive MPN patients (9). However, in the few studies examining CALRMUT fragment immunogenicity, T cells from CALRMUT MPN patients had less immunoreactivity to CALRMUT-derived peptides compared to healthy individuals (10-14), even though many immunogenic peptides are predicted (14, 15). Interestingly, T cells from healthy donors display a stronger and more frequent response to CALRMUT peptides compared to T cells from patients with CALRMUT MPN (13). Additionally, several of the healthy donor T cell responses were elicited by memory T cells (13). This indicates that CALRMUT peptides are immunogenic in normal donors and suggest that CALRMUT-specific immune responses may be a mechanism of immunosurveillance eliminating the early tumor before its clinical manifestation. However, it is not clear how the tumor could escape this level of control in patients with clinical disease and why T cells from these patients did not respond to the CALRMUT fragment. 
     For antigens to be recognized by CD8+ T cells they must first be processed into smaller peptides, translocate to the endoplasmic reticulum (ER) and, if conditions are met, bind to the class I major histocompatibility complex (MHC-I) to form a peptide:MHC-I complex (pMHC-I) capable of reaching the cell surface to be recognized by the T cell receptor (TCR). However, not all peptides fit the stoichiometric requirements for MHC-I binding. Successful pMHC-I binding requires peptides to be the correct length and to have the appropriate anchor residues at specific locations to stabilize binding to pockets of the MHC-I allele (16). However, MHC genes have evolved to be extremely polymorphic across populations. Humans encode two copies of three different MHC-I genes, human leukocytes antigens (HLA)-A, HLA-B and HLA-C, with each having hundreds or thousands of polymorphisms (17). Importantly, different polymorphic residues alter the anchor residues such that peptides that bind to some MHC-I alleles may not bind to others (18, 19). As a result, the sum of presented peptides can vary greatly across individuals. We therefore hypothesized that some individuals possess the ability to present CALRMUT-derived peptides and eliminate early CALRMUT-positive MPNs, while other individuals do not and are more likely to develop the disease. 
     Since many peptides possess some but not all stoichiometric requirements for pMHC-I binding, affinities can range from strong to weak with many intermediate affinities possible. This affinity affects the number of surface-bound pMHC-I available for recognition by CD8+ T cells (20) and ultimately their activation. Notably, naïve T cells require higher levels of antigen stimulation than antigen-experienced T cells to potentiate T cell activation (21-26) and some antigens may be unable to activate naïve T cells. However, heteroclitic peptides (also known as anchor-optimized or anchor-improved peptides) are peptides in which one or two residues are specifically altered in order to increase MHC binding affinity. These resulting strong MHC-binding peptides robustly activate naïve T cells, and are similar enough to the original peptides that the activated T cells cross react with the original antigen (27-30), assuming that the original antigen has an adequate intermediate binding affinity. 
     In this study, we investigated two independent MPN patient cohorts and found that six MHC-I alleles predicted to efficiently bind to multiple CALRMUT-derived peptides were less frequently observed in CALRMUT MPN patients. This strongly pointed to a higher risk of developing CALRMUT MPN in patients lacking these MHC-I alleles and, at the same time, suggested to us that individuals with these MHC-I alleles could potentially control primordial CALRMUT-expressing tumors as part of the immunoediting process. In addition, this suggested to us that CALRMUT positive MPN patients are unlikely to respond to cancer vaccines composed of the CALRMUT fragment. Therefore, we analyzed the CALRMUT fragment for peptides that could be modified into heteroclitic peptides and serve as more potent anti-CALRMUT vaccines. We first tested this approach in in vitro assays using peripheral blood mononuclear cells (PBMCs) from healthy donors unable to respond to CALRMUT peptides and found that the same T cells could be induced to release IFNγ when primed using heteroclitic peptides. Then, to verify whether heteroclitic CALRMUT peptides can control the growth of CALRMUT tumors in vivo, we tested them as a vaccine in a pre-clinical mouse model. We established that C57BL/6J mice, which were unable to mount an immune response against the original CALRMUT fragment, had significantly delayed tumor growth when given a heteroclitic peptide vaccine of the same specificity and that this was further enhanced by PD1 blockade. 
     Results &amp; Discussion 
     CALRMUT MPN Patients Demonstrate Skewed MHC Allele Frequencies 
     We investigated MHC-I and MHC-II allele frequencies in CALRMUT and JAK2 V617F  MPN patients using haplotypes collected from two medical centers in the Northeastern United States (NEUS). In parallel, we assessed MHC-I allele frequencies of patients with MPN from eight medical centers in Denmark in order to independently validate the results observed in the NEUS cohort. MHC-II haplotypes were unavailable for the Danish cohort. As MHC allele frequencies vary greatly by geographic location (31), we analyzed each cohort separately. Furthermore, since the NEUS cohort is 88% Caucasian, we also compared MHC-I and MHC-II allele frequencies to those found in the US Caucasian population from the National Marrow Donor Program (17). To test for MHC-I and MHC-II allele frequency differences, we performed a principal component analysis comparing MHC-I and MHC-II allele frequencies from both NEUS MPN groups and the general US Caucasian population. We observed that the JAK2 V617F  MPN group clusters in proximity to the US Caucasian group, while the CALRMUT MPN group is isolated for both MHC-I ( FIG.  1 A ) and MHC-II ( FIG.  6   ) allele frequencies, suggesting distinct MHC-I and MHC-II allele representation in the CALRMUT MPN patients. We then examined the MHC-I alleles with frequencies that were different in CALRMUT MPN patients compared to both JAK2 V617F  MPN patients and the US Caucasian population in the NEUS cohort ( FIG.  1 B ), and likewise those in CALRMUT MPN patients compared to the JAK2 V617F  MPN patients from the Danish cohort ( FIG.  1 C ). No single MHC-I allele reached statistical significance in both cohorts using Barnard&#39;s unconditional test (used for moderate numbers) or the chi-square test ( FIG.  7 A ,B), and we, therefore, opted to analyze alleles with a fold change above or below a set threshold of ±0.2 fold frequency change in the NEUS cohort and ±0.125 fold frequency change in the Danish cohort ( FIG.  1 B ,C). We observed that in CALRMUT MPN patients from both cohorts only HLA-B*51:01 is over-represented, while six MHC-I alleles are under-represented: HLA-A*11:01, HLA-B*08:01, HLA-B*44:02, HLA-C*07:01, HLA-C*07:02 and HLA-C*06:02, and this trend is also reflected in the fraction of patients which are positive for these alleles. For HLA-II alleles, no single allele had a statistically significant decrease in frequency in CALRMUT MPN patients ( FIG.  6 B ) and the same fold-change threshold approach as for HLA-I alleles was therefore applied. We observed that HLA-DRB1*03:01, HLA-DRB1*04:01, HLA-DRB1*07:01, HLA-DRB1*13:01, HLA-DQB1*02:01 and HLA-DQB1*06:03 were less frequent in CALRMUT MPN patients compared to JAK2 V617F  MPN patients and US individuals of European descent, while HLA-DRB1*11:01 and HLA-DRB1*11:04 were more frequent ( FIG.  6 C ). Collectively our data suggest that CALRMUT MPN patients have skewed MHC-I and MHC-II haplotypes, whereas this does not appear to be the case for JAK2 V617F  MPN patients. 
