SEQUENCE ARRANGEMENTS AND SEQUENCES FOR NEOEPITOPE PRESENTATION

Abstract

Systems and methods are presented that allow for selection of tumor neoepitopes that are then used to generate a recombinant polytope that is optimized for proper trafficking and processing. In preferred methods, the polytope is encoded in a viral expression system that is used as a therapeutic agent.

Description

[0001] This application is a divisional application of co-pending U.S. patent application Ser. No. 15/468,046, filed Mar. 23, 2017, which claims priority to US provisional applications with the Ser. Nos. 62/313,621, filed 25 Mar. 2016, 62/312,974, filed 24 Mar. 2016, and 62/314,366, filed 28 Mar. 2016, all of which are incorporated by reference herein.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which is named SequenceListing_102402-27PCT_ST25 and is 76 kilo bytes in size, and was created on Mar. 22, 2017.

FIELD OF THE INVENTION

[0003] The field of the invention is compositions and methods of improved neoepitope-based immune therapeutics, especially as it relates to preparation of recombinant viral therapeutics for cancer therapy.

BACKGROUND OF THE INVENTION

[0004] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0005] All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0006] Cancer immunotherapies targeting certain antigens common to a specific cancer have led to remarkable responses in some patients. Unfortunately, many patients failed to respond to such immunotherapy despite apparent expression of the same antigen. One possible reason for such failure could be that various effector cells of the immune system may not have been present in sufficient quantities, or may have been exhausted. Moreover, intracellular antigen processing and HLA variability among patients may have led to insufficient processing of the antigen and/or antigen display, leading to a therapeutically ineffective or lacking response.

[0007] To increase the selection of targets for immune therapy, random mutations have more recently been considered since some random mutations in tumor cells may give rise to unique tumor specific antigens (neoepitopes). As such, and at least conceptually, neoepitopes may provide a unique precision target for immunotherapy. Additionally, it has been shown that cytolytic T-cell responses can be triggered by very small quantities of peptides (e.g., Sykulev et al., Immunity, Volume 4, Issue 6, p 565-571, 1 Jun. 1996). Moreover, due to the relatively large number of mutations in many cancers, the number of possible targets is relatively high. In view of these findings, the identification of cancer neoepitopes as therapeutic targets has attracted much attention. Unfortunately, current data appear to suggest that all or almost all cancer neoepitopes are unique to a patient and specific tumor and fail to provide any specific indication as to which neoepitope may be useful for an immunotherapeutic agent that is therapeutically effective.

[0008] To overcome at least some of the problems associated with large numbers of possible targets for immune therapy, the neoepitopes can be filtered for the type of mutation (e.g., to ascertain missense or nonsense mutation), the level of transcription to confirm transcription of the mutated gene, and to confirm protein expression. Moreover, the so filtered neoepitope may be further analyzed for specific binding to the patient's HLA system as described in WO 2016/172722. While such system advantageously reduces the relatively large number of potential neoepitopes, the significance of these neoepitopes with respect to treatment outcome remains uncertain. Still further, and especially where multiple peptides are to be expressed in an antigen presenting cell (e.g., dendritic cell), processing of precursor proteins to generate the neoepitopes is not fully understood and contributes as such to the lack of predictability of therapeutic success.

[0009] Thus, even though multiple methods of identification and delivery of neoepitopes to various cells are known in the art, all or almost all of them suffer from various disadvantages. Consequently, it would be desirable to have improved systems and methods for neoepitope selection and production that increases the likelihood of a therapeutic response in immune therapy.

SUMMARY OF THE INVENTION

[0010] The inventive subject matter is directed to various immune therapeutic compositions and methods, and especially recombinant viral expression systems, in which multiple selected neoepitopes are combined to form a rational-designed polypeptide with a trafficking signal to increase antigen processing and presentation and to so maximize therapeutic effect.

[0011] In one aspect of the inventive subject matter, the inventors contemplate a method of generating an expression vector for immune therapy. In preferred aspects, such method includes a step of constructing a recombinant nucleic acid that has a sequence that encodes a polytope that is operably linked to a promoter to drive expression of the polytope, wherein the polytope comprises multiple filtered neoepitope sequences. Most typically, the polytope includes one or more trafficking elements to directs the expressed polytope to a specific sub-cellular location, such as the cytoplasm, the proteasome, the recycling endosome, the sorting endosome, the lysosome, or the extracellular membrane. Alternatively, the trafficking element may also direct the polytope to the extracellular space for secretion.

[0012] Most preferably, the viral expression vector is an adenoviral expression vector having E1 and E2b genes deleted, and the promoter is a constitutive promoter. Alternatively, the promoter may also be an inducible promoter, and especially inducible under conditions that are common in a tumor microenvironment. Thus, suitable promoters may be inducible by hypoxia, IFN-gamma, and/or IL-8.

[0013] With respect to appropriate trafficking element it is contemplated that all know trafficking elements are suitable, however, preferred trafficking elements include cleavable ubiquitin, non-cleavable ubiquitin, a CD1b leader sequence, a CD1a tail, a CD1c tail, and a LAMP1-transmembrane sequence. Furthermore, it is contemplated that the filtered neoepitope sequences are filtered by comparing tumor versus matched normal of the same patient, by determination to have binding affinity to an MHC complex of equal or less than 200 nM, and/or the filtered neoepitope sequences are filtered against known human SNP and somatic variations.

[0014] It is further contemplated that the filtered neoepitope sequences have an arrangement within the polytope such that the polytope has a likelihood of a presence and/or strength of hydrophobic sequences or signal peptides that is below a predetermined threshold. Contemplated filtered neoepitope sequences may bind to MHC-I (typically with an affinity of less than 200 nM) while the trafficking element directs the polytope to the cytoplasm or proteasome, or may bind to MHC-I (typically with an affinity of less than 200 nM) while the trafficking element directs the polytope to the recycling endosome, sorting endosome, or lysosome, or may bind to MHC-II (typically with an affinity of less than 200 nM) while the trafficking element directs the polytope to the recycling endosome, sorting endosome, or lysosome.

[0015] In still further contemplated aspects, the recombinant nucleic acid may further include a sequence that encodes a second polytope, wherein the second polytope comprises a second trafficking element that directs the second polytope to a different sub-cellular location and wherein the second polytope comprises a second plurality of filtered neoepitope sequences (e.g., at least some of the plurality of filtered neoepitope sequences and some of the second plurality of filtered neoepitope sequences are identical).

[0016] Where desired, the recombinant nucleic acid may also comprises a sequence that encodes one or more co-stimulatory molecules (e.g., CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, OX40L, 4-1BBL, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1, LFA3), one or more immune stimulatory cytokines (e.g., IL-2, IL-12, IL-15, IL-15 super agonist (ALT803), IL-21, IPS1, and LMP1), and/or one or more proteins that interferes with or down-regulates checkpoint inhibition (e.g., antibody or an antagonist of CTLA-4, PD-1, TIM1 receptor, 2B4, or CD160).

[0017] Viewed from a different perspective, the inventors also contemplate a recombinant expression vector for immune therapy that comprises a sequence that encodes a polytope (e.g., comprising a plurality of filtered neoepitope sequences) operably linked to a promoter to drive expression of the polytope. Most typically, the polytope includes a trafficking element that directs the polytope to a sub-cellular location (e.g., cytoplasm, recycling endosome, sorting endosome, lysosome, extracellular membrane), or the trafficking element directs the polytope to an extracellular space for secretion of the polytope.

[0018] Preferred recombinant expression vectors are adenoviral expression vectors, especially having the E1 and E2b genes deleted. Such and other expression vectors may have a constitutive promoter, or an inducible promoter (preferably induced by hypoxia, IFN-gamma, or IL-8). As noted above it is contemplated that the trafficking element is a cleavable ubiquitin, a non-cleavable ubiquitin, a CD1b leader sequence, a CD1a tail, a CD1c tail, and/or a LAMP1-transmembrane sequence.

[0019] With respect to the filtered neoepitope sequences it is contemplated that the sequences are filtered by comparing tumor versus matched normal of the same patient, filtered to have binding affinity to an MHC complex of equal or less than 200 nM, and/or filtered against known human SNP and somatic variations. Furthermore, the filtered neoepitope sequences have preferably an arrangement within the polytope such that the polytope has a likelihood of a presence and/or strength of hydrophobic sequences or signal peptides that is below a predetermined threshold to so avoid mis-directing the polytope.

[0020] For example, the filtered neoepitope sequences may bind to MHC-I (typically with an affinity of less than 200 nM) while the trafficking element directs the polytope to the cytoplasm or proteasome, or the filtered neoepitope sequences may bind to MHC-I (typically with an affinity of less than 200 nM) while the trafficking element directs the polytope to the recycling endosome, sorting endosome, or lysosome, or the filtered neoepitope sequences may bind to MHC-II (typically with an affinity of less than 200 nM) and wherein the trafficking element directs the polytope to the recycling endosome, sorting endosome, or lysosome.

[0021] Additionally, it is contemplated that the recombinant nucleic acid may further include a sequence that encodes a second polytope, wherein the second polytope comprises a second trafficking element that directs the second polytope to a different sub-cellular location and wherein the second polytope comprises a second plurality of filtered neoepitope sequences. For example, at least some of the plurality of filtered neoepitope sequences and some of the second plurality of filtered neoepitope sequences are identical. Where desired, the recombinant nucleic acid may further comprise a sequence that encodes one or more co-stimulatory molecules (e.g., CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, OX40L, 4-1BBL, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1, LFA3), one or more immune stimulatory cytokines (e.g., IL-2, IL-12, IL-15, IL-15 super agonist (ALT803), IL-21, IPS1, and LMP1), and/or one or more proteins that interferes with or down-regulates checkpoint inhibition (e.g., antibody or an antagonist of CTLA-4, PD-1, TIM1 receptor, 2B4, or CD160).

[0022] Therefore, the inventors also contemplate a recombinant virus that includes the recombinant vector as described herein. Most typically, the virus is a replication deficient virus (e.g., adenovirus, preferably with deleted E1 and E2b gene). Such virus may then be included in a pharmaceutical composition that is typically formulated for injection or intranasal administration. While not limiting to the inventive subject matter, such recombinant virus may then be used in the treatment of cancer, and/or in the manufacture of a medicament for treatment of cancer.

[0023] Consequently, the inventors also contemplate a method of treating an individual that typically includes a step of transfecting a cell of the individual with a recombinant virus, wherein the recombinant virus comprises a sequence that encodes a polytope operably linked to a promoter to drive expression of the polytope in the cell. The polytope comprises a trafficking element that directs the polytope to a sub-cellular location selected from the group consisting of cytoplasm, recycling endosome, sorting endosome, lysosome, and extracellular membrane, or the trafficking element directs the polytope to an extracellular space. Most typically, the polytope in such methods comprises a plurality of filtered neoepitope sequences.

[0024] With respect to the recombinant virus, the promoter, the trafficking element, and the filtered neoepitope sequences, the same considerations as noted above apply. As also noted above it is contemplated that the recombinant nucleic acid further may comprise a sequence that encodes a second polytope, wherein the second polytope comprises a second trafficking element that directs the second polytope to a different sub-cellular location and that the second polytope comprises a second plurality of filtered neoepitope sequences.

[0025] In further contemplated methods, a step of transfecting another cell of the individual with a second recombinant virus may be included, wherein the second recombinant virus comprises a second sequence that encodes a second polytope operably linked to a promoter to drive expression of the second polytope in the another cell, wherein the second polytope comprises a second trafficking element that directs the polytope to a second sub-cellular location selected from the group consisting of cytoplasm, recycling endosome, sorting endosome, lysosome, and extracellular membrane, or wherein the trafficking element directs the polytope to an extracellular space, and wherein the sub-cellular location and the second sub-cellular location are distinct, and wherein the second polytope comprises a second plurality of filtered neoepitope sequences. For example, at least some of the plurality of filtered neoepitope sequences and some of the second plurality of filtered neoepitope sequences may be identical.

[0026] Contemplated recombinant viruses in such methods may further comprises a sequence that encodes one or more co-stimulatory molecules (e.g., CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, OX40L, 4-1BBL, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1, LFA3), one or more immune stimulatory cytokines (e.g., IL-2, IL-12, IL-15, IL-15 super agonist (ALT803), IL-21, IPS1, and LMP1), and/or one or more proteins that interferes with or down-regulates checkpoint inhibition (e.g., antibody or an antagonist of CTLA-4, PD-1, TIM1 receptor, 2B4, or CD160).

[0027] Viewed from yet another perspective, the inventors also contemplate a method of biasing an immune response against a neoepitope in an individual towards a CD4+ biased immune response. Such method will typically include a step of transfecting a cell of the individual with a recombinant virus, wherein the recombinant virus comprises a sequence that encodes a polytope operably linked to a promoter to drive expression of the polytope in the cell, wherein the polytope comprises the neoepitope. Most typically, the polytope comprises a trafficking element that directs the polytope to a sub-cellular location selected from the group consisting of a recycling endosome, a sorting endosome, and a lysosome. In a further step, the polytope is expressed from the sequence, trafficked, and proteolytically processed to present the neoepitope on a MHC-II complex and to thereby stimulate CD4+ T-cells.

[0028] Suitable neoepitope may be calculated to bind to MHC-I (typically with an affinity of less than 200 nM) or to bind to MHC-II (typically with an affinity of less than 200 nM), and/or suitable trafficking elements include a CD1b leader sequence, a CD1a tail, a CD1c tail, and a LAMP1-transmembrane sequence. As noted above, the recombinant virus may further comprise a sequence that encodes at least one of a co-stimulatory molecule, an immune stimulatory cytokine, and a protein that interferes with or down-regulates checkpoint inhibition.

[0029] Likewise, the inventors also contemplate a method of biasing an immune response against a neoepitope in an individual towards a CD8+ biased immune response. Such method will typically include a step of transfecting a cell of the individual with a recombinant virus, wherein the recombinant virus comprises a sequence that encodes a polytope operably linked to a promoter to drive expression of the polytope in the cell, wherein the polytope comprises the neoepitope. Most typically, the polytope comprises a trafficking element that directs the polytope to the proteasome. In a further step, the polytope is expressed from the sequence, trafficked, and proteolytically processed to present the neoepitope on a MHC-I complex and to thereby stimulate CD8+ T-cells.

[0030] Suitable neoepitope may be calculated to bind to MHC-I (typically with an affinity of less than 200 nM) or to bind to MHC-II (typically with an affinity of less than 200 nM), and/or suitable trafficking elements include a CD1b leader sequence, a CD1a tail, a CD1c tail, and a LAMP1-transmembrane sequence. As noted above, the recombinant virus may further comprise a sequence that encodes at least one of a co-stimulatory molecule, an immune stimulatory cytokine, and a protein that interferes with or down-regulates checkpoint inhibition.

[0031] In yet another aspect of the inventive subject matter, the inventors contemplate a method of generating a polytope for immune therapy. Such method preferably comprises a step of obtaining a plurality of neoepitope sequences, and generating a set of polytope sequences, each having a distinct linear arrangement of the plurality of neoepitope sequences. In another step, for each of the polytope sequences a score is calculated that is representative for a likelihood of presence and/or strength of hydrophobic sequences or signal peptides, and in yet another step, the score is used to rank the polytope sequences. The linear arrangement of the plurality of neoepitope sequences is then selected on the basis of the rank.

[0032] Most preferably, the score is calculated using at least one of a weight matrix and a neural network prediction, and/or the neoepitope sequences are filtered by at least one of MHC binding strength, known human SNP and somatic variations, and comparing tumor versus matched normal of the same patient. Typically, each of the polytope sequences comprises at least five neoepitope sequences.

[0033] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWING

[0034] FIG. 1 is a schematic representation of various neoepitope arrangements.