     Skewing of MHC-I Allele Frequencies is Associated with CALRMUT Peptide Binding Affinity 
     To determine if there are any correlations between MHC-I and MHC-II allele frequency skewing to the binding of the peptides derived from the 44 amino-acid mutant protein fragment, we compared the predicted binding affinity of the CALRMUT-derived peptides to each MHC-I and MHC-II allele with over- or under-represented frequencies using NetMHCpan 3.0 and NetMHCIIpan 3.2, respectively. Five of the six under-represented MHC-I alleles (except HLA-B*44:02) had a moderate predicted affinity (&lt;10000 nM) to approximately a quarter of all 9-mer peptides of which many had &lt;500 nM predicted affinity ( FIG.  1 D ). In addition, multiple 10-mer peptides also had a high predicted affinity to these MHC-I alleles ( FIG.  8   ). On the other hand, all MHC-I alleles that were over-represented in CALRMUT MPN patients had poor binding to almost all CALRMUT-derived peptides with the exception of HLA-B*15:01 and HLA-C*12:03 which only had a high frequency in the Danish cohort ( FIG.  1 D ). When the NEUS cohort was separated in the two original cohorts (Memorial Sloan Kettering (MSK) and Dana Farber Harvard Cancer Center (DFHCC)) the same MHC-I skewing was confirmed except for HLA-C*06:02 which was slightly elevated in patients from MSK ( FIG.  1 E ). To test whether these six under-represented MHC-I alleles were more likely to potentiate an immune response against the CALRMUT fragment, PBMCs from 7 healthy donors positive for at least one of the under-represented MHC-I alleles and PBMCs from 4 healthy donors that were negative for these MHC-I alleles were stimulated in vitro with peptides covering the entire CALRMUT fragment and examined for reactivity by IFNγ ELISpot following a final peptide restimulation. In the patients positive for the under-represented MHC-I alleles, 7/7 (100%) responded while only ¼ (25%) of patients negative for the MHC-I alleles ( FIG.  1 F ). Therefore, while we did not test each MHC-I allele individually, we can conclude that, as a group, these six under-represented MHC-I alleles can potentiate an immune response against the CALRMUT fragment. 
     We did not observe the same trend for MHC-II alleles. Both MHC-II alleles found at higher frequency in CALRMUT MPN patients were predicted to bind strongly to more than half of 15-mer peptides, whereas only one of the four MHC-II alleles found at lower frequency appears to do so ( FIG.  9   ). Interestingly, when we examined the MHC-II alleles of the healthy donors from which PBMCs were used to generate memory CD4+ T cell T cell lines against long CALRMUT peptides in a previous study (13), both donors were positive for HLA-DRB1*13:01 and one of these was further positive for HLA-DRB1*04:01. 
     Thus, here we show that patients with CALRMUT-positive MPNs were less likely to possess an MHC-I allele predicted to bind to peptides derived from the CALRMUT fragment. This may be due to the fact that individuals with MHC-I alleles that can bind to CALR-derived peptides are less likely to develop CALRMUT MPN. 
     MHC-I Skewing in CALRMUT MPN is Specific for CALRMUT-Derived Peptides 
     Prediction algorithms for pMHC-I binding based on neural networks like NetMHC are generally accepted to be useful yet imperfect tools, and their biases are typically hard to capture. To control for the prediction algorithm, we hypothesized that MHC-I allele frequency bias should not be observed for proteins or protein fragments that are not under selective immune pressure. To test this, we scored the predicted binding affinity of each CALRMUT-derived peptide or other irrelevant proteins in each individual MPN patient to generate what we have termed the Patient:Peptide Score (PPS). Briefly, the PPS of a peptide in a patient is equal to the binding score (nM) of that peptide against the MHC-I allele with the highest predicted binding affinity of the six possible MHC-I alleles for that peptide ( FIG.  2 A ). As expected based on our initial findings, the average PPS of CALRMUT-derived peptides is elevated in CALRMUT MPN patients from both cohorts compared to control groups, where a higher score is associated with worse predicted binding ( FIG.  2 B ,C). However, the average PPS of peptides derived from the wild-type portion of CALR is barely changed in the NEUS cohort and completely unchanged in the Danish cohort for the CALRMUT MPN patients compared to control groups ( FIG.  2 B ,C). Similarly, the capacity to bind peptides derived from the irrelevant foreign protein neuraminidase (NA) from the influenza virus does not substantially change comparing CALRMUT, JAK V617F  patients and the general US Caucasian populations ( FIG.  2 B ,C). Furthermore, when CALRMUT-derived peptides are subdivided based on predicted binding (&lt;10000 nM) and non-binding (&gt;10000 nM) scores, the greatest shift in average PPS occurs in predicted binding peptides (&lt;10000 nM), suggesting selection pressure against the capacity to efficiently bind those peptides in CALRMUT patients ( FIG.  2 B ,C). A generous cutoff of 10000 nM was used here to account for any bias in the prediction algorithm. Together, our findings suggest CALRMUT MPN patients have a skewed MHC-I allele repertoire that is less immunologically responsive to the CALRMUT protein fragment. The implication of this observation also suggests that these patients would be less likely to respond to cancer vaccines consisting of the CALRMUT fragment. 
     Therefore, we hypothesized that one approach to elicit an immune response against CALRMUT in these patients is to use a vaccine consisting of MHC-I binding-optimized heteroclitic peptides. To test this hypothesis, we examined the peptides with the lowest PPS in the CALRMUT MPN patients as possible candidates. We observe that the top peptide in both cohorts is the 9mer CALRMUT peptide starting at position 2 (CALR9p2) RMMRTKMRM (SEQ ID NO. 263) ( FIG.  2 D ), which is predominantly a function of its predicted binding to frequently observed MHC-I alleles such as HLA-A*02:01 and HLA-A*03:01 ( FIG.  2 E ,F). Therefore, based on the mean PPS of CALR9p2 in both cohorts, we hypothesized that this peptide is not able to potentiate naïve CD8+ T cells, but could be targeted by antigen-experienced CD8+ T cells. 
     Non-Responding Human PBMCs can Cross-React with CALR9p2 if First Primed with Heteroclitic Peptides 
     We next investigated whether heteroclitic peptides could be used to induce cross-reactivity against CALRMUT-derived peptides in human samples. To identify the best candidate CALR9p2 heteroclitic peptide, we examined the mean predicted PPS score of the NEUS CALRMUT MPN cohort to every possible CALR9p2 peptide variant containing a single amino acid substitution ( FIG.  3 A ). Seven of the top heteroclitic CALR9p2 peptide variants were selected for testing based on their predicted binding to HLA-A*02:01 ( FIG.  3 B ). Notably, five of those selected had a substitution at position 6 (K6) and two were selected with a substitution at position 1 (R1). A closer examination of binding predictions of each heteroclitic candidate to the top ten most frequent MHC-I alleles in CALRMUT MPN patients reveals that the K6 heteroclitic peptides could target multiple MHC-I alleles with predicted binding of the CALR9p2 ranging from 500-5000 nM, while the R1 heteroclitic peptides mostly affect HLA-A*02:01 binding affinity ( FIG.  3 C ). We also observed that alterations of the residues at positions 1 and 6 were predicted to affect HLA-A*02:01 binding through its minor anchor sites instead of the main anchor sites at positions 2 and 9, which are predicted to already have non-ideal but still adequate residues at these positions (32, 33). Each heteroclitic CALR9p2 peptide was tested for its ability to bind to HLA-A*02:01 and confirmed to have greater binding then native CALR9p2 peptide, which had a weak binding signal compared to DMSO control ( FIG.  10   ). However, all of the heteroclitic CALR9p2 peptides had weaker binding potential than the MART1-A2 peptide positive control. 
     PBMCs from six healthy HLA-A*02:01 individuals with known MHC-I haplotypes and PPS were stimulated for 10 days with a cytokine cocktail in the presence of: CALR9p2, each heteroclitic peptide individually, all heteroclitic peptides pooled, or a positive control peptide mixture of T cell epitopes from Cytomegalovirus, Epstein-Barr virus, Influenza and  Clostridium Tetani  (CEFT). Cells were then restimulated with control peptides, initial priming peptides, or in the case of heteroclitic peptide stimulation conditions, the CALR9p2 peptide and tested for IFNγ production ( FIG.  3 D ,E). As controls, some cells were unstimulated (DMSO), or stimulated with PMA and ionomycin ( FIG.  11   ). Four of six samples responded to at least one heteroclitic peptide ( FIG.  3 D ,E). Interestingly, three of the six healthy donors had significant (P&lt;0.05) or trending (P&lt;0.30) CD8+ T cells responses to CALR9p2 alone (Donors 2, 3 and 4), and two of them also showed significant CD8+ T cells cross-reactivity against CALR9p2 if primed with a heteroclitic peptide (Donors 3 and 4). Importantly, in two of the samples that did not respond to CALR9p2 alone (Donors 1 and 6), there was significantly detectable CD8+ T cell cross-reactivity with CALR9p2 when the PBMCs were primed with heteroclitic peptides. To test whether the heteroclitic peptides were indeed promoting cross-reactivity through HLA-A*02:01, we again activated healthy donor HLA-A*02:01-positive PBMCs using pooled heteroclitic peptides but the final restimulation was provided using peptide-pulsed K562 cells (HLA-null) transduced to only express HLA-A*02:01. In the four additional donors tested, two had a cross-reactive response against the CALR9p2 peptides, demonstrating that HLA-A*02:01 was at least one of the HLA alleles through which the heteroclitic peptides were providing a cross-reactive response ( FIG.  12   ). Together, our results suggest that cross-reactive immunity to CALRMUT can be achieved in human cells, especially if multiple different heteroclitic peptides are utilized. 
     Modeling CALRMUT MPN Patient MHC-I Allele Skewing in Mice 
     To determine whether a heteroclitic peptide cancer vaccine is a viable strategy against the CALRMUT fragment, we tested this approach in a pre-clinical mouse model mimicking CALRMUT MPN MHC-I allele skewing. We analyzed the predicted binding of all CALRMUT-derived peptides against all murine MHC-I alleles for which predictions are possible. We found no strong binding peptide (&lt;500 nM) to all murine MHC-I alleles but did observe that H-2Kb has a weakly binding predicted affinity to CALR9p2. When tested for its ability to stabilize MHC-I in the H-2Kb-expressing TAP-deficient RMA/S cell line, CALR9p2 did not elicit detectable H-2Kb stabilization compared to the control strong binding chicken ovalbumin (OVA)-derived peptide SIINFEKL (SEQ ID NO. 287) ( FIG.  4 B ). However, when serum was omitted from the assay, H-2Kb was stabilized only when the highest concentration of peptide (100 μg/mL), suggesting poor but still detectable binding ( FIG.  4 B ). To investigate whether the CALRMUT protein fragment is immunogenic in vivo, we immunized H2-Kb-expressing C57BL/6J (B6) mice with a DNA vaccine ( FIG.  4 C ) encoding the full-length 52 base pair deletion variant of the CALRMUT sequence (4-6) and analyzed CALR9p2 peptide-specific CD8+ T cells from draining lymph nodes. Compared to the immunogenic CD8+ T cell response against the SIINFEKL (SEQ ID NO. 287) peptide in OVA-immunized mice, mice immunized with the full-length CALRMUT sequence did not elicit any CD8+ T cell response against CALR9p2 ( FIG.  4 D ). Likewise, mice immunized in the footpad ( FIG.  4 E ) with the CALR9p2 peptide emulsified in the Titermax® adjuvant also did not elicit any CALR9p2-specific CD8+ T cell response compared to SIINFEKL(SEQ ID NO. 287)-immunized mice ( FIG.  4 F ). 
     We posited that this mouse model is a good preclinical model candidate of CALRMUT MPN patients mimicking an MHC-I skewed haplotype because we observe poor but detectable binding of CALR9p2 to H-2Kb but no vaccine-induced CALR9p2-specific CD8+ T cell responses in B6 mice. 
     Full-Length CALRMUT Variant does not have Dominant-Negative Activity 
     We wanted to investigate whether the CALRMUT fragment itself could inhibit antigen presentation. Wildtype CALR is required for antigen-presentation in healthy cells (34, 35) and the full-length CALRMUT is reported to be non-functional with respect to peptide loading (35). However, nearly all CALRMUT-positive MPN tumors are heterozygous (4, 5) and therefore have one wild-type copy of CALR, yet it is unknown whether CALRMUT acts as a dominant-negative with respect to its role in antigen presentation. To exclude this possibility, we co-transfected the murine B16F10 cells with the DNA sequences encoding OVA and either CALRWT-mCherry, CALRMUT-mCherry or the mCherry constructs, and measured surface expression of H-2Kb-presented SIINFEKL peptide. We observed that cells transfected with the CALRMUT variant had an equal percentage of H-2Kb-SIINFEKL (SEQ ID NO. 287) peptides expressing cells ( FIG.  13 B ), suggesting that the CALRMUT variant does not inhibit antigen presentation. Interestingly, we observe a marginal decrease in total surface MHC-I expression ( FIG.  12 C ), suggesting some effect of the CALRMUT variant, although it is unclear whether such a small reduction in MHC-I would have any functional effect. As a result, we conclude that CALRMUT does not prevent tumor cells from presenting antigens and suggest that CALRMUT tumors could respond to cancer vaccines, if the correct antigen is selected and is expressed by the tumor. 