[0035] FIG. 2 is an exemplary and partial schematic for selecting preferred arrangements of neoepitopes.

[0036] Prior Art FIG. 3 is a schematic illustration of antigen processing in the cytoplasm and MHC-I presentation.

[0037] Prior Art FIG. 4 is a schematic illustration of antigen processing in the lysosomal and endosomal compartment and MHC-II presentation.

[0038] FIGS. 5A-5C are exemplary sequence arrangements for class I antigen processing in the cytoplasm and MHC-I presentation.

[0039] FIGS. 6A-6C are exemplary sequence arrangements for class I antigen processing in the cytoplasm and MHC-II presentation.

[0040] FIGS. 7A-7C are exemplary sequence arrangements for class II antigen processing in the cytoplasm and MHC-II presentation.

[0041] FIG. 8 is an exemplary prophylactic vaccination schedule for B16-F10 melanoma.

[0042] FIGS. 9A-9C are graphs depicting exemplary results for the anti-tumor vaccination using subcutaneous injection of the vaccine.

[0043] FIGS. 10A-10C are graphs depicting exemplary results for the anti-tumor vaccination using intravenous injection of the vaccine.

DETAILED DESCRIPTION

[0044] The inventors have discovered that neoepitope-based immune therapy can be further improved by targeting expressed patient- and tumor specific neoepitopes towards processing and/or specific cell surface presentation or even secretion, and that neoepitope-based therapy can still further be augmented using checkpoint inhibition, immune stimulation via cytokines, and/or inhibitors of myeloid derived suppressor cells (MDCS), T-regulatory cells (Tregs), or M2 macrophages. Most preferably, such therapeutic entities will be expressed in vivo from a recombinant nucleic acid, and especially suitable recombinant nucleic acid include plasmids and viral nucleic acids. Where a viral nucleic acid is employed, it is particularly preferred that the nucleic acid is delivered via infection of the patient or patient cells by the virus.

[0045] Viewed from a different perspective, it should be appreciated that the compositions and methods presented herein will include one or more neoepitopes that are specific to the patient and the tumor in the patient to allow for targeted treatment. Moreover, such treatment may advantageously be tailored to achieve one or more specific immune reactions, including a CD4.sup.+ biased immune response, a CD8.sup.+ biased immune response, antibody biased immune response, and/or a stimulated immune response (e.g., reducing checkpoint inhibition and/or by activation of immune competent cells using cytokines). Most typically, such effects are in achieved in the context of the neoepitopes originating from the recombinant nucleic acid.

[0046] Neoepitopes can be characterized as expressed random mutations in tumor cells that created unique and tumor specific antigens. Therefore, viewed from a different perspective, neoepitopes may be identified by considering the type (e.g., deletion, insertion, transversion, transition, translocation) and impact of the mutation (e.g., non-sense, missense, frame shift, etc.), which may as such serve as a content filter through which silent and other non-relevant (e.g., non-expressed) mutations are eliminated. It should also be appreciated that neoepitope sequences can be defined as sequence stretches with relatively short length (e.g., 8-12 mers or 14-20mers) wherein such stretches will include the change(s) in the amino acid sequences. Most typically, but not necessarily, the changed amino acid will be at or near the central amino acid position. For example, a typical neoepitope may have the structure of A.sub.4-N-A.sub.4, or A.sub.3-N-A.sub.5, or A.sub.2-N-A.sub.7, or A.sub.5-N-A.sub.3, or A.sub.7-N-A.sub.2, where A is a proteinogenic wild type or normal (i.e., from corresponding healthy tissue of the same patient) amino acid and N is a changed amino acid (relative to wild type or relative to matched normal). Therefore, the neoepitope sequences contemplated herein include sequence stretches with relatively short length (e.g., 5-30 mers, more typically 8-12 mers, or 14-20 mers) wherein such stretches include the change(s) in the amino acid sequences. Where desired, additional amino acids may be placed upstream or downstream of the changed amino acid, for example, to allow for additional antigen processing in the various compartments (e.g., for proteasome processing in the cytosol, or specific protease processing in the endosomal and/or lysosomal compartments) of a cell.

[0047] Thus, it should be appreciated that a single amino acid change may be presented in numerous neoepitope sequences that include the changed amino acid, depending on the position of the changed amino acid. Advantageously, such sequence variability allows for multiple choices of neoepitopes and as such increases the number of potentially useful targets that can then be selected on the basis of one or more desirable traits (e.g., highest affinity to a patient HLA-type, highest structural stability, etc.). Most typically, neoepitopes will be calculated to have a length of between 2-50 amino acids, more typically between 5-30 amino acids, and most typically between 8-12 amino acids, or 14-20 amino acids, with the changed amino acid preferably centrally located or otherwise situated in a manner that improves its binding to MHC. For example, where the epitope is to be presented by the MHC-I complex, a typical neoepitope length will be about 8-12 amino acids, while the typical neoepitope length for presentation via MHC-II complex will have a length of about 14-20 amino acids. As will be readily appreciated, since the position of the changed amino acid in the neoepitope may be other than central, the actual peptide sequence and with that actual topology of the neoepitope may vary considerably, and the neoepitope sequence with a desired binding affinity to the MHC-I or MHC-II presentation and/or desired protease processing will typically dictate the particular sequence.

[0048] Of course, it should be appreciated that the identification or discovery of neoepitopes may start with a variety of biological materials, including fresh biopsies, frozen, or otherwise preserved tissue or cell samples, circulating tumor cells, exosomes, various body fluids (and especially blood), etc. Therefore, suitable methods of omics analysis include nucleic acid sequencing, and particularly NGS methods operating on DNA (e.g., Illumina sequencing, ion torrent sequencing, 454 pyrosequencing, nanopore sequencing, etc.), RNA sequencing (e.g., RNAseq, reverse transcription based sequencing, etc.), and in some cases protein sequencing or mass spectroscopy based sequencing (e.g., SRM, MRM, CRM, etc.).

[0049] As such, and particularly for nucleic acid based sequencing, it should be particularly recognized that high-throughput genome sequencing of a tumor tissue will allow for rapid identification of neoepitopes. However, it must be appreciated that where the so obtained sequence information is compared against a standard reference, the normally occurring inter-patient variation (e.g., due to SNPs, short indels, different number of repeats, etc.) as well as heterozygosity will result in a relatively large number of potential false positive neoepitopes. Notably, such inaccuracies can be eliminated where a tumor sample of a patient is compared against a matched normal (i.e., non-tumor) sample of the same patient.

[0050] In one especially preferred aspect of the inventive subject matter, DNA analysis is performed by whole genome sequencing and/or exome sequencing (typically at a coverage depth of at least 10.times., more typically at least 20.times.) of both tumor and matched normal sample. Alternatively, DNA data may also be provided from an already established sequence record (e.g., SAM, BAM, FASTA, FASTQ, or VCF file) from a prior sequence determination of the same patient. Therefore, data sets suitable for use herein include unprocessed or processed data sets, and exemplary preferred data sets include those having BAM format, SAM format, GAR format, FASTQ format, or FASTA format, as well as BAMBAM, SAMBAM, and VCF data sets. However, it is especially preferred that the data sets are provided in BAM format or as BAMBAM diff objects as is described in US2012/0059670A1 and US2012/0066001A1. Moreover, it should be noted that the data sets are reflective of a tumor and a matched normal sample of the same patient. Thus, genetic germ line alterations not giving rise to the tumor (e.g., silent mutation, SNP, etc.) can be excluded. Of course, it should be recognized that the tumor sample may be from an initial tumor, from the tumor upon start of treatment, from a recurrent tumor and/or metastatic site, etc. In most cases, the matched normal sample of the patient is blood, or a non-diseased tissue from the same tissue type as the tumor.

[0051] Likewise, the computational analysis of the sequence data may be performed in numerous manners. In most preferred methods, however, analysis is performed in silico by location-guided synchronous alignment of tumor and normal samples as, for example, disclosed in US 2012/0059670 and US 2012/0066001 using BAM files and BAM servers. Such analysis advantageously reduces false positive neoepitopes and significantly reduces demands on memory and computational resources.

[0052] It should be noted that any language directed to a computer should be read to include any suitable combination of computing devices, including servers, interfaces, systems, databases, agents, peers, engines, controllers, or other types of computing devices operating individually or collectively. One should appreciate the computing devices comprise a processor configured to execute software instructions stored on a tangible, non-transitory computer readable storage medium (e.g., hard drive, solid state drive, RAM, flash, ROM, etc.). The software instructions preferably configure the computing device to provide the roles, responsibilities, or other functionality as discussed below with respect to the disclosed apparatus. Further, the disclosed technologies can be embodied as a computer program product that includes a non-transitory computer readable medium storing the software instructions that causes a processor to execute the disclosed steps associated with implementations of computer-based algorithms, processes, methods, or other instructions. In especially preferred embodiments, the various servers, systems, databases, or interfaces exchange data using standardized protocols or algorithms, possibly based on HTTP, HTTPS, AES, public-private key exchanges, web service APIs, known financial transaction protocols, or other electronic information exchanging methods. Data exchanges among devices can be conducted over a packet-switched network, the Internet, LAN, WAN, VPN, or other type of packet switched network; a circuit switched network; cell switched network; or other type of network.

[0053] Viewed from a different perspective, a patient- and cancer-specific in silico collection of sequences can be established that encode neoepitopes having a predetermined length of, for example, between 5 and 25 amino acids and include at least one changed amino acid. Such collection will typically include for each changed amino acid at least two, at least three, at least four, at least five, or at least six members in which the position of the changed amino acid is not identical. Such collection advantageously increases potential candidate molecules suitable for immune therapy and can then be used for further filtering (e.g., by sub-cellular location, transcription/expression level, MHC-I and/or II affinity, etc.) as is described in more detail below.

[0054] For example, and using synchronous location guided analysis to tumor and matched normal sequence data, the inventors previously identified various cancer neoepitopes from a variety of cancers and patients, including the following cancer types: BLCA, BRCA, CESC, COAD, DLBC, GBM, HNSC, KICH, KIRC, KIRP, LAML, LGG, LIHC, LUAD, LUSC, OV, PRAD, READ, SARC, SKCM, STAD, THCA, and UCEC. Exemplary neoepitope data for these cancers can be found in International application PCT/US16/29244, incorporated by reference herein.

[0055] Depending on the type and stage of the cancer, as well as the patient's immune status it should be recognized that not all of the identified neoepitopes will necessarily lead to a therapeutically equally effective reaction in a patient. Indeed, it is well known in the art that only a fraction of neoepitopes will generate an immune response. To increase likelihood of a therapeutically desirable response, the initially identified neoepitopes can be further filtered. Of course, it should be appreciated that downstream analysis need not take into account silent mutations for the purpose of the methods presented herein. However, preferred mutation analyses will provide in addition to the particular type of mutation (e.g., deletion, insertion, transversion, transition, translocation) also information of the impact of the mutation (e.g., non-sense, missense, etc.) and may as such serve as a first content filter through which silent mutations are eliminated. For example, neoepitopes can be selected for further consideration where the mutation is a frame-shift, non-sense, and/or missense mutation.

[0056] In a further filtering approach, neoepitopes may also be subject to detailed analysis for sub-cellular location parameters. For example, neoepitope sequences may be selected for further consideration if the neoepitopes are identified as having a membrane associated location (e.g., are located at the outside of a cell membrane of a cell) and/or if an in silico structural calculation confirms that the neoepitope is likely to be solvent exposed, or presents a structurally stable epitope (e.g., J Exp Med 2014), etc.

[0057] With respect to filtering neoepitopes, it is generally contemplated that neoepitopes are especially suitable for use herein where omics (or other) analysis reveals that the neoepitope is actually expressed. Identification of expression and expression level of a neoepitope can be performed in all manners known in the art and preferred methods include quantitative RNA (hnRNA or mRNA) analysis and/or quantitative proteomics analysis. Most typically, the threshold level for inclusion of neoepitopes will be an expression level of at least 20%, at least 30%, at least 40%, or at least 50% of expression level of the corresponding matched normal sequence, thus ensuring that the (neo)epitope is at least potentially `visible` to the immune system. Consequently, it is generally preferred that the omics analysis also includes an analysis of gene expression (transcriptomic analysis) to so help identify the level of expression for the gene with a mutation.

[0058] There are numerous methods of transcriptomic analysis known in the art, and all of the known methods are deemed suitable for use herein. For example, preferred materials include mRNA and primary transcripts (hnRNA), and RNA sequence information may be obtained from reverse transcribed polyAtRNA, which is in turn obtained from a tumor sample and a matched normal (healthy) sample of the same patient. Likewise, it should be noted that while poly A.sup.+-RNA is typically preferred as a representation of the transcriptome, other forms of RNA (hn-RNA, non-polyadenylated RNA, siRNA, miRNA, etc.) are also deemed suitable for use herein. Preferred methods include quantitative RNA (hnRNA or mRNA) analysis and/or quantitative proteomics analysis, especially including RNAseq. In other aspects, RNA quantification and sequencing is performed using RNAseq, qPCR and/or rtPCR based methods, although various alternative methods (e.g., solid phase hybridization-based methods) are also deemed suitable. Viewed from another perspective, transcriptomic analysis may be suitable (alone or in combination with genomic analysis) to identify and quantify genes having a cancer- and patient-specific mutation.

[0059] Similarly, proteomics analysis can be performed in numerous manners to ascertain actual translation of the RNA of the neoepitope, and all known manners of proteomics analysis are contemplated herein. However, particularly preferred proteomics methods include antibody-based methods and mass spectroscopic methods. Moreover, it should be noted that the proteomics analysis may not only provide qualitative or quantitative information about the protein per se, but may also include protein activity data where the protein has catalytic or other functional activity. One exemplary technique for conducting proteomic assays is described in U.S. Pat. No. 7,473,532, incorporated by reference herein. Further suitable methods of identification and even quantification of protein expression include various mass spectroscopic analyses (e.g., selective reaction monitoring (SRM), multiple reaction monitoring (MRM), and consecutive reaction monitoring (CRM)). Consequently, it should be appreciated that the above methods will provide patient and tumor specific neoepitopes, which may be further filtered by sub-cellular location of the protein containing the neoepitope (e.g., membrane location), the expression strength (e.g., overexpressed as compared to matched normal of the same patient), etc.

[0060] In yet another aspect of filtering, the neoepitopes may be compared against a database that contains known human sequences (e.g., of the patient or a collection of patients) to so avoid use of a human-identical sequence. Moreover, filtering may also include removal of neoepitope sequences that are due to SNPs in the patient where the SNPs are present in both the tumor and the matched normal sequence. For example, dbSNP (The Single Nucleotide Polymorphism Database) is a free public archive for genetic variation within and across different species developed and hosted by the National Center for Biotechnology Information (NCBI) in collaboration with the National Human Genome Research Institute (NHGRI). Although the name of the database implies a collection of one class of polymorphisms only (single nucleotide polymorphisms (SNPs)), it in fact contains a relatively wide range of molecular variation: (1) SNPs, (2) short deletion and insertion polymorphisms (indels/DIPs), (3) microsatellite markers or short tandem repeats (STRs), (4) multinucleotide polymorphisms (MNPs), (5) heterozygous sequences, and (6) named variants. The dbSNP accepts apparently neutral polymorphisms, polymorphisms corresponding to known phenotypes, and regions of no variation. Using such database and other filtering options as described above, the patient and tumor specific neoepitopes may be filtered to remove those known sequences, yielding a sequence set with a plurality of neoepitope sequences having substantially reduced false positives.