     C57BL/6J-Optimized Heteroclitic CALR9p2 Peptide Elicits Cross-Reactive Immunity in Mice 
     To identify the best candidate CALR9p2 heteroclitic peptide in C57BL/6J (B6) mice, we examined the predicted binding affinity of H-2Kb to every possible CALR9p2 peptide variant containing a single amino acid substitution. We observed that the variant with the strongest predicted affinity has a threonine (T) to phenylalanine (F) substitution at position 5 (T5F) of the CALR9p2 peptide ( FIG.  5 A ). The CALR9p2-T5F peptide has the amino acid sequence of SEQ ID NO. 40. This is consistent with previous studies showing that this site is a major anchor residue for H-2Kb ( FIG.  5 B ) (19, 36, 37). When investigated for its ability to stabilize H-2Kb in RMA/S cells, CALR9p2(T5F) demonstrated approximately a tenfold greater H-2Kb stabilization compared to CALR9p2 ( FIG.  5 C ). We then tested whether CALR9p2(T5F) could elicit a cross-reactive CALR9p2 immune response. Mice immunized with a single dose of CALR9p2(T5F) elicited a CD8+ T cell capable of cross-reacting with CALR9p2 in vitro ( FIG.  5 D ) and killing tumor cells pulsed with the CALR9p2 peptide ( FIG.  5 E ). Likewise, mice immunized by DNA vaccine against CALRMUT encoding the CALR9p2(T5F) variant also elicited a cross-reactive response against CALR9p2 ( FIG.  14 A ). CALR9p2(T5F)-specific CD8+ T cells by tetramer staining ( FIG.  14 D ) had higher levels of the activation markers CD44, Tim3 and Pd1. Importantly, the ability to mount an antigen specific response against the full-length antigen demonstrated that the full-length CALRMUT sequence can be endogenously processed and presented, which had not previously been proven directly. 
     To confirm that the same TCR clones were recognizing both CALR9p2(T5F) and CALR9p2, CD8+ T cells from CALR9p2(T5F)-immunized mice were restimulated in vivo with CALR9p2, and CALR9p2(T5F)-tetramer-specific CD8+ T cells were examined for IFNγ restimulation. The only CALR9p2-potentiated CD8+ T cells were those also staining for the CALR9p2(T5F)-tetramer ( FIG.  15 A-D ). Notably, both the CALR9p2-specific and CALR9p2(T5F)-specific CD8+ T cells had equal levels of Tim3 and Pd1 after restimulation. 
     To test the ability of a CALRMUT heteroclitic peptide vaccine to elicit a cross-reactive anti-tumor response in vivo, we used the newly developed PresentER antigen minigene system (38). Here, the nucleotide sequence of the CALR9p2 peptide is cloned downstream of an ER signal sequence (SS) and virally transduced into TAP-deficient RMA/S cells (RMA/SpER-CALR9p2). Once expressed, the peptide-SS is shuttled into the ER and the peptide is cleaved from the SS, releasing CALR9p2 into the ER where it can be loaded into MHC-I, assuming binding is possible. When mice were given three doses of the heteroclitic peptide vaccine prior to tumor implantation, RMA/SpER-CALR9p2 tumors grew significantly slower than those injected into mice given adjuvant alone or with the CALR9p2 peptide, which both grew at the same rate ( FIG.  5 F ,G) although differences in survival were not significant ( FIG.  5 H ). However, when RMA/SpER-CALR9p2 tumors were allowed to grow before mice received multiple therapeutic doses of the heteroclitic peptide vaccine ( FIG.  5 I ), tumors grew significantly slower ( FIG.  5 J ,K) and mice had improved survival ( FIG.  5 L ) compared to the adjuvant alone condition. Importantly, the effect of the vaccine was even more prominent when the immunization is administered with immune checkpoint blockade using a PD-1 antibody ( FIG.  5 I-K ). 
     Interestingly, the therapeutic vaccine had greater efficacy than the prophylactic vaccine. As this was unexpected, we hypothesized that CALR9p2-specific cross-reactive CD8+ T cells were diminishing in efficacy over time and that the available CALR9p2 antigen present in the tumor cells was not generating a strong memory response. To investigate this further, we immunized mice with three doses of the heteroclitic CALR9p2(T5F) peptide vaccine and compared cross-reactive potential in conditions where mice instead received CALR9p2 peptide boosts following an initial CALR9p2(T5F) priming dose. Consistent with the prophylactic vaccine results, mice that received an initial CALR9p2(T5F) followed by two CALR9p2 boosts had no detectable cross-reactive CD8+ T cell responses to CALR9p2 in vitro and a very small response to the CALR9p2(T5F) peptide ( FIG.  16   ). On the other hand, mice that received two doses of CALR9p2(T5F) and a final CALR9p2 boost had no detectable cross-reactive CD8+ T cells but an intermediate response to CALR9p2(T5F). Therefore, it appears that cross-reactive immunity to the CALR9p2 peptide in the context of B6 mice is a function of time-from-last CALR9p2(T5F) dose. 
     Together, the results of this study provide proof of principle for the use of a heteroclitic peptide cancer vaccine strategy for tumor cells expressing CALRMUT antigens. Such a vaccine could provide a valuable non-redundant benefit in CALRMUT MPN patients, as there are currently no rationally designed treatments specially targeting the CALR mutation. While the JAK1/JAK2 inhibitor, ruxolitinib is approved by the FDA for the treatment of patients with MPN, this approval was granted primarily based on symptomatic benefits (53, 54). Although CALRMUT MPN patients demonstrate clinical responses to ruxolitinib, there is no reduction in CALRMUT allele burden following JAK2 inhibition and as a result ruxolitinib does not have substantial disease-modifying activity in MPN and is not curative (55). Mutations in CALR are disease-initiating in MPN and often occur as the sole mutation (4). 