[0061] Once the desired level of filtering for the neoepitope is accomplished (e.g., neoepitope filtered by tumor versus normal, and/or expression level, and/or sub-cellular location, and/or patient specific HLA-match, and/or known variants), a further filtering step is contemplated that takes into account the gene type that is affected by the neoepitope. For example, suitable gene types include cancer driver genes, genes associated with regulation of cell division, genes associated with apoptosis, and genes associated with signal transduction. However, in especially preferred aspects, cancer driver genes are particularly preferred (which may span by function a variety of gene types, including receptor genes, signal transduction genes, transcription regulator genes, etc.). In further contemplated aspects, suitable gene types may also be known passenger genes and genes involved in metabolism.

[0062] With respect to the identification or other determination (e.g., prediction) of a gene as being a cancer driver gene, various methods and prediction algorithms are known in the art, and are deemed suitable for use herein. For example, suitable algorithms include MutsigCV (Nature 2014, 505(7484):495-501), ActiveDriver (Mol Syst Biol 2013, 9:637), MuSiC (Genome Res 2012, 22(8):1589-1598), OncodriveClust (Bioinformatics 2013, 29(18):2238-2244), OncodriveFM (Nucleic Acids Res 2012, 40(21):e169), OncodriveFML (Genome Biol 2016, 17(1):128), Tumor Suppressor and Oncogenes (TUSON) (Cell 2013, 155(4):948-962), 20/20+(https://github.com/KarchinLab/2020plus), and oncodriveROLE (Bioinformatics (2014) 30 (17): i549-i555). Alternatively, or additionally, identification of cancer driver genes may also employ various sources for known cancer driver genes and their association with specific cancers. For example, the Intogen Catalog of driver mutations (2016.5; URL: www.intogen.org) contains the results of the driver analysis performed by the Cancer Genome Interpreter across 6,792 exomes of a pan-cancer cohort of 28 tumor types.

[0063] Nevertheless, despite filtering, it should be recognized that not all neoepitopes will be visible to the immune system as the neoepitopes also need to be processed where present in a larger context (e.g., within a polytope) and presented on the MHC complex of the patient. In that context, it must be appreciated that only a fraction of all neoepitopes will have sufficient affinity for presentation. Consequently, and especially in the context of immune therapy it should be apparent that neoepitopes will be more likely effective where the neoepitopes are properly processed, bound to, and presented by the MHC complexes. Viewed from another perspective, treatment success will be increased with an increasing number of neoepitopes that can be presented via the MHC complex, wherein such neoepitopes have a minimum affinity to the patient's HLA-type. Consequently, it should be appreciated that effective binding and presentation is a combined function of the sequence of the neoepitope and the particular HLA-type of a patient. Therefore, HLA-type determination of the patient tissue is typically required. Most typically, the HLA-type determination includes at least three MHC-I sub-types (e.g., HLA-A, HLA-B, HLA-C) and at least three MHC-II sub-types (e.g., HLA-DP, HLA-DQ, HLA-DR), preferably with each subtype being determined to at least 2-digit or at least 4-digit depth. However, greater depth (e.g., 6 digit, 8 digit) is also contemplated.

[0064] Once the HLA-type of the patient is ascertained (using known chemistry or in silico determination), a structural solution for the HLA-type is calculated and/or obtained from a database, which is then used in a docking model in silico to determine binding affinity of the (typically filtered) neoepitope to the HLA structural solution. As will be further discussed below, suitable systems for determination of binding affinities include the NetMHC platform (see e.g., Nucleic Acids Res. 2008 Jul. 1; 36(Web Server issue): W509-W512.). Neoepitopes with high affinity (e.g., less than 100 nM, less than 75 nM, less than 50 nM) for a previously determined HLA-type are then selected for therapy creation, along with the knowledge of the patient's MHC-I/II subtype.

[0065] HLA determination can be performed using various methods in wet-chemistry that are well known in the art, and all of these methods are deemed suitable for use herein. However, in especially preferred methods, the HLA-type can also be predicted from omics data in silico using a reference sequence containing most or all of the known and/or common HLA-types. For example, in one preferred method according to the inventive subject matter, a relatively large number of patient sequence reads mapping to chromosome 6p21.3 (or any other location near/at which HLA alleles are found) is provided by a database or sequencing machine. Most typically the sequence reads will have a length of about 100-300 bases and comprise metadata, including read quality, alignment information, orientation, location, etc. For example, suitable formats include SAM, BAM, FASTA, GAR, etc. While not limiting to the inventive subject matter, it is generally preferred that the patient sequence reads provide a depth of coverage of at least 5.times., more typically at least 10.times., even more typically at least 20.times., and most typically at least 30.times..

[0066] In addition to the patient sequence reads, contemplated methods further employ one or more reference sequences that include a plurality of sequences of known and distinct HLA alleles. For example, a typical reference sequence may be a synthetic (without corresponding human or other mammalian counterpart) sequence that includes sequence segments of at least one HLA-type with multiple HLA-alleles of that HLA-type. For example, suitable reference sequences include a collection of known genomic sequences for at least 50 different alleles of HLA-A. Alternatively, or additionally, the reference sequence may also include a collection of known RNA sequences for at least 50 different alleles of HLA-A. Of course, and as further discussed in more detail below, the reference sequence is not limited to 50 alleles of HLA-A, but may have alternative composition with respect to HLA-type and number/composition of alleles. Most typically, the reference sequence will be in a computer readable format and will be provided from a database or other data storage device. For example, suitable reference sequence formats include FASTA, FASTQ, EMBL, GCG, or GenBank format, and may be directly obtained or built from data of a public data repository (e.g., IMGT, the International ImMunoGeneTics information system, or The Allele Frequency Net Database, EUROSTAM, URL: www.allelefrequencies.net). Alternatively, the reference sequence may also be built from individual known HLA-alleles based on one or more predetermined criteria such as allele frequency, ethnic allele distribution, common or rare allele types, etc.

[0067] Using the reference sequence, the patient sequence reads can now be threaded through a de Bruijn graph to identify the alleles with the best fit. In this context, it should be noted that each individual carries two alleles for each HLA-type, and that these alleles may be very similar, or in some cases even identical. Such high degree of similarity poses a significant problem for traditional alignment schemes. The inventor has now discovered that the HLA alleles, and even very closely related alleles can be resolved using an approach in which the de Bruijn graph is constructed by decomposing a sequence read into relatively small k-mers (typically having a length of between 10-20 bases), and by implementing a weighted vote process in which each patient sequence read provides a vote ("quantitative read support") for each of the alleles on the basis of k-mers of that sequence read that match the sequence of the allele. The cumulatively highest vote for an allele then indicates the most likely predicted HLA allele. In addition, it is generally preferred that each fragment that is a match to the allele is also used to calculate the overall coverage and depth of coverage for that allele.

[0068] Scoring may further be improved or refined as needed, especially where many of the top hits are similar (e.g., where a significant portion of their score comes from a highly shared set of k-mers). For example, score refinement may include a weighting scheme in which alleles that are substantially similar (e.g., >99%, or other predetermined value) to the current top hit are removed from future consideration. Counts for k-mers used by the current top hit are then re-weighted by a factor (e.g., 0.5), and the scores for each HLA allele are recalculated by summing these weighted counts. This selection process is repeated to find a new top hit. The accuracy of the method can be even further improved using RNA sequence data that allows identification of the alleles expressed by a tumor, which may sometimes be just 1 of the 2 alleles present in the DNA. In further advantageous aspects of contemplated systems and methods, DNA or RNA, or a combination of both DNA and RNA can be processed to make HLA predictions that are highly accurate and can be derived from tumor or blood DNA or RNA. Further aspects, suitable methods and considerations for high-accuracy in silico HLA typing are described in WO 2017/035392, incorporated by reference herein.

[0069] Once patient and tumor specific neoepitopes and HLA-type are identified, further computational analysis can be performed by in silico docking neoepitopes to the HLA and determining best binders (e.g., lowest K.sub.D, for example, less than 500 nM, or less than 250 nM, or less than 150 nM, or less than 50 nM), for example, using NetMHC. It should be appreciated that such approach will not only identify specific neoepitopes that are genuine to the patient and tumor, but also those neoepitopes that are most likely to be presented on a cell and as such most likely to elicit an immune response with therapeutic effect. Of course, it should also be appreciated that thusly identified HLA-matched neoepitopes can be biochemically validated in vitro prior to inclusion of the nucleic acid encoding the epitope as payload into the virus as is further discussed below.

[0070] Of course, it should be appreciated that matching of the patient's HLA-type to the patient- and cancer-specific neoepitope can be done using systems other than NetMHC, and suitable systems include NetMHC II, NetMHCpan, IEDB Analysis Resource (URL immuneepitope.org), RankPep, PREDEP, SVMHC, Epipredict, HLABinding, and others (see e.g., J Immunol Methods 2011; 374:1-4). In calculating the highest affinity, it should be noted that the collection of neoepitope sequences in which the position of the altered amino acid is moved (supra) can be used. Alternatively, or additionally, modifications to the neoepitopes may be implemented by adding N- and/or C-terminal modifications to further increase binding of the expressed neoepitope to the patient's HLA-type. Thus, neoepitopes may be native as identified or further modified to better match a particular HLA-type. Moreover, where desired, binding of corresponding wild type sequences (i.e., neoepitope sequence without amino acid change) can be calculated to ensure high differential affinities. For example, especially preferred high differential affinities in MHC binding between the neoepitope and its corresponding wild type sequence are at least 2-fold, at least 5-fold, at least 10-fold, at least 100-fold, at least 500-fold, at least 1000-fold, etc.).

[0071] Binding affinity, and particularly differential binding affinity may also be determined in vitro using various systems and methods. For example, antigen presenting cells of a patient or cells with matched HLA-type can be transfected with a nucleic acid (e.g., viral, plasmid, linear DNA, RNA, etc.) to express one or more neoepitopes using constructs as described in more detail below. Upon expression and antigen processing, the neoepitopes can then be identified in the MHC complex on the outside of the cell, either using specific binders to the neoepitope or using a cell based system (e.g., PBMC of the patient) in which T cell activation or cytotoxic NK cell activity can be observed in vitro. Neoepitopes with differential activity (elicit a stronger signal or immune response as compared to the corresponding wild type epitope) will then be selected for therapy creation.

[0072] Upon identification of desired neoepitopes, one or more immune therapeutic agents may be prepared using the sequence information of the neoepitope. Among other agents, it is especially preferred that the patient may be treated with a virus that is genetically modified with a nucleic acid construct as further discussed below that leads to expression of at least one of the identified neoepitopes to initiate an immune response against the tumor. For example, suitable viruses include adenoviruses, adeno-associated viruses, alphaviruses, herpes viruses, lentiviruses, etc. However, adenoviruses are particularly preferred. Moreover, it is further preferred that the virus is a replication deficient and non-immunogenic virus, which is typically accomplished by targeted deletion of selected viral proteins (e.g., E1, E3 proteins). Such desirable properties may be further enhanced by deleting E2b gene function, and high titers of recombinant viruses can be achieved using genetically modified human 293 cells as has been recently reported (e.g., J Virol. 1998 February; 72(2): 926-933).

[0073] Regardless of the type of recombinant virus it is contemplated that the virus may be used to infect patient (or non-patient) cells ex vivo or in vivo. For example, the virus may be injected subcutaneously or intravenously, or may be administered intranasaly or via inhalation to so infect the patients cells, and especially antigen presenting cells. Alternatively, immune competent cells (e.g., NK cells, T cells, macrophages, dendritic cells, etc.) of the patient (or from an allogeneic source) may be infected in vitro and then transfused to the patient. Alternatively, immune therapy need not rely on a virus but may be effected with nucleic acid transfection or vaccination using RNA or DNA, or other recombinant vector that leads to the expression of the neoepitopes (e.g., as single peptides, tandem mini-gene, etc.) in desired cells, and especially immune competent cells.

[0074] Most typically, the desired nucleic acid sequences (for expression from virus infected cells) are under the control of appropriate regulatory elements well known in the art. For example, suitable promoter elements include constitutive strong promoters (e.g., SV40, CMV, UBC, EF1A, PGK, CAGG promoter), but inducible promoters are also deemed suitable for use herein, particularly where induction conditions are typical for a tumor microenvironment. For example, inducible promoters include those sensitive to hypoxia and promoters that are sensitive to TGF-.beta. or IL-8 (e.g., via TRAF, JNK, Erk, or other responsive elements promoter). In other examples, suitable inducible promoters include the tetracycline-inducible promoter, the myxovirus resistance 1 (Mx1) promoter, etc.

[0075] In this context, it should be appreciated that the inventors have discovered that the manner of neoepitope arrangement and rational-designed trafficking of the neoepitopes can have a substantial impact on the efficacy of various immune therapeutic compositions. For example, single neoepitopes can be expressed individually from the respective recombinant constructs that are delivered as a single plasmid, viral expression construct, etc. Alternatively, multiple neoepitopes can be separately expressed from individual promoters t form individual mRNA that are then individually translated into the respective neoepitopes, or from a single mRNA comprising individual translation starting points for each neoepitope sequence (e.g., using 2A or IRES signals). Notably, while such arrangements are generally thought to allow for controlled delivery of proper neoepitope peptide, efficacy of such expression systems has been less than desirable (data not shown).

[0076] In contrast, where multiple neoepitopes were expressed from a single transcript to so form a single transcript that is then translated into a single polytope (i.e., polypeptide with a series of concatemerically linked neoepitopes, optionally with intervening linker sequences) expression, processing, and antigen presentation was found to be effective. Notably, the expression of polytopes requires processing by the appropriate proteases (e.g., proteasome, endosomal proteases, lysosomal proteases) within a cell to yield the neoepitope sequences, and polytopes led to improved antigen processing and presentation for most neoepitopes as compared to expression of individual neoepitopes, particularly where the individual neoepitopes had a relatively short length (e.g., less than 25 amino acids; results not shown). Moreover, such approach also allows rational design of protease sensitive sequence motifs between the neoepitope peptide sequences to so assure or avoid processing by specific proteases as the proteasome, endosomal proteases, and lysosomal proteases have distinct cleavage preferences. Therefore, polytopes may be designed that include not only linker sequences to spatially separate neoepitopes, but also sequence portions (e.g., between 3-15 amino acids) that will be preferentially cleaved by a specific protease.

[0077] Therefore, the inventors contemplate recombinant nucleic acids and expression vectors (e.g., viral expression vectors) that comprise a nucleic acid segment that encodes a polytope wherein the polytope is operably coupled to a desired promoter element, and wherein individual neoepitopes are optionally separated by a linker and/or protease cleavage or recognition sequence. For example, FIG. 1 exemplarily illustrates various contemplated arrangements for neoepitopes for expression from an adenoviral expression system (here: AdV5, with deletion of E1 and E2b genes). Here, Construct 1 exemplarily illustrates a neoepitope arrangement that comprises eight neoepitopes (`minigene`) with a total length of 15 amino acids in concatemeric series without intervening linker sequences, while Construct 2 shows the arrangement of Construct 1 but with inclusion of nine amino acid linkers between each neoepitope sequence. Of course, and as already noted above, it should be recognized that the exact length of the neoepitope sequence is not limited to 15 amino acids, and that the exact length may vary considerably. However, in most cases, where neoepitope sequences of between 8-12 amino acids are flanked by additional amino acids, the total length will typically not exceed 25 amino acids, or 30 amino acids, or 50 amino acids. Likewise, it should be noted that while FIG. 1 denotes G-S linkers, various other linker sequences are also suitable for use herein. Such relatively short neoepitopes are especially beneficial where presentation of the neoepitope is intended to be via the MHC-I complex.

[0078] In this context, it should be appreciated that suitable linker sequences will provide steric flexibility and separation of two adjacent neoepitopes. However, care must be taken to as to not chose amino acids for the linker that could be immunogenic/form an epitope that is already present in a patient. Consequently, it is generally preferred that the polytope construct is filtered once more for the presence of epitopes that could be found in a patient (e.g., as part of normal sequence or due to SNP or other sequence variation). Such filtering will apply the same technology and criteria as already discussed above.

[0079] Similarly, Construct 3 exemplarily illustrates a neoepitope arrangement that includes eight neoepitopes in concatemeric series without intervening linker sequences, and Construct 4 shows the arrangement of Construct 3 with inclusion of nine amino acid linkers between each neoepitope sequence. As noted above, it should be recognized that the exact length of such neoepitope sequences is not limited to 25 amino acids, and that the exact length may vary considerably. However, in most cases, where neoepitope sequences of between 14-20 amino acids are flanked by additional amino acids, the total length will typically not exceed 30 amino acids, or 45 amino acids, or 60 amino acids. Likewise, it should be noted that while FIG. 1 denotes G-S linkers for these constructs, various other linker sequences are also suitable for use herein. Such relatively long neoepitopes are especially beneficial where presentation of the neoepitope is intended to be via the MHC-II complex.

[0080] In this example, it should be appreciated that the 15-aa minigenes are MHC Class I targeted tumor mutations selected with 7 amino acids of native sequence on either side, and that the 25-aa minigenes are MHC Class II targeted tumor mutations selected with 12 amino acids of native sequence on either side. The exemplary 9 amino acid linkers are deemed to have sufficient length such that "unnatural" MHC Class I epitopes will not form between adjacent minigenes. Polytope sequences tended to be processed and presented more efficiently than single neoepitopes (data not shown), and addition of amino acids beyond 12 amino acids for MHC-I presentation and addition of amino acids beyond 20 amino acids for MHC-I presentation appeared to allow for somewhat improved protease processing.

[0081] To maximize the likelihood that customized protein sequences remain intracellular for processing and presentation by the HLA complex, neoepitope sequences may be arranged in a manner to minimize hydrophobic sequences that may direct trafficking to the cell membrane or into the extracellular space. Most preferably, hydrophobic sequence or signal peptide detection is done either by comparison of sequences to a weight matrix (see e.g., Nucleic Acids Res. 1986 Jun. 11; 14(11): 4683-4690) or by using neural networks trained on peptides that contain signal sequences (see e.g., Journal of Molecular Biology 2004, Volume 338, Issue 5, 1027-1036). FIG. 2 depicts an exemplary scheme of arrangement selection in which a plurality of polytope sequences are analyzed. Here, all positional permutations of all neoepitopes are calculated to produce a collection of arrangements. This collection is then processed through a weight matrix and/or neural network prediction to generate a score representing the likelihood of presence and/or strength of hydrophobic sequences or signal peptides. All positional permutations are then ranked by score, and the permutation(s) with a score below a predetermined threshold or lowest score for likelihood of presence and/or strength of hydrophobic sequences or signal peptides is/are used to construct a customized neoepitope expression cassette.

[0082] With respect to the total number of neoepitope sequences in a polytope it is generally preferred that the polytope comprise at least two, or at least three, or at least five, or at least eight, or at least ten neoepitope sequences. Indeed, the payload capacity of the recombinant DNA is generally contemplated the limiting factor, along with the availability of filtered and appropriate neoepitopes. Therefore, adenoviral expression vectors, and particularly Adv5 are especially preferred as such vectors can accommodate up to 14 kb in recombinant payload.

[0083] In still further contemplated aspects of the inventive subject matter, it should be noted that the neoepitopes/polytopes can be directed towards a specific sub-cellular compartment (e.g., cytosol, endosome, lysosome), and with that, towards a particular MHC presentation type. Such directed expression, processing, and presentation is particularly advantageous as contemplated compositions may be prepared that direct an immune response towards a CD8.sup.+ type response (where the polytope is directed to the cytoplasmic space) or towards a CD4.sup.+ type response (where the polytope is directed to the endosomal/lysosomal compartment). Moreover, it should be recognized that polytopes that would ordinarily be presented via the MHC-I pathway can be presented via the MHC-II pathway (and thereby mimick cross-presentation of neoepitopes). Therefore, it should be appreciated that neoepitope and polytope sequences may be designed and directed to one or both MHC presentation pathways using suitable sequence elements. With respect to routing the so expressed neoepitopes to the desired MHC-system, it is noted that the MHC-I presented peptides will typically arise from the cytoplasm via proteasome processing and delivery through the endoplasmatic reticulum. Thus, expression of the epitopes intended for MHC-I presentation will generally be directed to the cytoplasm as is further discussed in more detail below. On the other hand, MHC-II presented peptides will typically arise from the endosomal and lysosomal compartment via degradation and processing by acidic proteases (e.g., legumain, cathepsin L and cathepsin S) prior to delivery to the cell membrane.

[0084] Moreover, it is contemplated that proteolytic degradation of the polytope can also be enhanced using various methods, and especially contemplated methods include addition of a cleavable or non-cleavable ubiquitin moiety to the N-terminus, and/or placement of one or more destabilizing amino acids (e.g., N, K, C, F, E, R, Q) to the N-terminus of the polytope where the presentation is directed towards MHC-I. On the other hand, where presentation is directed towards MHC-II, cleavage sites for particular endosomal or lysosomal proteases can be engineered into the polytope to so facilitate of promote antigen processing.

[0085] Therefore, in contemplated aspects of the inventive subject matter, signal and/or leader peptides may be used for trafficking neoepitopes and/or polytopes to the endosomal and lysosomal compartment, or for retention in the cytoplasmic space. For example, where the polytope is to be exported to the endosomal and lysosomal compartment, a leader peptide such as the CD1b leader peptide may be employed to sequester the (nascent) protein from the cytoplasm. Additionally, or alternatively, targeting presequences and/or targeting peptides can be employed. The presequences of the targeting peptide may be added to the N-terminus and/or C-terminus and typically comprise between 6-136 basic and hydrophobic amino acids. In case of peroxisomal targeting, the targeting sequence may be at the C-terminus. Other signals (e.g., signal patches) may be used and include sequence elements that are separate in the peptide sequence and become functional upon proper peptide folding. In addition, protein modifications like glycosylations can induce targeting. Among other suitable targeting signals, the inventors contemplate peroxisome targeting signal 1 (PTS1), a C-terminal tripeptide, and peroxisome targeting signal 2 (PTS2), which is a nonapeptide located near the N-terminus.

[0086] In addition, sorting of proteins to endosomes and lysosomes may also be mediated by signals within the cytosolic domains of the proteins, typically comprising short, linear sequences. Some signals are referred to as tyrosine-based sorting signals and conform to the NPXY or YXXO consensus motifs. Other signals known as dileucine-based signals fit [DE]XXXL[LI] or DXXLL consensus motifs. All of these signals are recognized by components of protein coats peripherally associated with the cytosolic face of membranes. YXXO and [DE]XXXL[LI] signals are recognized with characteristic fine specificity by the adaptor protein (AP) complexes AP-1, AP-2, AP-3, and AP-4, whereas DXXLL signals are recognized by another family of adaptors known as GGAs. Also FYVE domain can be added, which has been associated with vacuolar protein sorting and endosome function. In still further aspects, endosomal compartments can also be targeted using human CD1 tail sequences (see e.g., Immunology, 122, 522-531). For example, lysosomal targeting can be achieved using a LAMP1-TM (transmembrane) sequence, while recycling endosomes can be targeted via the CD1a tail targeting sequence, and sorting endosomes can be targeted via the CD1c tail targeting sequence as is shown in more detail further below.

[0087] In still further contemplated aspects, the polytope may also be designed as a chimeric polytope that includes at least a portion of, and more typically an entire tumor associated antigen (e.g., CEA, PSMA, PSA, MUC1, AFP, MAGE, HER2, HCC1, p62, p90, etc.). Most notably, tumor associated antigens are generally processed and presented via the MHC-II pathway. Therefore, instead of using compartment specific signal sequences and/or leader sequences, the processing mechanism for tumor associated antigens can be employed for MHC-II targeting.

[0088] Trafficking to or retention in the cytosolic compartment may not necessarily require one or more specific sequence elements. However, in at least some aspects, N- or C-terminal cytoplasmic retention signals may be added, including a membrane-anchored protein or a membrane anchor domain of a membrane-anchored protein such that the protein is retained in the cell facing the cytosol. For example, membrane-anchored proteins include SNAP-25, syntaxin, synaptoprevin, synaptotagmin, vesicle associated membrane proteins (VAMPs), synaptic vesicle glycoproteins (SV2), high affinity choline transporters, Neurexins, voltage-gated calcium channels, acetylcholinesterase, and NOTCH.

[0089] In still further contemplated aspects of the inventive subject matter, the polytope may also comprise one or more transmembrane segments that will direct the neoepitope after processing to the outside of the cell membrane to so be visible to immune competent cells. There are numerous transmembrane domains known in the art, and all of those are deemed suitable for use herein, including those having a single alpha helix, multiple alpha helices, alpha/beta barrels, etc. For example, contemplated transmembrane domains can comprise comprises the transmembrane region(s) of the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, or PAG/Cbp. Where a fusion protein is desired, it is contemplated that the recombinant chimeric gene has a first portion that encodes the transmembrane region(s), wherein the first portion is cloned in frame with a second portion that encodes the inhibitory protein. It should be noted that such presentation will not result in MHC-complex presentation and as such provides a neoepitope presentation independent of MHC/T-cell receptor interaction, which may further open additional avenues for immune recognition and trigger antibody production against the neoepitopes.

[0090] Alternatively, or additionally, the polytope may also be designed to include signal sequences for protein export of one or more neoepitope to thereby force a transfected cell to produce and secrete one or more neoepitopes. For example, the SPARC leader sequence may be added to a neoepitope or polytope sequence, leading to in vivo secretion of the neoepitope or polytope sequence into the extracellular space. Advantageously, such secreted neoepitopes or polytopes are then taken up by immune competent cells, and especially antigen presenting cells and dendritic cells, that in turn process and display the neoepitopes, typically via MHC-II pathways.

[0091] Alternatively, or additionally, the neoepitope or polytope may also be administered as peptide, optionally bound to a carrier protein to so act as a peptide vaccine. Among other suitable carrier proteins, human albumin or lactoferrin are particularly preferred. Such carrier proteins may be in native conformation, or pretreated to form nanoparticles with exposed hydrophobic domains (see e.g., Adv Protein Chem Struct Biol. 2015; 98:121-43) to which the neoepitope or polytope can be coupled. Most typically, coupling of the neoepitope or polytope to the carrier protein will be non-covalent. Similar to the secreted neoepitopes or polytopes, carrier protein-bound neoepitopes or polytopes will be taken up by the immune competent cells, and especially antigen presenting cells and dendritic cells, that in turn process and display the neoepitopes, typically via MHC-II pathways.

[0092] Therefore, it should be appreciated that immune therapeutic compositions may be prepared that can deliver one or more neoepitopes to various sub-cellular locations, and with that generate distinct immune responses. For example, Prior Art FIG. 3 schematically illustrates a scenario where the polytope is predominantly processed in the proteasome of the cytoplasm and presented via the MHC-I complex, which is recognized by the T-cell receptor of a CD8.sup.+ T-cell. Consequently, targeting polytope processing to the cytosolic compartment will skew the immune response towards a CD8.sup.+ type response. On the other hand, Prior Art FIG. 4 schematically illustrates a scenario where the polytope is predominantly processed in the endosomal compartment and presented via the MHC-II complex, which is recognized by the T-cell receptor of a CD4.sup.+ T-cell. Consequently, targeting polytope processing to the endosomal or lysosomal compartment will skew the immune response towards a CD4.sup.+ type response. In addition, it should be appreciated that such targeting methods allow for specific delivery of a polytope or neoepitope peptide to an MHC subtype having the highest affinity with the peptide, even if that peptide would otherwise not be presented by that MHC subtype. Therefore, and as noted earlier, peptides for MHC-I presentation will generally be designed to have 8-12 amino acids (plus additional amino acids for flexibility in protease processing), while peptides for MHC-II presentation will be designed to have 14-20 amino acids (plus additional amino acids for flexibility in protease processing). In the examples below, further amino acids were added to allow for processing flexibility in the cytoplasmic, proteasome, or endosomal compartments.

[0093] In still further contemplated aspects of the inventive subject matter, it should be noted that trafficking modes of the neoepitope or polytope may be combined to accommodate one or more specific purposes. For example, sequential administration of the same neoepitopes or polytope with different targeting may be particularly beneficial in a prime-boost regimen where in a first administration the patient in inoculated with a recombinant virus to infect the patients cells, leading to antigen expression, processing, and presentation (e.g., predominantly MHC-I presentation) that will result in a first immune response originating from within a cell. The second administration of the same neoepitopes bound to albumin may then be employed as a boost as the so delivered protein is taken up by antigen presenting cells, leading in most cases to a distinct antigen presentation (e.g., predominantly MHC-II presentation). Where the same neoepitopes or polytope is trafficked to the cell surface for cell surface bound MHC-independent presentation, ADCC responses or NK mediated cell killing may be promoted. In still further contemplated aspects, and as illustrated in the examples below, immunogenicity of neoepitopes may be enhanced by cross presentation or MHC-II directed presentation. Notably, as cancer cell neoepitopes are typically internally generated and recycled, and with that preferentially presented via the MHC-I system, contemplated systems and methods now allow for presentation of such neoepitopes via MHC-II, which may be more immunogenic as is shown in more detail below. In addition, multiple and distinct trafficking of the same neoepitopes or polytopes may advantageously increase or supplement an immune response due to the stimulation of various and distinct components of the cellular and humoral immune system.

[0094] Of course, it should be appreciated that multiple and distinct trafficking of the same neoepitopes or polytopes may be achieved in numerous manners. For example, differently trafficked neoepitopes or polytopes may be administered separately using the same (e.g., viral expression vector) or different (e.g., viral expression vector and albumin bound) modality. Similarly, and especially where the therapeutic agent is an expression system (e.g., viral or bacterial), the recombinant nucleic acid may include two distinct portions that encode the same, albeit differently trafficked neoepitope or polytope (e.g., first portion trafficked to first location (e.g., cytosol or endosomal or lysosomal), second portion trafficked to a second, distinct location (e.g., cytosol or endosomal or lysosomal, secreted, membrane bound)). Likewise, a first administration may employ viral delivery of cytoplasm targeted neoepitopes or polytope, while a second administration is typically at least a day, two days, four days, a week, or two weeks after the first administration and may employ viral delivery of endosomal or lysosomal targeted or secreted neoepitopes or polytope.

[0095] Additionally, it is contemplated that the expression construct (e.g., recombinant viral expression vector or plasmid) may further encode at least one, more typically at least two, even more typically at least three, and most typically at least four co-stimulatory molecules to enhance the interaction between the infected cells (e.g., antigen presenting cells) and T-cells. For example, suitable co-stimulatory molecules include CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, OX40L, 4-1BBL, while other stimulatory molecules with less defined (or understood) mechanism of action include GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1, LFA3, and members of the SLAM family. However, especially preferred molecules for coordinated expression with the cancer-associated sequences include CD80 (B7-1), CD86 (B7-2), CD54 (ICAM-1) and CD11 (LFA-1). In addition to co-stimulatory molecules, the inventors also contemplate that one or more cytokines or cytokine analogs may be expressed from the recombinant nucleic acid, and especially preferred cytokines and cytokine analogs include IL-2, IL-15, and IL-a5 superagonist (ALT-803). Moreover, it should be appreciated that expression of the co-stimulatory molecules and/or cytokines will preferably be coordinated such that the neoepitopes or polytope are expressed contemporaneously with one or more co-stimulatory molecules and/or cytokines. Thus, it is typically contemplated that the co-stimulatory molecules and/or cytokines are produced from a single transcript (which may or may not include the sequence portion encoding the polytope), for example, using an internal ribosome entry site or 2A sequence, or from multiple transcripts.