     We plan to perform clinical trials. Initial clinical trials of heterolytic CALRMUT directed vaccination approaches as described herein will focus primarily on safety and will be performed in patients with more advanced MPN. A longer-term goal will be to treat CALRMUT MPN patients early in the course of their disease before genetic and clonal evolution has occurred. By directing autologous immune responses specifically against CALRMUT, peptide vaccination offers the potential to preferentially target the disease-initiating MPN stem cell in patients, which is a deficiency of current MPN drugs, including JAK inhibitors. Accordingly, CALRMUT targeted peptide vaccination offers the potential to definitely eradicate MPN to cure the disease. 
     Importantly, while our work suggests that certain patients are not likely to respond to a cancer vaccine composed solely of the CALRMUT fragment due to MHC-I skewing, we believe that these patients are likely to respond to a heteroclitic peptide cancer vaccine as demonstrated in our pre-clinical model. Furthermore, as our data shows that this strategy can be enhanced with immune checkpoint blockade (anti-PD-1/PD-L1), an off-the-shelf vaccine composed of one or more of the heteroclitic CALRMUT peptides described herein in combination with anti-PD-1 or other checkpoint inhibitor therapies, can be a viable strategy for CALRMUT MPN patients. 
     Methods 
     Patient Samples 
     Approval was obtained for the use of patient-derived specimens and access to clinical data extracted from patient charts by the Institutional Review Boards at Memorial Sloan Kettering Cancer Center, the Dana-Farber Cancer Institute and the Massachusetts General Hospital, as well as by the Danish Regional Science Ethics Committee. All patients analyzed in this study were diagnosed with MPN and tested positive for the CALRMUT or JAK V617F  mutations. 
     Mice 
     C57BL/6J mice were purchased from The Jackson Laboratory (Sacramento, Calif.). Mouse experiments were performed in accordance with institutional guidelines under a protocol approved by the Memorial Sloan-Kettering Cancer Center Institutional Animal Care and Use Committee. All mice were maintained in a pathogen-free facility according to the National Institutes of Health Animal Care guidelines. 
     MHC Allele Frequencies 
     For the NEUS cohort, MHC genotypes were manually extracted from patient charts. For the Danish cohort, HLA class I genotypes were determined by next-generation sequencing performed on bio-banked samples collected for a previous study (56) using recently described methods (57). In cases where NMDP allele codes were used instead of the World Health Organization nomenclature, the conversion was done according to https://bioinformatics.bethematchclinical.org/hla-resources/allele-codes/allele-code-lists/. If multiple alleles were plausible for a given NMDP allele code, we selected the most likely allele based on ethnicity (typically around 99% confidence, based on known frequencies in the general population (17)). MHC allele frequency for each HLA gene (A, B, C, DQ, and DR) was broadly calculated as the number of each specific allele divided by the number of the total allele in that cohort (2n per individual). In rare samples, certain patients had incomplete haplotype information where one or more alleles were unknown or incomplete. If the allele was missing, it was censored from the number of total alleles. If locus and group were known (ex: HLA-A*02) but the exact protein was unknown (ex: HLA-A*02:XX), this allele was censored from the frequency calculation only for alleles from the same group. For an MHC allele to be considered positively skewed, it was required to have an allele frequency of &gt;0.05 in CALRMUT MPN cases and have &gt;0.2 fold frequency increase compared to both the JAK2 V617F  and US Caucasian population groups allele frequency in the NEUS cohort, or a &gt;0.125 fold frequency increase compared to the JAK2 V617F  group allele frequency in the Danish cohort. Likewise, for an MHC allele to be negatively skewed, it was required to have a frequency of at least 0.05 in both the JAK2 V617F  and US Caucasian population groups for the NEUS cohort, or just the JAK2 V617F  group for the Danish cohort, and have &gt;0.2 fold decrease in the CALRMUT MPN compared to both the JAK2 V617F  and US Caucasian population groups allele frequency in the NEUS cohort, or a &gt;0.125 fold frequency decrease compared to the JAK2 V617F  group allele frequency in the Danish cohort. Principal component analysis was calculated in R and plotted in Graphpad Prism 7. All data processing and analysis were performed using the R version 3.3.2 Sincere Pumpkin Patch and GraphPad Prism v7. 
     Binding Affinity Predictions and Patient:Peptide Score (PPS) 
     pMHC-I binding predictions were collected using NetMHCpan v3 (32) for human MHC-I alleles and NetMHC v4 (36) for murine MHC-I alleles. pMHC-II binding predictions were collected using NetMHCIIpan v3.2 (58). To calculate the PPS, peptide affinities for all six possible MHC-I alleles were identified and only the lowest affinity value was retained. The protein fragment RRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTEA (SEQ ID NO. 288) was used for the CALRMUT. For the CALRWT sequence, amino acids 1-361 of the UniProt sequence P27797 was used. For the Influenza neuraminidase sequence, the full UniProt sequence D7ED91 was used. All subsequent data processing and analyses were performed using the R version 3.3.2 Sincere Pumpkin Patch and GraphPad Prism v7. 
     MHC-I Stabilization Assay 
     To test peptide binding to murine H-2Kb or human HLA-A*02:01, TAP-deficient RMA/S and T2 cells were used, respectively, as previously reported (59, 60). Briefly, RMA/S cells (61) were serum-starved in serum-free RPMI media overnight at 31° C., and peptides were added at indicated final concentrations, followed by 30 minutes at 31° C. and another 3 hours at 37° C. before measuring H-2Kb by flow cytometry (BD; Clone AF6-88.5). T2 cells (62) were serum-starved in serum-free RPMI media overnight and cells were added at indicated final concentrations for 16 hours before measuring HLA-A*02:01 levels by flow cytometry (BD; Clone BB7.2). The SIINFEKL (SEQ ID NO. 287) peptide was acquired as a custom order from Genscript. The MART1-A2 peptide (ELAGIGILTV, SEQ ID NO. 289) was purchased from JPT Peptide Technologies. 