[0096] Likewise, it is contemplated that the viral vector may also include a sequence portion that encodes one or more peptide ligands that bind to a checkpoint receptor. Most typically, binding will inhibit or at least reduce signaling via the receptor, and particularly contemplated receptors include CTLA-4 (especially for CD8.sup.+ cells), PD-1 (especially for CD4.sup.+ cells), TIM1 receptor, 2B4, and CD160. For example, suitable peptide binders can include antibody fragments and especially scFv, but also small molecule peptide ligands (e.g., isolated via RNA display or phage panning) that specifically bind to the receptors. Once more, it should be appreciated that expression of the peptide molecules will preferably be coordinated such that the neoepitopes or polytope are expressed contemporaneously with one or more of the peptide ligands. Thus, it is typically contemplated that the peptide ligands are produced from a single transcript (which may or may not include the sequence portion encoding the polytope), for example, using an internal ribosome entry site or 2A sequence, or from multiple transcripts.

[0097] It should be appreciated that all of the above noted co-stimulatory genes and genes coding for inhibitory proteins that interfere with/down-regulate checkpoint inhibition are well known in the art, and sequence information of these genes, isoforms, and variants can be retrieved from various public resources, including sequence data bases accessible at the NCBI, EMBL, GenBank, RefSeq, etc. Moreover, while the above exemplary stimulating molecules are preferably expressed in full length form as expressed in human, modified and non-human forms are also deemed suitable so long as such forms assist in stimulating or activating T-cells. Therefore, muteins, truncated forms and chimeric forms are expressly contemplated herein.

[0098] Consequently, contemplated expression constructs will preferably include a sequence portion that encodes one or more polytopes, wherein at least one, and more typically at least two, or all of the polytopes will include a trafficking signal that will result in preferential trafficking of the polytope to at least one, and more typically at least two different sub-cellular locations. For example, the first polytope may be directed towards the cytoplasm (and may include an additional cleavable or non-cleavable ubiquitin) while the second polytope may be directed towards the endosomal or lysosomal compartment. Or the first polytope may be directed towards the endosomal or lysosomal compartment while the second polytope may be directed towards the cell membrane or be secreted. As noted before, the encoded polytope will comprise at least two neoepitopes, optionally separated by a linker. Moreover, such contemplated expression constructs will also include a sequence portion that encodes one or more co-stimulatory molecules and/or cytokines, and may also include one or more inhibitory proteins that interfere with/down-regulate checkpoint inhibition. Most typically, the expression construct will also include regulatory sequences operably coupled to the above sequence portions to drive contemporaneous expression of the polytope and the co-stimulatory molecules, cytokines, and/or inhibitory proteins. Suitable promoter elements are known in the art, and especially preferred promoters include the constitutive and inducible promoters discussed above.

[0099] Where the expression construct is a viral expression construct (e.g., an adenovirus, and especially AdV with E1 and E2b deleted), it is contemplated that the recombinant viruses may then be individually or in combination used as a therapeutic vaccine in a pharmaceutical composition, typically formulated as a sterile injectable composition with a virus titer of between 10.sup.6-10.sup.13 virus particles, and more typically between 10.sup.9-10.sup.12 virus particles per dosage unit. Alternatively, virus may be employed to infect patient (or other HLA matched) cells ex vivo and the so infected cells are then transfused to the patient. In further examples, treatment of patients with the virus may be accompanied by allografted or autologous natural killer cells or T cells in a bare form or bearing chimeric antigen receptors expressing antibodies targeting neoepitope, neoepitopes, tumor associated antigens or the same payload as the virus. The natural killer cells, which include the patient-derived NK-92 cell line, may also express CD16 and can be coupled with an antibody.

[0100] Where desired, additional therapeutic modalities may be employed which may be neoepitope based (e.g., synthetic antibodies against neoepitopes as described in WO 2016/172722), alone or in combination with autologous or allogenic NK cells, and especially haNK cells or taNK cells (e.g., both commercially available from NantKwest, 9920 Jefferson Blvd. Culver City, Calif. 90232). Where haNK or taNK cells are employed, it is particularly preferred that the haNK cell carries a recombinant antibody on the CD16 variant that binds to a neoepitope of the treated patient, and where taNK cells are employed it is preferred that the chimeric antigen receptor of the taNK cell binds to a neoepitope of the treated patient. The additional treatment modality may also be independent of neoepitopes, and especially preferred modalities include cell-based therapeutics such as activated NK cells (e.g., aNK cells, commercially available from NantKwest, 9920 Jefferson Blvd. Culver City, Calif. 90232), and non cell-based therapeutics such as chemotherapy and/or radiation. In still further contemplated aspects, immune stimulatory cytokines, and especially IL-2, IL15, and IL-21 may be administered, alone or in combination with one or more checkpoint inhibitors (e.g., ipilimumab, nivolumab, etc.). Similarly, it is still further contemplated that additional pharmaceutical intervention may include administration of one or more drugs that inhibit immune suppressive cells, and especially MDSCs Tregs, and M2 macrophages. Thus, suitable drugs include IL-8 or interferon-y inhibitors or antibodies binding IL-8 or interferon-.gamma., as well as drugs that deactivate MDSCs (e.g., NO inhibitors, arginase inhibitors, ROS inhibitors), that block development of or differentiation of cells to MDSCs (e.g., IL-12, VEGF-inhibitors, bisphosphonates), or agents that are toxic to MDSCs (e.g., gemcitabine, cisplatin, 5-FU). Likewise, drugs like cyclophosphamide, daclizumab, and anti-GITR or anti-OX40 antibodies may be used to inhibit Tregs.

[0101] To trigger overexpression or transcription of stress signals, it is also contemplated that the chemotherapy and/or radiation for the patient may be done using a low-dose regimen, preferably in a metronomic fashion. For example, it is generally preferred that such treatment will use doses effective to affect at least one of protein expression, cell division, and cell cycle, preferably to induce apoptosis or at least to induce or increase the expression of stress-related genes (and particularly NKG2D ligands). Thus, in further contemplated aspects, such treatment will include low dose treatment using one or more chemotherapeutic agents. Most typically, low dose treatments will be at exposures that are equal or less than 70%, equal or less than 50%, equal or less than 40%, equal or less than 30%, equal or less than 20%, equal or less than 10%, or equal or less than 5% of the LD.sub.50 or IC.sub.50 for the chemotherapeutic agent. Additionally, where advantageous, such low-dose regimen may be performed in a metronomic manner as described, for example, in U.S. Pat. Nos. 7,758,891, 7,771,751, 7,780,984, 7,981,445, and 8,034,375.

[0102] With respect to the particular drug used in such low-dose regimen, it is contemplated that all chemotherapeutic agents are deemed suitable. Among other suitable drugs, kinase inhibitors, receptor agonists and antagonists, anti-metabolic, cytostatic and cytotoxic drugs are all contemplated herein. However, particularly preferred agents include those identified to interfere or inhibit a component of a pathway that drives growth or development of the tumor. Suitable drugs can be identified using pathway analysis on omics data as described in, for example, WO 2011/139345 and WO 2013/062505. Most notably, so achieved expression of stress-related genes in the tumor cells will result in surface presentation of NKG2D, NKP30, NKP44, and/or NKP46 ligands, which in turn activate NK cells to specifically destroy the tumor cells. Thus, it should be appreciated that low-dose chemotherapy may be employed as a trigger in tumor cells to express and display stress related proteins, which in turn will trigger NK-cell activation and/or NK-cell mediated tumor cell killing. Additionally, NK-cell mediated killing will be associated with release of intracellular tumor specific antigens, which is thought to further enhance the immune response.

[0103] Consequently, the inventors contemplate various methods of treatment of cancer in a patient in which a recombinant nucleic acid is administered to the patient (preferably in form of viral transfection in vivo) wherein the recombinant nucleic acid comprises a sequence portion that encodes one or more polytopes. Most preferably, at least one, and more typically at least two, or all of the polytopes include a trafficking signal that will result in preferential trafficking (e.g., at least 70%, more typically at least 80%, and most typically at least 90% of all expressed polytope is found in the targeted sub-cellular compartment) of the polytope to at least one, or at least two different sub-cellular locations. For example, the first polytope may be directed towards the cytoplasm (and may include an additional cleavable or non-cleavable ubiquitin) while the second polytope may be directed towards the endosomal or lysosomal compartment. Or the first polytope may be directed towards the endosomal or lysosomal compartment while the second polytope may be directed towards the cell membrane or be secreted. As discussed above, the encoded polytope will comprise at least two neoepitopes, optionally separated by a linker.

[0104] In at least some of the contemplated methods, contemplated expression constructs will also include a sequence portion that encodes one or more co-stimulatory molecules and/or cytokines, and may also include one or more inhibitory proteins that interfere with/down-regulate checkpoint inhibition. Therefore, expression of the polytope may be accompanied by expression of the co-stimulatory molecules, cytokines, and/or inhibitory proteins. Thus, the expression construct will include regulatory sequences operably coupled to the above sequence portions to drive contemporaneous expression of the polytope and the co-stimulatory molecules, cytokines, and/or inhibitory proteins.

[0105] As will be readily appreciated, the same (or different) polytopes may be targeted in contemplated methods to different sub-cellular compartments at the same time or in subsequent administrations, for example, using different expression constructs that are administered contemporaneously or temporally spaced apart. Consequently, and due to the possibility of targeting different compartments (and even extracellular space), contemplated compositions and methods may also be employed to trigger a CD8.sup.+ biased immune response (stimulation of cytotoxic T cells), to trigger a CD4.sup.+ biased immune response (producing Th1 and/or Th2 cytokine to stimulate B-cells, antibody production, and memory cells), an antibody biased immune response, and/or a stimulated immune response (at least partial reversal of checkpoint inhibition and/or increased activation of NK and T cell cytotoxicity). Thus, and viewed from a different perspective, where a patient has insufficient activity of NK and T cell cytotoxicity against a tumor, contemplated compositions and methods may be employed to support or increase such activity by targeting polytopes towards the cytoplasmic antigen presentation pathway to so increase MHC-I presentation and enhance a CD8.sup.+ biased immune response. On the other hand, where a patient has an insufficient CD4.sup.+ type immune response, contemplated compositions and methods may be employed to support or increase such activity by targeting polytopes towards the endosomal and/or lysosomal antigen presentation pathway to so increase MHC-II presentation, and with that enhance a CD4.sup.+ biased immune response.

[0106] As used herein, the term "administering" a pharmaceutical composition or drug refers to both direct and indirect administration of the pharmaceutical composition or drug, wherein direct administration of the pharmaceutical composition or drug is typically performed by a health care professional (e.g., physician, nurse, etc.), and wherein indirect administration includes a step of providing or making available the pharmaceutical composition or drug to the health care professional for direct administration (e.g., via injection, infusion, oral delivery, topical delivery, etc.). Most preferably, the recombinant virus is administered via subcutaneous or subdermal injection. However, in other contemplated aspects, administration may also be intravenous injection. Alternatively, or additionally, antigen presenting cells may be isolated or grown from cells of the patient, infected in vitro, and then transfused to the patient. Therefore, it should be appreciated that contemplated systems and methods can be considered a complete drug discovery system (e.g., drug discovery, treatment protocol, validation, etc.) for highly personalized cancer treatment.

Examples

[0107] Exemplary Sequence Arrangements

[0108] Neoepitope sequences were determined in silico by location-guided synchronous alignment of tumor and normal samples as, for example, disclosed in US 2012/0059670 and US 2012/0066001 using BAM files and BAM servers. Specifically, DNA analysis of the tumor was from the B16-F10 mouse melanoma line and matched normal was blood from C57bl/6 parental mouse DNA. The results were filtered for expression by RNA sequencing of this tumor cell line. Neoepitopes that were found expressed were further analyzed for binding affinity towards murine MHC-I (here: Kb) and MHC-II (here: I-Ab). Selected binders (with affinity of equal or less than 200 nM) were further analyzed after a further step of dbSNP filtering using positional permutations of all neoepitopes that were then processed through a weight matrix and neural network prediction to generate a score representing the likelihood of presence and/or strength of hydrophobic sequences or signal peptides. The best scoring arrangement (lowest likelihood of hydrophobic sequences or signal peptides) for the polytope (not shown) was used for further experiments. Neoepitopes were prioritized by detection in RNAseq or other quantitative system that yielded expression strength for a specific gene harboring the neoepitope mutation.

[0109] Table 1 shows exemplary neoepitopes that were expressed as determined by RNAseq along with gene name and mutated amino acid and position of the mutated amino acid. The neoepitope listed with * was discarded after dbSNP filtering as that neoepitope occurred as variant Rs71257443 in 28% of the population.

TABLE-US-00001 TABLE 1 Neoepitope- Neoepitope- Gene Position a b VIPR2 V73M GETVTMPCP LILRB3 T187N VGPVNPSHR* FCRL1 R286C GLGAQCSEA FAT4 S1613L RKLTTELTI PERRKLTTE PIEZO2 T2356M MDWVWMDTT VWMDTTLSL SIGLEC14 A292T GKTLNPSQT REGKTLNPS SIGLEC1 D1143N VRNATSYRC NVTVRNATS SLC4A11 Q678P FAMAQIPSL AQIPSLSLR

[0110] Table 2 shows further examples of neoepitopes in which the position of the mutated amino acid was changed, and shows further alternate sequences for MHC-I presentation (9-mer) and MHC-II presentation (15-mer). The neoepitope sequence for MHC-II presentation was back-translated to the corresponding nucleic acid sequence, which is also shown in Table 2.

TABLE-US-00002 TABLE 2 Gene Change Neoepitope-a Neoepitope-b Extended 15 mer Nucleotide Sequence SLC4A11 Q678P FAMAQIPSL AQIPSLSLR PFAMAQIPSLSLRAV CCCTTCGCCATGGCCC AGATCCCCAGCCTGA GCCTGAGGGCCGTG SIGLEC1 D1143N VRNATSYRC NVTVRNATS LPNVTVRNATSYRCG CTGCCCAACGTGACC GTGAGGAACGCCACC AGCTACAGGTGCGGC SIGLEC14 A292T GKTLNPSQT REGKTLNPS SWFREGKTLNPSQTS AGCTGGTTCAGGGAG GGCAAGACCCTGAAC CCCAGCCAGACCAGC PIEZO2 T2356M MDWVWMDTT VWMDTTLSL AVMDWVWMDTTLSLS GCCGTGATGGACTGG GTGTGGATGGACACC ACCCTGAGCCTGAGC FAT4 S1613L RKLTTELTI PERRKLTTE LGPERRKLTTELTII CTGGGCCCCGAGAGG AGGAAGCTGACCACC GAGCTGACCATCATC FCRL1 R286C GLGAQCSEA NNGLGAQCSEAVTLN AACAACGGCCTGGGC GCCCAGTGCAGCGAG GCCGTGACCCTGAAC VIPR2 V73M GETVTMPCP NVGENTMPCPKVFS AACGTGGGCGAGACC GTGACCATGCCCTGC CCCAAGGTGTTCAGC FLRT2 R346W EQVWGMAVR CQGPEQVWGMAVREL TGCCAGGGCCCCGAG CAGGTGTGGGGCATG GCCGTGAGGGAGCT G

[0111] Sequence Trafficking

[0112] Model cancer: Murine B16-F10 melanoma (derived from C57/B16 mouse) was used tumors were screened in a tumor versus normal manner as described above, and expressed mutant epitopes were identified in the B16F10 melanoma cell line. Candidate neoepitopes were further filtered as described above using sequencing data analysis and binding analysis to murine MHC I (H2-Kb, H2-Dd) and MHC II (I-Ab). Nine distinct polytope constructs were then prepared for testing various trafficking schemes, and each construct was prepared as the corresponding recombinant nucleic acid under the control of a CMV promoter. Each construct was cloned into an AdV5 expression vector that had deleted E1 and E2b genes, and the resulting recombinant virus was then used for transfection of mice as is further discussed below.