     Murine Immunizations and IFNγ ELISpot Assay 
     All peptides were purchased as custom peptide synthesis orders from GenScript at a purity of &gt;98% and resuspend at 10 mg/mL in DMSO (Sigma). For peptide vaccines, peptides were diluted in PBS and emulsified with Titermax® (Titermax USA, Inc) at a 1:1 ratio immediately prior to immunization, such that each dose was composed of 10 μg peptide in a total volume of 25 uL. In control (DMSO) immunization, an equivalent volume of DMSO is substituted for the diluted peptide in the TiterMax® emulsion. For subsequent in vitro assays, draining inguinal and popliteal lymph nodes were collected at indicated time points and CD8+ T cells were isolated using positive magnetic sorting using mouse CD8a (Ly-2) MicroBeads (Miltenyi Biotec). DNA vaccines were performed using a gene gun as previously described (27, 63) according to indicated time points. Briefly, mice received four injections (400 lbs/inch2) of DNA-coated gold particles into the abdominal region of the skin for a total 4 μg of DNA per dose. DNA plasmids encoding the wildtype or 52 base-pair deletion CALRMUT sequences fused with the flag sequence are as previously described (64). For pING-OVA, the full-length chicken ovalbumin sequence was cloned into the pING plasmid (65). CD8+ T cells were collected as before, but only from draining inguinal lymph nodes. To test for antigen-specificity, the mouse IFNγ ELISpot set (BD) was used according to the manufacturer&#39;s instructions. Briefly, CD8+ T cells were frozen immediately after purification in FBS containing 10% DMSO, thawed one day prior to restimulation and allowed to recover overnight in 20 U/mL IL-2 (Peprotech) RPMI-1640 medium containing 10% fetal bovine serum (FBS), Na-Pyruvate, L-glutamine and Penicillin/Streptomycin. As a source of antigen-presenting cells (APCs), splenocytes from naïve mice were depleted of T cells using magnetic microbeads for CD8a (Ly2) and CD4 (L3T4) (Miltenyi Biotec), pulsed for one hour with 100 μg/mL peptide at 37° C. followed by a wash. For each well, 105 CD8+ T cells were co-culture with 1×105-3×105 peptide-pulsed APCs and incubated for approximately 18 hours. Spots were counted using the ImmunoSpot analyzer (Cellular Technology Limited). 
     SIINFEKL (SEQ ID NO. 287) H-2Kb Expression 
     B16F10 were co-transfected with equal parts pING-OVA and pCMV-Sport6-CALR constructs fused to mCherry, which are previously described (64), using the Megatran 1.0 transfection reagent (Origene). Each construct was mixed with the transfection reagent separately such that all cells received the same amount of pING-OVA construct. B16F10 cells were originally obtained from I. Fidler (M. D. Anderson Cancer Center) and cultured in RPMI 1640 medium supplemented with 7.5% inactivated FBS, 1×non-essential amino acids and 2 mM L-glutamine. One day after transfection, cells were stained by flow cytometry with H-2Kb (BD; Clone AF6-88.5) and H-2Kb-SIINFEKL (SEQ ID NO. 287) (Biolegend; Clone 25-D1.16). 
     Tumor Growth Experiments 
     RMA/S cells were maintained in RPMI 1640 medium supplemented with 7.5% inactivated FBS, 1×non-essential amino acids and 2 mM L-glutamine. The DNA sequence encoding the CALR9p2 peptide (bold) was cloned into the PresentER-IRES-GFP (38) construct using the following oligo: 
                            (SEQ ID NO. 290)           5′GGCCGTATTGGCCCCGCCACCTGTGAGCGG                       GAGGATGATGAGGACAAAGATGAGGATGTAAGGCC                       AAACAGGCC-3′            
following SfiI digestion and T4 ligation (New England Biolabs). The resulting construct was used to generate retrovirus by co-transfection with pCL-Ampho into ecotropic Pheonix cells (ATCC). Viral supernatants were collected at 48 and 72 hours, pooled and Retro-X Concentrator (Takara Bio USA)-concentrated retrovirus was used to transduce RMA/S cells by spinoculation using polybrene (Sigma). GFP-positive cells were FACS sorted (BD FACSAria III) and cultured in 4 μg/mL puromycin (Gibco) media. A total of 5×106 cells were injected subcutaneously in the flank of mice. For anti-PD1 treatment, 250 μg of RMP1-14 was injected intra-peritoneally in PBS at indicated time points.
 
     Human PBMC In Vitro Restimulation 
     Freshly isolated or thawed cryopreserved healthy donor PBMCs were restimulated with cytokines and peptides as previously described (14). Briefly, on day 0, PBMCs were resuspended in X-VIVO15 media (Lonza) and seeded at 105 per well of a 96 U-bottom plate with 1000 IU/mL GM-CSF (Sanofi), 500 IU/mL IL-4 (R&amp;D Systems) and 50 ng/mL Flt3L (R&amp;D Systems). On day 1, media was refreshed with 0.1 μg/mL LPS (Invivogen), 10 μM R848 (Invivogen), 5 μg/mL IL-10 (R&amp;D Systems) and 1 μg/mL of indicated peptides, and incubated for 24 hours. The CMV, EBV, Flu, Tetanus (CEFT) and the myelin oligodendrocyte glycoprotein (MOG) peptide pools (JPT Technologies) were used as positive and negative controls, respectively. On days 2 and 5, half the media was refreshed with RPMI (Gibco) containing 10% human serum (Gemini Bio-Products), 10 ug/ml gentamycin (Gibco), HEPES (Gibco), GlutaMAX (Gibco) and hIL-2 and hIL-7 to a final concentration of 10 IU/mL and 10 ng/mL, respectively (R&amp;D Systems). On day 8, the media was refreshed without cytokines. On day 10, PBMCs were restimulated with corresponding peptides in the presence of 1 μg/mL of anti-hCD28 and anti-hCD49d (BD Biosciences). As controls, some cells were stimulated with PMA (Sigma-Aldrich, 50 ng/mL) and ionomycin (Sigma-Aldrich, 1 μg/mL). For intracellular staining, monensin and brefeldin A (BD Biosciences) were added 1 hour after restimulation cells and culture left to incubate for another 12 hours. Cells were then stained for CD3 (Clone: OKT3, FITC), CD4 (Clone: RPA-T8, APC) and CD8a (Clone: RPA-T4, BV785), permeabilized and fixed with BD Cytofix/Cytoperm™ reagents according to manufacturer&#39;s protocol and subsequently stained for IFNγ (Clone: B27, PE) and TNFα (Clone: Mab11, PE/Cy7). All antibodies were purchased BioLegend. LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit by Thermo Fischer Scientific was used for live and dead cell discrimination. Data was acquired using the BD Fortessa and the data was analyzed on FlowJo V10 (TreeStar). For the ELISpot, methods are as previously described (14) using the follower 15-mer peptides that cover the entire CALRMUT fragment: RTRRMMRTKMRMRRM (SEQ ID NO. 291), MMRTKMRMRRMRRTR (SEQ ID NO. 292), TKMRMRRMRRTRRK (SEQ ID NO. 293), RMRRMRRTRRKMRRK (SEQ ID NO. 294), MRRTRRKMRRKMSPA (SEQ ID NO. 295), RRKMRRKMSPARPRT (SEQ ID NO. 296), RRKMSPARPRTSCRE (SEQ ID NO. 297), SPARPRTSCREACLQ (SEQ ID NO. 298), PRTSCREACLQGWTE (SEQ ID NO. 299), TSCREACLQGWTEA (SEQ ID NO. 300). 