[0113] More specifically, three polytope constructs included MHC I binding neoepitopes for MHC-I presentation and were therefore targeted to the cytoplasmic compartment. While one construct had an unmodified N-terminus, another construct had an N-terminal non-cleavable ubiquitin, and yet another construct had an N-terminal cleavable ubiquitin. Ubiquitination was used to target the proteasome in the cytosol. Three further polytope constructs included MHC I binding neoepitopes for MHC-II presentation and were therefore targeted to the lysosomal/endosomal compartments compartment. While one construct had lysosomal targeting sequence, another construct had a recycling endosomal targeting sequence, and yet another construct had a sorting endosomal targeting sequence. Three additional polytope constructs included MHC II binding neoepitopes for MHC-II presentation and were also targeted to the lysosomal/endosomal compartments compartment. Once more, one construct had lysosomal targeting sequence, another construct had a recycling endosomal targeting sequence, and yet another construct had a sorting endosomal targeting sequence. These nine constructs had sequence arrangements as follows.

[0114] In the following exemplary sequences, for MHC-I presentation, ubiquitin (cleavable and non-cleavable) were used for proteasome targeting, while the CD1b leader peptide was used as an export leader peptide for trafficking the polypeptide out of the cytosol for all MHC-II directed sequences. LAMP1-TM/cytoplasmic tail was used as a lysosomal targeting sequence, while LAMP1-TM/CD1a tail was used as a recycling endosomes targeting sequence, and LAMP1-TM/CD1c tail was used as a sorting endosomes targeting domain.

[0115] It should further be noted that various internal controls were also used in the above polypeptides to account for expression and presentation. More specifically, the SIINFEKL peptide was used as an internal control for a MHC I restricted (Kb) peptide epitope, while the Esat6 peptide was used as an internal control for a secreted protein for MHC II presentation. FLAG-tag was used as an internal control epitope for detection of expression, and cMYC used as an internal control Tag for simple protein detection.

[0116] Exemplary Constructs for MHC-I Epitopes Directed to MHC-I Presentation (Traffic Through Proteasome, Cytoplasmic Targeting):

[0117] Polyepitope only: 12aa-A.sup.m-12aa-AAAA-12aa-B.sup.m-12aa-AAAA-(12aa-X.sup.m-12aa-AAAA).su- b.n-SIINFEKL-AAAA-Esat6-cMYC. FIG. 5A exemplarily depicts the polypeptide structure of such arrangement where the SIINFEKL motif is underlined, the Esat6 motif is in italics, and where the cMY motif is in bold type font. The nucleotide sequence for FIG. 5A is SEQ ID NO: 9, and the polypeptide sequence for FIG. 5A is SEQ ID NO: 12.

[0118] Polyepitope and cleavable ubiquitin GGR N-terminus: Ubiquitin-GGR-12aa-A.sup.m-12aa-AAAA-12aa-B.sup.m-12aa-AAAA-(12aa-X.sup.m- -12aa-AAAA).sub.n-SIINFEKL-AAAA-Esat6-cMYC. FIG. 5B exemplarily depicts the polypeptide structure of such arrangement where the cleavable ubiquitin moiety is italics and underlined, the SIINFEKL motif is underlined, the Esat6 motif is in italics, and where the cMY motif is in bold type font. The nucleotide sequence for FIG. 5B is SEQ ID NO: 10, and the polypeptide sequence for FIG. 5B is SEQ ID NO: 13.

[0119] Polyepitope and non-cleavable ubiquitin G N-terminus: Ubiquitin-G-12aa-A.sup.m-12aa-AAAA-12aa-B.sup.m-12aa-AAAA-(12aa-X.sup.m-1- 2aa-AAAA).sub.n-SIINFEKL-AAAA-Esat6-cMYC. FIG. 5C exemplarily depicts the polypeptide structure of such arrangement where the cleavable ubiquitin moiety is italics and underlined, the SIINFEKL motif is underlined, the Esat6 motif is in italics, and where the cMY motif is in bold type font. The nucleotide sequence for FIG. 5C is SEQ ID NO: 11, and the polypeptide sequence for FIG. 5C is SEQ ID NO: 14.

[0120] Exemplary Constructs for MHC-I Epitopes Directed to MHC-II Presentation (Export from Cytoplasm, Traffic Through Endo/Lysosome):

[0121] Lysosomal targeting of Kb epitope peptides: (CD1b leader peptide)-20aa-A.sup.m-20aa-GPGPG-20aa B.sup.m-20aa-GPGPG-(20aa-X.sup.m-20aa-GPGPG-).sub.n-Esat6-FlagTag-LAMP1TM- /cytoplasmic tail. FIG. 6A exemplarily depicts the polypeptide structure of such arrangement where the CD1b leader peptide moiety is bold, the Esat6 motif is in underlined, the Flag-tag motif is italics, and where the LAMP1TM/cytoplasmic tail is in bold/underline type font. The nucleotide sequence for FIG. 6A is SEQ ID NO: 15, and the polypeptide sequence for FIG. 6A is SEQ ID NO: 18.

[0122] Recycling lysosome targeting of Kb epitope peptides: (CD1b leader peptide)-20aa-A.sup.m-20aa-GPGPG-20aa B.sup.m-20aa-GPGPG-(20aa-X.sup.m-20aa-GPGPG-).sub.n-Esat6-FlagTag-LAMP1TM- /CD1a tail. FIG. 6B exemplarily depicts the polypeptide structure of such arrangement where the CD1b leader peptide moiety is bold, the Esat6 motif is in underlined, the Flag-tag motif is italics, and where the LAMP1TM motif is in bold/underline type font, and the CD1a targeting motif is underline/italics. The nucleotide sequence for FIG. 6B is SEQ ID NO: 16, and the polypeptide sequence for FIG. 6B is SEQ ID NO: 19.

[0123] Sorting endosome targeting of Kb epitope peptides: (CD1b leader peptide)-20aa-A.sup.m-20aa-GPGPG-20aa B.sup.m-20aa-GPGPG-(20aa-X.sup.m-20aa-GPGPG-).sub.n-Esat6-FlagTag-LAMP1TM- /CD1c tail. FIG. 6C exemplarily depicts the polypeptide structure of such arrangement where the CD1b leader peptide moiety is bold, the Esat6 motif is in underlined, the Flag-tag motif is italics, and where the LAMP1TM motif is in bold/underline type font, and the CD1c targeting motif is underline/italics. The nucleotide sequence for FIG. 6C is SEQ ID NO: 17, and the polypeptide sequence for FIG. 6C is SEQ ID NO: 20.

[0124] Exemplary Constructs for MHC-II Epitopes Directed to MHC-II Presentation (Export from Cytoplasm, Traffic Through Endo/Lysosome):

[0125] Lysosomal targeting of IAb epitope peptides: (CD1b leader peptide)-20aa-A.sup.m-20aa-GPGPG-20aa B.sup.m-20aa-GPGPG-(20aa-X.sup.m-20aa-GPGPG-).sub.n-Esat6-FlagTag-LAMP1TM- /cytoplasmic tail. FIG. 7A exemplarily depicts the polypeptide structure of such arrangement where the CD1b leader peptide moiety is bold, the SIINFEKL and Esat6 motifs are underlined, the Flag-tag motif is italics, and where the LAMP1TM/cytoplasmic tail is in bold/underline type font. The nucleotide sequence for FIG. 7A is SEQ ID NO: 21, and the polypeptide sequence for FIG. 7A is SEQ ID NO: 24.

[0126] Recycling lysosome targeting of IAb epitope peptides: (CD1b leader peptide)-20aa-A.sup.m-20aa-GPGPG-20aa B.sup.m-20aa-GPGPG-(20aa-X.sup.m-20aa-GPGPG-).sub.n-Esat6-FlagTag-LAMP1TM- /CD1a tail. FIG. 7B exemplarily depicts the polypeptide structure of such arrangement where the CD1b leader peptide moiety is bold, the SIINFEKL and Esat6 motifs are underlined, the Flag-tag motif is italics, where the LAMP1TM motif is in bold/underline type font, and where the CD1a tail is in bold/italics. The nucleotide sequence for FIG. 7B is SEQ ID NO: 22, and the polypeptide sequence for FIG. 7B is SEQ ID NO: 25.

[0127] Sorting endosome targeting of IAb epitope peptides: (CD1b leader peptide)-20aa-A.sup.m-20aa-GPGPG-20aa B.sup.m-20aa-GPGPG-(20aa-X.sup.m-20aa-GPGPG-).sub.n-Esat6-FlagTag-LAMP1TM- /CD1c tail. FIG. 7C exemplarily depicts the polypeptide structure of such arrangement where the CD1b leader peptide moiety is bold, the SIINFEKL and Esat6 motifs are underlined, the Flag-tag motif is italics, where the LAMP1TM motif is in bold/underline type font, and where the CD1c tail is in bold/italics. The nucleotide sequence for FIG. 7C is SEQ ID NO: 23, and the polypeptide sequence for FIG. 7C is SEQ ID NO: 26.

[0128] In Vivo Vaccination

[0129] FIG. 8 depicts an exemplary in vivo vaccination experiment where nine groups of C57bl/6 mice were immunized with nine distinct recombinant Ad5 viruses comprising the sequence arrangements substantially as described above. Immunization followed a biweekly schedule of administration with distinct routes for separate groups of animals (subcutaneous and intravenous) as is schematically shown in FIG. 8. Tumor challenge with B16-F10 melanoma cells was at day 42, followed by administration of an M2 macrophage suppressive drug (RP 182) and IL-15 superagonist (Alt-803). FIGS. 9A-9C depict exemplary results for subcutaneous administration, while FIGS. 10A-10C depict exemplary results for intravenous administration.

[0130] Notably, subcutaneous injection of adenovirus encoding Class I polytopes directed to the cytoplasm and MHC-I presentation did not provide a significant immune protection, regardless of the presence or absence of ubiquitination as can be taken from FIG. 9A. On the other hand, where the Class I polytopes were directed to the endosomal and lysosomal compartments for processing and presentation via MHC-II, some protective immunity was observed for direction to the recycling endosomal compartment and lysosomal compartment as is evident from FIG. 9B. Even stronger immune protection was observed when Class II polytopes were directed to the endosomal and lysosomal compartments for processing and presentation via MHC-II. Here, the strongest protection was observed for lysosomal and sorting endosomal compartments as is shown in FIG. 9C.

[0131] When immunization was performed with the same viral constructs, albeit via intravenous injection, protective effect of neoepitope vaccination was observed for Class I neoepitopes directed to the cytoplasm where the polytope included cleavable ubiquitin, and some protective effect was observed where the polytope included non-cleavable ubiquitin as can be seen from FIG. 10A. Notably, when the Class I polytopes were directed to the endosomal and lysosomal compartment, stronger protective effect was observed in all vaccinations as is shown in FIG. 10B. Moreover, strong protective effect was observed when Class II polytopes were directed to the endosomal and lysosomal compartments for processing and presentation via MHC-II. Here, the strongest protection was observed for recycling and sorting endosomal compartments as is shown in the graph of FIG. 10C.

[0132] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0133] It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the scope of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refers to at least one of something selected from the group consisting of A, B, C . . . and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.

Sequence CWU 1

1

261402DNAArtificial SequenceRecombinant construct 1 1ctcgaggaag cttgccgcca ccatgccatt tgccatggcc cagatcccca gcctgagcct 60gagagctgtg ctgcctaatg tgaccgtgcg gaacgccacc agctacagat gtggcagctg 120gttcagagag ggcaagaccc tgaaccccag ccagaccagc gccgtgatgg actgggtgtg 180gatggacacc accctgtccc tgagcctggg ccccgagaga agaaagctga ccaccgagct 240gacaatcatc aacaatggcc tgggcgctca gtgtagcgag gccgtgaccc tgaataatgt 300gggcgagaca gtgaccatgc cctgccccaa ggtgttcagc tgccagggcc ccgaacaagt 360gtggggaatg gctgtgcgcg agctgtgaga tatcgcggcc gc 4022591DNAArtificial SequenceArtificial construct 2 2ctcgaggaag cttgccgcca ccatgccatt tgccatggcc cagatcccca gcctgagcct 60gagagctgtg ggaagcggga gtggctcagg ttcaggactg cctaatgtga ccgtgcggaa 120cgccaccagc tacagatgtg gcggaagtgg gtcaggctcc ggttctggaa gctggttcag 180agagggcaag accctgaacc ccagccagac cagcgggtca ggaagtggta gcggctccgg 240ggccgtgatg gactgggtgt ggatggacac caccctgtcc ctgagcggaa gtggatcagg 300ttccggctct gggctgggcc ccgagagaag aaagctgacc accgagctga caatcatcgg 360atccgggtct ggcagtggtt caggcaacaa tggcctgggc gctcagtgta gcgaggccgt 420gaccctgaat ggatcagggt ccggcagcgg tagtggcaat gtgggcgaga cagtgaccat 480gccctgcccc aaggtgttca gcgggtccgg atctggtagt ggctcaggtt gccagggccc 540cgaacaagtg tggggaatgg ctgtgcgcga gctgtgagat atcgcggccg c 5913642DNAArtificial SequenceArtificial construct 3 3ctcgaggaag cttgccgcca ccatgcctag cgagagccct ccatttgcca tggcccagat 60ccccagcctg agcctgagag ctgtgtctgg cgctatggga gcccacagca tccccctgcc 120taatgtgacc gtgcggaacg ccaccagcta cagatgtggc gtgggacctc ctggccctcc 180tgcttccctg agctggttca gagagggcaa gaccctgaac cccagccaga ccagcatgag 240cggcaccctg ctgacagagc tgagagccgt gatggactgg gtgtggatgg acaccaccct 300gtccctgagc agctggatct gtgtggtgtc cgccacagac ctgggccccg agagaagaaa 360gctgaccacc gagctgacaa tcatcctgca gggcctggac tacagctgcg aggccaacaa 420tggcctgggc gctcagtgta gcgaggccgt gaccctgaat ttcaccgtgc ccacctgttg 480gaggcccgcc aatgtgggcg agacagtgac catgccctgc cccaaggtgt tcagcaactt 540ctacagcaaa gtgcggggct tcatgtgcca gggccccgaa caagtgtggg gaatggctgt 600gcgcgagctg aacatgaacc tgctgtgaga tatcgcggcc gc 6424831DNAArtificial SequenceArtificial Construct 4 4ctcgaggaag cttgccgcca ccatgcctag cgagagccct ccatttgcca tggcccagat 60ccccagcctg agcctgagag ctgtgtctgg cgctatggga ggaagcggga gtggctcagg 120ttcaggagcc cacagcatcc ccctgcctaa tgtgaccgtg cggaacgcca ccagctacag 180atgtggcgtg ggacctcctg gcggaagtgg gtcaggctcc ggttctggac ctcctgcttc 240cctgagctgg ttcagagagg gcaagaccct gaaccccagc cagaccagca tgagcggcac 300cctggggtca ggaagtggta gcggctccgg gctgacagag ctgagagccg tgatggactg 360ggtgtggatg gacaccaccc tgtccctgag cagctggatc tgtgtgggaa gtggatcagg 420ttccggctct ggggtgtccg ccacagacct gggccccgag agaagaaagc tgaccaccga 480gctgacaatc atcctgcagg gcctggacgg atccgggtct ggcagtggtt caggctacag 540ctgcgaggcc aacaatggcc tgggcgctca gtgtagcgag gccgtgaccc tgaatttcac 600cgtgcccacc ggatcagggt ccggcagcgg tagtggctgt tggaggcccg ccaatgtggg 660cgagacagtg accatgccct gccccaaggt gttcagcaac ttctacagca aagggtccgg 720atctggtagt ggctcaggtg tgcggggctt catgtgccag ggccccgaac aagtgtgggg 780aatggctgtg cgcgagctga acatgaacct gctgtgagat atcgcggccg c 8315121PRTArtificial SequenceArtificial construct 1 protein sequence 5Met Pro Phe Ala Met Ala Gln Ile Pro Ser Leu Ser Leu Arg Ala Val1 5 10 15Leu Pro Asn Val Thr Val Arg Asn Ala Thr Ser Tyr Arg Cys Gly Ser 20 25 30Trp Phe Arg Glu Gly Lys Thr Leu Asn Pro Ser Gln Thr Ser Ala Val 35 40 45Met Asp Trp Val Trp Met Asp Thr Thr Leu Ser Leu Ser Leu Gly Pro 50 55 60Glu Arg Arg Lys Leu Thr Thr Glu Leu Thr Ile Ile Asn Asn Gly Leu65 70 75 80Gly Ala Gln Cys Ser Glu Ala Val Thr Leu Asn Asn Val Gly Glu Thr 85 90 95Val Thr Met Pro Cys Pro Lys Val Phe Ser Cys Gln Gly Pro Glu Gln 100 105 110Val Trp Gly Met Ala Val Arg Glu Leu 115 1206184PRTArtificial SequenceArtificial construct 2 protein sequence 6Met Pro Phe Ala Met Ala Gln Ile Pro Ser Leu Ser Leu Arg Ala Val1 5 10 15Gly Ser Gly Ser Gly Ser Gly Ser Gly Leu Pro Asn Val Thr Val Arg 20 25 30Asn Ala Thr Ser Tyr Arg Cys Gly Gly Ser Gly Ser Gly Ser Gly Ser 35 40 45Gly Ser Trp Phe Arg Glu Gly Lys Thr Leu Asn Pro Ser Gln Thr Ser 50 55 60Gly Ser Gly Ser Gly Ser Gly Ser Gly Ala Val Met Asp Trp Val Trp65 70 75 80Met Asp Thr Thr Leu Ser Leu Ser Gly Ser Gly Ser Gly Ser Gly Ser 85 90 95Gly Leu Gly Pro Glu Arg Arg Lys Leu Thr Thr Glu Leu Thr Ile Ile 100 105 110Gly Ser Gly Ser Gly Ser Gly Ser Gly Asn Asn Gly Leu Gly Ala Gln 115 120 125Cys Ser Glu Ala Val Thr Leu Asn Gly Ser Gly Ser Gly Ser Gly Ser 130 135 140Gly Asn Val Gly Glu Thr Val Thr Met Pro Cys Pro Lys Val Phe Ser145 150 155 160Gly Ser Gly Ser Gly Ser Gly Ser Gly Cys Gln Gly Pro Glu Gln Val 165 170 175Trp Gly Met Ala Val Arg Glu Leu 1807201PRTArtificial SequenceArtificial construct 3 protein sequence 7Met Pro Ser Glu Ser Pro Pro Phe Ala Met Ala Gln Ile Pro Ser Leu1 5 10 15Ser Leu Arg Ala Val Ser Gly Ala Met Gly Ala His Ser Ile Pro Leu 20 25 30Pro Asn Val Thr Val Arg Asn Ala Thr Ser Tyr Arg Cys Gly Val Gly 35 40 45Pro Pro Gly Pro Pro Ala Ser Leu Ser Trp Phe Arg Glu Gly Lys Thr 50 55 60Leu Asn Pro Ser Gln Thr Ser Met Ser Gly Thr Leu Leu Thr Glu Leu65 70 75 80Arg Ala Val Met Asp Trp Val Trp Met Asp Thr Thr Leu Ser Leu Ser 85 90 95Ser Trp Ile Cys Val Val Ser Ala Thr Asp Leu Gly Pro Glu Arg Arg 100 105 110Lys Leu Thr Thr Glu Leu Thr Ile Ile Leu Gln Gly Leu Asp Tyr Ser 115 120 125Cys Glu Ala Asn Asn Gly Leu Gly Ala Gln Cys Ser Glu Ala Val Thr 130 135 140Leu Asn Phe Thr Val Pro Thr Cys Trp Arg Pro Ala Asn Val Gly Glu145 150 155 160Thr Val Thr Met Pro Cys Pro Lys Val Phe Ser Asn Phe Tyr Ser Lys 165 170 175Val Arg Gly Phe Met Cys Gln Gly Pro Glu Gln Val Trp Gly Met Ala 180 185 190Val Arg Glu Leu Asn Met Asn Leu Leu 195 2008264PRTArtificial SequenceArtificial construct 4 protein sequence 8Met Pro Ser Glu Ser Pro Pro Phe Ala Met Ala Gln Ile Pro Ser Leu1 5 10 15Ser Leu Arg Ala Val Ser Gly Ala Met Gly Gly Ser Gly Ser Gly Ser 20 25 30Gly Ser Gly Ala His Ser Ile Pro Leu Pro Asn Val Thr Val Arg Asn 35 40 45Ala Thr Ser Tyr Arg Cys Gly Val Gly Pro Pro Gly Gly Ser Gly Ser 50 55 60Gly Ser Gly Ser Gly Pro Pro Ala Ser Leu Ser Trp Phe Arg Glu Gly65 70 75 80Lys Thr Leu Asn Pro Ser Gln Thr Ser Met Ser Gly Thr Leu Gly Ser 85 90 95Gly Ser Gly Ser Gly Ser Gly Leu Thr Glu Leu Arg Ala Val Met Asp 100 105 110Trp Val Trp Met Asp Thr Thr Leu Ser Leu Ser Ser Trp Ile Cys Val 115 120 125Gly Ser Gly Ser Gly Ser Gly Ser Gly Val Ser Ala Thr Asp Leu Gly 130 135 140Pro Glu Arg Arg Lys Leu Thr Thr Glu Leu Thr Ile Ile Leu Gln Gly145 150 155 160Leu Asp Gly Ser Gly Ser Gly Ser Gly Ser Gly Tyr Ser Cys Glu Ala 165 170 175Asn Asn Gly Leu Gly Ala Gln Cys Ser Glu Ala Val Thr Leu Asn Phe 180 185 190Thr Val Pro Thr Gly Ser Gly Ser Gly Ser Gly Ser Gly Cys Trp Arg 195 200 205Pro Ala Asn Val Gly Glu Thr Val Thr Met Pro Cys Pro Lys Val Phe 210 215 220Ser Asn Phe Tyr Ser Lys Gly Ser Gly Ser Gly Ser Gly Ser Gly Val225 230 235 240Arg Gly Phe Met Cys Gln Gly Pro Glu Gln Val Trp Gly Met Ala Val 245 250 255Arg Glu Leu Asn Met Asn Leu Leu 2609993DNAArtificial SequenceMHC-I nucleic acid for MHC-I presentation 9atggataaaa tcacgcagta cgaaaagtca ttgtactatt gttctttcct ggaggccctc 60gttagagatg tatgcatcgc tgcagctgcc tgtgtcgact gtcttgacag aaccaacacc 120gcccaggtaa tggttggaaa atgtgctctg gcctaccagc tgtatgcagc cgccgcagtg 180gtcaaagcat acctgcctgt caatgaatct ttcgccttca ctgccgattt gaggagcaat 240actggcggcc aggcagcagc cgcaaacatc ctggctgtgt ccttcgcacc tctggtgcaa 300ctcagtaaaa acgacaacgg aactcctgac tccgtgggag cagccgccgc acagagtaac 360taccagcaca tcacgaattt tgagtggtgc atttccatac tggttgaact gacgcgcctg 420gagggtgcag ctgccgctta ttacacagtg ttcgatcggg acaacaatcg ggtctctttt 480gctaatgctg tggtgctggc cgctgccgct cacagcggac tggtcacttt ccaagccttt 540attgatgtga tgtcaaggga aacaactgac acagacactg cagacgctgc cgcagccctg 600gatctggccg ctttggaaga cgtctccgcc aactgtctca ctgagaccct ggaggacaag 660aatgaaggtg tggccgctgc cgctgtcctg tcattcgtgg gccagacgag ggtgttgatg 720atcaacgggg aagaagttga agaaacagaa ctgatgggtg ccgctgccgc agaggtgtcc 780ggactcgagc agctggaatc tataattaac tttgaaaagt tgacggaatg gacttcctct 840aacgtggccg ctgccgctat gacggagcaa caatggaact tcgcaggcat cgaggctgcc 900gccagcgcca tccaaggaaa cgtaacttca atccacagcc ttctggatgc tgctgctgcc 960gaacaaaagc tcatcagtga ggaggacttg taa 993101221DNAArtificial SequenceMHC-I nucleic acid for MHC-I presentation (Ubiquitin, cleavable) 10atgcagattt ttgtgaagac actgaccgga aaaactatca ccctcgaggt ggagccttcc 60gacactatcg aaaacgtcaa ggccaagatc caggacaagg aaggcattcc acccgatcag 120cagcgcctta tttttgcagg taagcagctg gaagacggaa ggaccctgag cgactataat 180atccagaaag aaagcacact ccacctcgtg ctcaggctcc gcgggggcag ggataaaatc 240acgcagtacg aaaagtcatt gtactattgt tctttcctgg aggccctcgt tagagatgta 300tgcatcgctg cagctgcctg tgtcgactgt cttgacagaa ccaacaccgc ccaggtaatg 360gttggaaaat gtgctctggc ctaccagctg tatgcagccg ccgcagtggt caaagcatac 420ctgcctgtca atgaatcttt cgccttcact gccgatttga ggagcaatac tggcggccag 480gcagcagccg caaacatcct ggctgtgtcc ttcgcacctc tggtgcaact cagtaaaaac 540gacaacggaa ctcctgactc cgtgggagca gccgccgcac agagtaacta ccagcacatc 600acgaattttg agtggtgcat ttccatactg gttgaactga cgcgcctgga gggtgcagct 660gccgcttatt acacagtgtt cgatcgggac aacaatcggg tctcttttgc taatgctgtg 720gtgctggccg ctgccgctca cagcggactg gtcactttcc aagcctttat tgatgtgatg 780tcaagggaaa caactgacac agacactgca gacgctgccg cagccctgga tctggccgct 840ttggaagacg tctccgccaa ctgtctcact gagaccctgg aggacaagaa tgaaggtgtg 900gccgctgccg ctgtcctgtc attcgtgggc cagacgaggg tgttgatgat caacggggaa 960gaagttgaag aaacagaact gatgggtgcc gctgccgcag aggtgtccgg actcgagcag 1020ctggaatcta taattaactt tgaaaagttg acggaatgga cttcctctaa cgtggccgct 1080gccgctatga cggagcaaca atggaacttc gcaggcatcg aggctgccgc cagcgccatc 1140caaggaaacg taacttcaat ccacagcctt ctggatgctg ctgctgccga acaaaagctc 1200atcagtgagg aggacttgta a 1221111215DNAArtificial SequenceMHC-I nucleic acid for MHC-I presentation (Ubiquitin, non-cleavable) 11atgcagattt ttgtgaagac actgaccgga aaaactatca ccctcgaggt ggagccttcc 60gacactatcg aaaacgtcaa ggccaagatc caggacaagg aaggcattcc acccgatcag 120cagcgcctta tttttgcagg taagcagctg gaagacggaa ggaccctgag cgactataat 180atccagaaag aaagcacact ccacctcgtg ctcaggctcc gcggggataa aatcacgcag 240tacgaaaagt cattgtacta ttgttctttc ctggaggccc tcgttagaga tgtatgcatc 300gctgcagctg cctgtgtcga ctgtcttgac agaaccaaca ccgcccaggt aatggttgga 360aaatgtgctc tggcctacca gctgtatgca gccgccgcag tggtcaaagc atacctgcct 420gtcaatgaat ctttcgcctt cactgccgat ttgaggagca atactggcgg ccaggcagca 480gccgcaaaca tcctggctgt gtccttcgca cctctggtgc aactcagtaa aaacgacaac 540ggaactcctg actccgtggg agcagccgcc gcacagagta actaccagca catcacgaat 600tttgagtggt gcatttccat actggttgaa ctgacgcgcc tggagggtgc agctgccgct 660tattacacag tgttcgatcg ggacaacaat cgggtctctt ttgctaatgc tgtggtgctg 720gccgctgccg ctcacagcgg actggtcact ttccaagcct ttattgatgt gatgtcaagg 780gaaacaactg acacagacac tgcagacgct gccgcagccc tggatctggc cgctttggaa 840gacgtctccg ccaactgtct cactgagacc ctggaggaca agaatgaagg tgtggccgct 900gccgctgtcc tgtcattcgt gggccagacg agggtgttga tgatcaacgg ggaagaagtt 960gaagaaacag aactgatggg tgccgctgcc gcagaggtgt ccggactcga gcagctggaa 1020tctataatta actttgaaaa gttgacggaa tggacttcct ctaacgtggc cgctgccgct 1080atgacggagc aacaatggaa cttcgcaggc atcgaggctg ccgccagcgc catccaagga 1140aacgtaactt caatccacag ccttctggat gctgctgctg ccgaacaaaa gctcatcagt 1200gaggaggact tgtaa 121512330PRTArtificial SequenceMHC-I polypeptide for MHC-I presentation 12Met Asp Lys Ile Thr Gln Tyr Glu Lys Ser Leu Tyr Tyr Cys Ser Phe1 5 10 15Leu Glu Ala Leu Val Arg Asp Val Cys Ile Ala Ala Ala Ala Cys Val 20 25 30Asp Cys Leu Asp Arg Thr Asn Thr Ala Gln Val Met Val Gly Lys Cys 35 40 45Ala Leu Ala Tyr Gln Leu Tyr Ala Ala Ala Ala Val Val Lys Ala Tyr 50 55 60Leu Pro Val Asn Glu Ser Phe Ala Phe Thr Ala Asp Leu Arg Ser Asn65 70 75 80Thr Gly Gly Gln Ala Ala Ala Ala Asn Ile Leu Ala Val Ser Phe Ala 85 90 95Pro Leu Val Gln Leu Ser Lys Asn Asp Asn Gly Thr Pro Asp Ser Val 100 105 110Gly Ala Ala Ala Ala Gln Ser Asn Tyr Gln His Ile Thr Asn Phe Glu 115 120 125Trp Cys Ile Ser Ile Leu Val Glu Leu Thr Arg Leu Glu Gly Ala Ala 130 135 140Ala Ala Tyr Tyr Thr Val Phe Asp Arg Asp Asn Asn Arg Val Ser Phe145 150 155 160Ala Asn Ala Val Val Leu Ala Ala Ala Ala His Ser Gly Leu Val Thr 165 170 175Phe Gln Ala Phe Ile Asp Val Met Ser Arg Glu Thr Thr Asp Thr Asp 180 185 190Thr Ala Asp Ala Ala Ala Ala Leu Asp Leu Ala Ala Leu Glu Asp Val 195 200 205Ser Ala Asn Cys Leu Thr Glu Thr Leu Glu Asp Lys Asn Glu Gly Val 210 215 220Ala Ala Ala Ala Val Leu Ser Phe Val Gly Gln Thr Arg Val Leu Met225 230 235 240Ile Asn Gly Glu Glu Val Glu Glu Thr Glu Leu Met Gly Ala Ala Ala 245 250 255Ala Glu Val Ser Gly Leu Glu Gln Leu Glu Ser Ile Ile Asn Phe Glu 260 265 270Lys Leu Thr Glu Trp Thr Ser Ser Asn Val Ala Ala Ala Ala Met Thr 275 280 285Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser Ala Ile 290 295 300Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Ala Ala Ala Ala305 310 315 320Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 325 33013406PRTArtificial SequenceMHC-I polypeptide for MHC-I presentation (Ubiquitin, cleavable) 13Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys 35 40 45Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly Arg Asp Lys Ile65 70 75 80Thr Gln Tyr Glu Lys Ser Leu Tyr Tyr Cys Ser Phe Leu Glu Ala Leu 85 90 95Val Arg Asp Val Cys Ile Ala Ala Ala Ala Cys Val Asp Cys Leu Asp 100 105 110Arg Thr Asn Thr Ala Gln Val Met Val Gly Lys Cys Ala Leu Ala Tyr 115 120 125Gln Leu Tyr Ala Ala Ala Ala Val Val Lys Ala Tyr Leu Pro Val Asn 130 135 140Glu Ser Phe Ala Phe Thr Ala Asp Leu Arg Ser Asn Thr Gly Gly Gln145 150 155 160Ala Ala Ala Ala Asn Ile Leu Ala Val Ser Phe Ala Pro Leu Val Gln 165 170 175Leu Ser Lys Asn Asp Asn Gly Thr Pro Asp Ser Val Gly Ala Ala Ala 180 185 190Ala Gln Ser Asn Tyr Gln His Ile Thr Asn Phe Glu Trp Cys Ile Ser 195 200 205Ile Leu Val Glu Leu Thr Arg Leu Glu Gly Ala Ala Ala Ala Tyr Tyr 210 215 220Thr Val Phe Asp Arg Asp Asn Asn Arg Val Ser Phe Ala Asn Ala Val225 230 235 240Val Leu Ala Ala Ala Ala His