     Statistical Analysis 
     Details of the study outline, sample size, and statistical analysis are shown in the main Example text, above and in, the Figures and Brief Description of the Figures. To calculate significance in distribution of MHC frequencies, Barnard&#39;s unconditional test and the chi-square test were used as indicated in R using the barnard.test function (two-tail) from the Barnard package and as well as the base chisq.test function. The R version 3.3.2 Sincere Pumpkin Patch was used. For unpaired Student&#39;s t tests, area under the curve calculations and log-rank survival test, GraphPad Prism v7 was used. 
     Example 2 
     Additional CALR MUT  heteroclitic peptides were designed utilizing a novel algorithm that we designed specifically to identify and select heteroclitic peptides likely to be useful for vaccination of as large a proportion of the general population as possible. In brief, this entailed first identifying native CALR MUT  peptides likely to be good starting points for the generation of heteroclitic mutants/derivatives based on their predicted utility for vaccination of the greatest number of patients (based on HLA-I allele diversity). Then mutations of these “native” peptides were evaluated based on certain criteria to identify heteroclitic mutants. The conditions for a mutant peptide to be considered heteroclitic in a given individual were: 1) that the native peptide from which it was derived had a predicted binding affinity to a given HLA-I expressed by that individual of between 500 nM and 2000 nM (i.e. intermediate binding HLA-I), and 2) that the mutation altered the predicted binding affinity of the peptide to at least one of the intermediate binding HLA-Is in that individual to &lt;500 nM. From that first subset of mutants identified as being heteroclitic using those two criteria, a second subset having a predicted HLA-I binding affinity of &lt;100 nM was identified—as these were predicted to have the best immunogenicity. Our algorithm assigned scores to all of the heteroclitic peptides based on various criteria including their likely utility across multiple ethnic groups (Caucasian, African, Asian and Hispanic) and across multiple HLA phenotypes. The amino acid sequences of the subset of 262 heteroclitic derivatives having the best scores as determined by this method are provided in Table 1 (SEQ ID NOs 1-262). An analysis of the amino acid sequences of these 262 peptides in comparison to the native CALR MUT  peptide from which they were derived, identified certain common features of the mutant heteroclitic derivatives—which common features are described by the various consensus amino acid sequences provided in Table 2. Seven of the mutant heteroclitic derivatives were tested functionally in living human cells and/or in mice, as described in Example 1, providing proof of concept for the design approach and for the utility of the designed mutant heteroclitic peptides. 
     REFERENCE LIST 
     
         
         1. R. L. Levine, D. G. Gilliland, Myeloproliferative disorders. Blood 112, 2190-2198 (2008). 
         2. P. J. Campbell, A. R. Green, The myeloproliferative disorders. N Engl J Med 355, 2452-2466 (2006). 
         3. Rampal et al., Integrated genomic analysis illustrates the central role of JAK-STAT pathway activation in myeloproliferative neoplasm pathogenesis. Blood 123, e123-133 (2014). 
         4. Nangalia et al., Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. N Engl J Med 369, 2391-2405 (2013). 
         5. Klampfl et al., Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med 369, 2379-2390 (2013). 
         6. Elf et al., Mutant Calreticulin Requires Both Its Mutant C-terminus and the Thrombopoietin Receptor for Oncogenic Transformation. Cancer Discov 6, 368-381 (2016). 
         7. Chachoua et al., S. N. Constantinescu, Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants. Blood 127, 1325-1335 (2016). 
         8. Araki et al., Activation of the thrombopoietin receptor by mutant calreticulin in CALR-mutant myeloproliferative neoplasms. Blood 127, 1307-1316 (2016). 
         9. How &amp; Hobbs, A. Mullally, Mutant calreticulin in myeloproliferative neoplasms. Blood 134, 2242-2248 (2019). 
         10. Holmstrom et al., The CALR exon 9 mutations are shared neoantigens in patients with CALR mutant chronic myeloproliferative neoplasms. Leukemia 30, 2413-2416 (2016). 
         11. Holmstrom et al., The calreticulin (CALR) exon 9 mutations are promising targets for cancer immune therapy. Leukemia 32, 429-437 (2018). 
         12. Tubb, et al., Isolation of T cell receptors targeting recurrent neoantigens in hematological malignancies. Journal for immunotherapy of cancer 6, 70 (2018). 
         13. Holmstrom et al., High frequencies of circulating memory T cells specific for calreticulin exon 9 mutations in healthy individuals. Blood Cancer J 9, 8 (2019). 
         14. Cimen Bozkus, et al., Immune Checkpoint Blockade Enhances Shared Neoantigen-Induced T Cell Immunity Directed against Mutated Calreticulin in Myeloproliferative Neoplasms. Cancer Discov, (2019). 
         15. Schischlik et al., Mutational Landscape of the Transcriptome Offers Putative Targets for Immunotherapy of Myeloproliferative Neoplasms. Blood, (2019). 
         16. Madden, The three-dimensional structure of peptide-MHC complexes. Annu Rev Immunol 13, 587-622 (1995). 
         17. Gragert et al., Six-locus high resolution HLA haplotype frequencies derived from mixed-resolution DNA typing for the entire US donor registry. Human immunology 74, 1313-1320 (2013). 
         18. Falk et al., Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules. Nature 351, 290-296 (1991). 
         19. Rammensee et al., MHC ligands and peptide motifs: first listing. Immunogenetics 41, 178-228 (1995). 
         20. Boulanger et al., Dalchau, A Mechanistic Model for Predicting Cell Surface Presentation of Competing Peptides by MHC Class I Molecules. Front Immunol 9, 1538 (2018). 
         21. Ericsson et al., Differential activation of phospholipase C-gamma 1 and mitogen-activated protein kinase in naive and antigen-primed CD4 T cells by the peptide/MHC ligand. Journal of immunology 156, 2045-2053 (1996). 
         22. Kimachi et al., The minimal number of antigen-major histocompatibility complex class II complexes required for activation of naive and primed T cells. Eur J Immunol 27, 3310-3317 (1997). 
         23. London et al., Functional responses and costimulator dependence of memory CD4+ T cells. Journal of immunology 164, 265-272 (2000). 
         24. Pihlgren et al., Resting memory CD8+ T cells are hyperreactive to antigenic challenge in vitro. The Journal of experimental medicine 184, 2141-2151 (1996). 
         25. Rogers et al., Qualitative changes accompany memory T cell generation: faster, more effective responses at lower doses of antigen. Journal of immunology 164, 2338-2346 (2000). 
         26. Slifka et al., Functional avidity maturation of CD8(+) T cells without selection of higher affinity TCR. Nat Immunol 2, 711-717 (2001). 
         27. Dyall et al., Heteroclitic immunization induces tumor immunity. The Journal of experimental medicine 188, 1553-1561 (1998). 
         28. Rosenberg, et al., White, Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nature medicine 4, 321-327 (1998). 
         29. England, et al., Molecular analysis of a heteroclitic T cell response to the immunodominant epitope of sperm whale myoglobin. Implications for peptide partial agonists. Journal of immunology 155, 4295-4306 (1995). 
         30. Boehncke, et al., The importance of dominant negative effects of amino acid side chain substitution in peptide-MHC molecule interactions and T cell recognition. Journal of immunology 150, 331-341 (1993). 
         31. Gonzalez-Galarza, et al., Allele frequency net 2015 update: new features for HLA epitopes, KIR and disease and HLA adverse drug reaction associations. Nucleic Acids Res 43, D784-788 (2015). 
         32. Nielsen &amp; Andreatta, NetMHCpan-3.0; improved prediction of binding to MHC class I molecules integrating information from multiple receptor and peptide length datasets. Genome Med 8, 33 (2016). 
         33. Hoof, et al., NetMHCpan, a method for MHC class I binding prediction beyond humans. Immunogenetics 61, 1-13 (2009). 
         34. Gao, et al., Assembly and antigen-presenting function of MHC class I molecules in cells lacking the ER chaperone calreticulin. Immunity 16, 99-109 (2002). 
         35. Arshad, et al., Tumor-associated calreticulin variants functionally compromise the peptide loading complex and impair its recruitment of MHC-I. J Biol Chem 293, 9555-9569 (2018). 
         36. Andreatta &amp; Nielsen, Gapped sequence alignment using artificial neural networks: application to the MHC class I system. Bioinformatics 32, 511-517 (2016). 
         37. Nielsen, et al., Reliable prediction of T-cell epitopes using neural networks with novel sequence representations. Protein Sci 12, 1007-1017 (2003). 
         38. Gejman, et al., Rejection of immunogenic tumor clones is limited by clonal fraction. Elife 7, (2018). 
         39. Dunn, et al., The three Es of cancer immunoediting. Annu Rev Immunol 22, 329-360 (2004). 
         40. Teng, et al., From mice to humans: developments in cancer immunoediting. The Journal of clinical investigation 125, 3338-3346 (2015). 
         41. Hanahan, et al., Hallmarks of cancer: the next generation. Cell 144, 646-674 (2011). 
         42. O&#39;Donnell, et al., Cancer immunoediting and resistance to T cell-based immunotherapy. Nature reviews. Clinical oncology 16, 151-167 (2019). 
         43. Efremova, et al., Targeting immune checkpoints potentiates immunoediting and changes the dynamics of tumor evolution. Nat Commun 9, 32 (2018). 
         44. Rizvi, et al., Molecular Determinants of Response to Anti-Programmed Cell Death (PD)-1 and Anti-Programmed Death-Ligand 1 (PD-L1) Blockade in Patients With Non-Small-Cell Lung Cancer Profiled With Targeted Next-Generation Sequencing. J Clin Oncol 36, 633-641 (2018). 
         45. Turajlic et al., Insertion-and-deletion-derived tumour-specific neoantigens and the immunogenic phenotype: a pan-cancer analysis. Lancet Oncol 18, 1009-1021 (2017). 
         46. Linnebacher, et al., Frameshift peptide-derived T-cell epitopes: a source of novel tumor-specific antigens. International journal of cancer. Journal international du cancer 93, 6-11 (2001). 
         47. Posthuma, et al., Niederwieser, HLA-B8 and HLA-A3 coexpressed with HLA-B8 are associated with a reduced risk of the development of chronic myeloid leukemia. The Chronic Leukemia Working Party of the EBMT. Blood 93, 3863-3865 (1999). 
         48. Kuzelova, et al., Altered HLA Class I Profile Associated with Type A/D Nucleophosmin Mutation Points to Possible Anti-Nucleophosmin Immune Response in Acute Myeloid Leukemia. PloS one 10, e0127637 (2015). 
         49. Sharma, et al., Primary, Adaptive, and Acquired Resistance to Cancer Immunotherapy. Cell 168, 707-723 (2017). 
         50. Rodig, et al., MHC proteins confer differential sensitivity to CTLA-4 and PD-1 blockade in untreated metastatic melanoma. Sci Transl Med 10, (2018). 
         51. Li, et al., T cell receptor signalling in the control of regulatory T cell differentiation and function. Nature reviews. Immunology 16, 220-233 (2016). 
         52. Klebanoff, et al., Therapeutic cancer vaccines: are we there yet? Immunological reviews 239, 27-44 (2011). 
         53. Harrison, et al., JAK inhibition with ruxolitinib versus best available therapy for myelofibrosis. N Engl J Med 366, 787-798 (2012). 
         54. Verstovsek, et al., A double-blind, placebo-controlled trial of ruxolitinib for myelofibrosis. N Engl J Med 366, 799-807 (2012). 
         55. Guglielmelli, et al., Investigators, Ruxolitinib is an effective treatment for CALR-positive patients with myelofibrosis. British journal of haematology 173, 938-940 (2016). 
         56. Knudsen et al, Long-Term Efficacy and Safety of Recombinant Interferon Alpha-2 Vs. Hydroxyurea in Polycythemia Vera: Preliminary Results from the Three-Year Analysis of the Daliah Trial—a Randomized Controlled Phase III Clinical Trial. Blood 132, 580-580 (2018). 
         57. Jan, et al., Recurrent genetic HLA loss in AML relapsed after matched unrelated allogeneic hematopoietic cell transplantation. Blood Adv 3, 2199-2204 (2019). 
         58. Jensen, et al., Improved methods for predicting peptide binding affinity to MHC class II molecules. Immunology 154, 394-406 (2018). 
         59. Houghton, et al., Immunological validation of the EpitOptimizer program for streamlined design of heteroclitic epitopes. Vaccine 25, 5330-5342 (2007). 
         60. Guevara-Patino, et al., A. N. Houghton, Optimization of a self antigen for presentation of multiple epitopes in cancer immunity. The Journal of clinical investigation 116, 1382-1390 (2006). 
         61. Hosken &amp; Bevan, An endogenous antigenic peptide bypasses the class I antigen presentation defect in RMA-S. The Journal of experimental medicine 175, 719-729 (1992). 
         62. Salter, et al., Cresswell, Genes regulating HLA class I antigen expression in T-B lymphoblast hybrids. Immunogenetics 21, 235-246 (1985). 
         63. Weber, et al., Tumor immunity and autoimmunity induced by immunization with homologous DNA. The Journal of clinical investigation 102, 1258-1264 (1998). 
         64. Elf, et al., Defining the requirements for the pathogenic interaction between mutant calreticulin and MPL in MPN. Blood 131, 782-786 (2018). 
         65. Wolchok, et al., DNA vaccines: an active immunization strategy for prostate cancer. Semin Oncol 30, 659-666 (2003).