Ser Gly Leu Val Thr Phe Gln Ala Phe 245 250 255Ile Asp Val Met Ser Arg Glu Thr Thr Asp Thr Asp Thr Ala Asp Ala 260 265 270Ala Ala Ala Leu Asp Leu Ala Ala Leu Glu Asp Val Ser Ala Asn Cys 275 280 285Leu Thr Glu Thr Leu Glu Asp Lys Asn Glu Gly Val Ala Ala Ala Ala 290 295 300Val Leu Ser Phe Val Gly Gln Thr Arg Val Leu Met Ile Asn Gly Glu305 310 315 320Glu Val Glu Glu Thr Glu Leu Met Gly Ala Ala Ala Ala Glu Val Ser 325 330 335Gly Leu Glu Gln Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu 340 345 350Trp Thr Ser Ser Asn Val Ala Ala Ala Ala Met Thr Glu Gln Gln Trp 355 360 365Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser Ala Ile Gln Gly Asn Val 370 375 380Thr Ser Ile His Ser Leu Leu Asp Ala Ala Ala Ala Glu Gln Lys Leu385 390 395 400Ile Ser Glu Glu Asp Leu 40514404PRTArtificial SequenceMHC-I polypeptide for MHC-I presentation (Ubiquitin, non-cleavable) 14Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys 35 40 45Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Asp Lys Ile Thr Gln65 70 75 80Tyr Glu Lys Ser Leu Tyr Tyr Cys Ser Phe Leu Glu Ala Leu Val Arg 85 90 95Asp Val Cys Ile Ala Ala Ala Ala Cys Val Asp Cys Leu Asp Arg Thr 100 105 110Asn Thr Ala Gln Val Met Val Gly Lys Cys Ala Leu Ala Tyr Gln Leu 115 120 125Tyr Ala Ala Ala Ala Val Val Lys Ala Tyr Leu Pro Val Asn Glu Ser 130 135 140Phe Ala Phe Thr Ala Asp Leu Arg Ser Asn Thr Gly Gly Gln Ala Ala145 150 155 160Ala Ala Asn Ile Leu Ala Val Ser Phe Ala Pro Leu Val Gln Leu Ser 165 170 175Lys Asn Asp Asn Gly Thr Pro Asp Ser Val Gly Ala Ala Ala Ala Gln 180 185 190Ser Asn Tyr Gln His Ile Thr Asn Phe Glu Trp Cys Ile Ser Ile Leu 195 200 205Val Glu Leu Thr Arg Leu Glu Gly Ala Ala Ala Ala Tyr Tyr Thr Val 210 215 220Phe Asp Arg Asp Asn Asn Arg Val Ser Phe Ala Asn Ala Val Val Leu225 230 235 240Ala Ala Ala Ala His Ser Gly Leu Val Thr Phe Gln Ala Phe Ile Asp 245 250 255Val Met Ser Arg Glu Thr Thr Asp Thr Asp Thr Ala Asp Ala Ala Ala 260 265 270Ala Leu Asp Leu Ala Ala Leu Glu Asp Val Ser Ala Asn Cys Leu Thr 275 280 285Glu Thr Leu Glu Asp Lys Asn Glu Gly Val Ala Ala Ala Ala Val Leu 290 295 300Ser Phe Val Gly Gln Thr Arg Val Leu Met Ile Asn Gly Glu Glu Val305 310 315 320Glu Glu Thr Glu Leu Met Gly Ala Ala Ala Ala Glu Val Ser Gly Leu 325 330 335Glu Gln Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu Trp Thr 340 345 350Ser Ser Asn Val Ala Ala Ala Ala Met Thr Glu Gln Gln Trp Asn Phe 355 360 365Ala Gly Ile Glu Ala Ala Ala Ser Ala Ile Gln Gly Asn Val Thr Ser 370 375 380Ile His Ser Leu Leu Asp Ala Ala Ala Ala Glu Gln Lys Leu Ile Ser385 390 395 400Glu Glu Asp Leu151218DNAArtificial SequenceMHC-I nucleic acid for MHC-II presentation (lysosomal target) 15atgctgctgc tgccattcca gctcttggcc gtgctgttcc ccggggggaa ttctgaggat 60aagatcaccc agtatgaaaa atccttgtat tattgtagct ttctggaagc cctggtgcga 120gatgtgtgta taggcccagg gccggggtgt gttgattgcc tcgacaggac aaataccgcc 180caggtaatgg ttggcaaatg cgcactcgca tatcaactct acggaccagg acccggcgtg 240gtaaaggctt atctgccagt taacgagagc tttgccttca ccgcagacct gcgctccaat 300actggtggtc aagggcccgg accaggtaat atcctcgccg tgagcttcgc ccctctggtc 360cagctgagta aaaatgacaa tggtactcct gatagtgtag gcggacccgg tccgggtcag 420tccaattacc agcacattac caatttcgaa tggtgcatca gtatactggt tgaattgacc 480agactcgagg ggggcccagg cccaggctac tacactgttt tcgatcgcga caataatcgg 540gttagcttcg ctaatgcagt agtgctgggc cccgggccag ggcactccgg tctggtgacg 600ttccaggcct tcatcgatgt aatgagccgc gagactacgg acaccgacac cgctgatggc 660cccgggcctg gtctggatct tgcagccctg gaggatgtgt ccgctaactg tttgactgag 720acactggagg acaagaacga gggcgtcgga ccagggcctg gtgtcctttc tttcgtcggt 780cagacaaggg tgttgatgat caatggagaa gaggtggaag agaccgaatt gatgggaggc 840ccagggcccg gcgaggttag cggcctggaa cagctggaga gtattatcaa ttttgaaaag 900ctgaccgagt ggacaagctc caatgtaggc ggacctggac ccgggatgac agagcagcag 960tggaattttg ccggtattga agctgcagcc agtgctatcc aaggcaacgt aacgagtatt 1020cacagcctgc tcgacgacta caaagacgac gacgacaagg gcagtgacta taaggaccat 1080gatggtgact ataaggacca cgacatcatg ctcatcccaa tcgctgtcgg aggtgctctg 1140gccggtctcg tattgatcgt tctgatcgct tatttgatcg ggaggaagag gagtcacgca 1200ggctaccaga ctatctag 1218161203DNAArtificial SequenceMHC-I nucleic acid for MHC-II presentation (recycling endosome target) 16atgctgctgc tgccattcca gctcttggcc gtgctgttcc ccggggggaa ttctgaggat 60aagatcaccc agtatgaaaa atccttgtat tattgtagct ttctggaagc cctggtgcga 120gatgtgtgta taggcccagg gccggggtgt gttgattgcc tcgacaggac aaataccgcc 180caggtaatgg ttggcaaatg cgcactcgca tatcaactct acggaccagg acccggcgtg 240gtaaaggctt atctgccagt taacgagagc tttgccttca ccgcagacct gcgctccaat 300actggtggtc aagggcccgg accaggtaat atcctcgccg tgagcttcgc ccctctggtc 360cagctgagta aaaatgacaa tggtactcct gatagtgtag gcggacccgg tccgggtcag 420tccaattacc agcacattac caatttcgaa tggtgcatca gtatactggt tgaattgacc 480agactcgagg ggggcccagg cccaggctac tacactgttt tcgatcgcga caataatcgg 540gttagcttcg ctaatgcagt agtgctgggc cccgggccag ggcactccgg tctggtgacg 600ttccaggcct tcatcgatgt aatgagccgc gagactacgg acaccgacac cgctgatggc 660cccgggcctg gtctggatct tgcagccctg gaggatgtgt ccgctaactg tttgactgag 720acactggagg acaagaacga gggcgtcgga ccagggcctg gtgtcctttc tttcgtcggt 780cagacaaggg tgttgatgat caatggagaa gaggtggaag agaccgaatt gatgggaggc 840ccagggcccg gcgaggttag cggcctggaa cagctggaga gtattatcaa ttttgaaaag 900ctgaccgagt ggacaagctc caatgtaggc ggacctggac ccgggatgac agagcagcag 960tggaattttg ccggtattga agctgcagcc agtgctatcc aaggcaacgt aacgagtatt 1020cacagcctgc tcgacgacta caaagacgac gacgacaagg gcagtgacta taaggaccat 1080gatggtgact ataaggacca cgacatcatg ctcatcccaa tcgctgtcgg aggtgctctg 1140gccggtctcg tattgatcgt tctgatcgct tatttgatcg ggaggaaacg ctgtttctgt 1200taa 1203171215DNAArtificial SequenceMHC-I nucleic acid for MHC-II presentation (sorting endosome target) 17atgctgctgc tgccattcca gctcttggcc gtgctgttcc ccggggggaa ttctgaggat 60aagatcaccc agtatgaaaa atccttgtat tattgtagct ttctggaagc cctggtgcga 120gatgtgtgta taggcccagg gccggggtgt gttgattgcc tcgacaggac aaataccgcc 180caggtaatgg ttggcaaatg cgcactcgca tatcaactct acggaccagg acccggcgtg 240gtaaaggctt atctgccagt taacgagagc tttgccttca ccgcagacct gcgctccaat 300actggtggtc aagggcccgg accaggtaat atcctcgccg tgagcttcgc ccctctggtc 360cagctgagta aaaatgacaa tggtactcct gatagtgtag gcggacccgg tccgggtcag 420tccaattacc agcacattac caatttcgaa tggtgcatca gtatactggt tgaattgacc 480agactcgagg ggggcccagg cccaggctac tacactgttt tcgatcgcga caataatcgg 540gttagcttcg ctaatgcagt agtgctgggc cccgggccag ggcactccgg tctggtgacg 600ttccaggcct tcatcgatgt aatgagccgc gagactacgg acaccgacac cgctgatggc 660cccgggcctg gtctggatct tgcagccctg gaggatgtgt ccgctaactg tttgactgag 720acactggagg acaagaacga gggcgtcgga ccagggcctg gtgtcctttc tttcgtcggt 780cagacaaggg tgttgatgat caatggagaa gaggtggaag agaccgaatt gatgggaggc 840ccagggcccg gcgaggttag cggcctggaa cagctggaga gtattatcaa ttttgaaaag 900ctgaccgagt ggacaagctc caatgtaggc ggacctggac ccgggatgac agagcagcag 960tggaattttg ccggtattga agctgcagcc agtgctatcc aaggcaacgt aacgagtatt 1020cacagcctgc tcgacgacta caaagacgac gacgacaagg gcagtgacta taaggaccat 1080gatggtgact ataaggacca cgacatcatg ctcatcccaa tcgctgtcgg aggtgctctg 1140gccggtctcg tattgatcgt tctgatcgct tatttgatcg ggaagaagca ctgctcatat 1200caggacatcc tgtga 121518405PRTArtificial SequenceMHC-I polypeptide for MHC-II presentation (lysosomal target) 18Met Leu Leu Leu Pro Phe Gln Leu Leu Ala Val Leu Phe Pro Gly Gly1 5 10 15Asn Ser Glu Asp Lys Ile Thr Gln Tyr Glu Lys Ser Leu Tyr Tyr Cys 20 25 30Ser Phe Leu Glu Ala Leu Val Arg Asp Val Cys Ile Gly Pro Gly Pro 35 40 45Gly Cys Val Asp Cys Leu Asp Arg Thr Asn Thr Ala Gln Val Met Val 50 55 60Gly Lys Cys Ala Leu Ala Tyr Gln Leu Tyr Gly Pro Gly Pro Gly Val65 70 75 80Val Lys Ala Tyr Leu Pro Val Asn Glu Ser Phe Ala Phe Thr Ala Asp 85 90 95Leu Arg Ser Asn Thr Gly Gly Gln Gly Pro Gly Pro Gly Asn Ile Leu 100 105 110Ala Val Ser Phe Ala Pro Leu Val Gln Leu Ser Lys Asn Asp Asn Gly 115 120 125Thr Pro Asp Ser Val Gly Gly Pro Gly Pro Gly Gln Ser Asn Tyr Gln 130 135 140His Ile Thr Asn Phe Glu Trp Cys Ile Ser Ile Leu Val Glu Leu Thr145 150 155 160Arg Leu Glu Gly Gly Pro Gly Pro Gly Tyr Tyr Thr Val Phe Asp Arg 165 170 175Asp Asn Asn Arg Val Ser Phe Ala Asn Ala Val Val Leu Gly Pro Gly 180 185 190Pro Gly His Ser Gly Leu Val Thr Phe Gln Ala Phe Ile Asp Val Met 195 200 205Ser Arg Glu Thr Thr Asp Thr Asp Thr Ala Asp Gly Pro Gly Pro Gly 210 215 220Leu Asp Leu Ala Ala Leu Glu Asp Val Ser Ala Asn Cys Leu Thr Glu225 230 235 240Thr Leu Glu Asp Lys Asn Glu Gly Val Gly Pro Gly Pro Gly Val Leu 245 250 255Ser Phe Val Gly Gln Thr Arg Val Leu Met Ile Asn Gly Glu Glu Val 260 265 270Glu Glu Thr Glu Leu Met Gly Gly Pro Gly Pro Gly Glu Val Ser Gly 275 280 285Leu Glu Gln Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu Trp 290 295 300Thr Ser Ser Asn Val Gly Gly Pro Gly Pro Gly Met Thr Glu Gln Gln305 310 315 320Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser Ala Ile Gln Gly Asn 325 330 335Val Thr Ser Ile His Ser Leu Leu Asp Asp Tyr Lys Asp Asp Asp Asp 340 345 350Lys Gly Ser Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 355 360 365Ile Met Leu Ile Pro Ile Ala Val Gly Gly Ala Leu Ala Gly Leu Val 370 375 380Leu Ile Val Leu Ile Ala Tyr Leu Ile Gly Arg Lys Arg Ser His Ala385 390 395 400Gly Tyr Gln Thr Ile 40519400PRTArtificial SequenceMHC-I polypeptide for MHC-II presentation (recycling endosome target) 19Met Leu Leu Leu Pro Phe Gln Leu Leu Ala Val Leu Phe Pro Gly Gly1 5 10 15Asn Ser Glu Asp Lys Ile Thr Gln Tyr Glu Lys Ser Leu Tyr Tyr Cys 20 25 30Ser Phe Leu Glu Ala Leu Val Arg Asp Val Cys Ile Gly Pro Gly Pro 35 40 45Gly Cys Val Asp Cys Leu Asp Arg Thr Asn Thr Ala Gln Val Met Val 50 55 60Gly Lys Cys Ala Leu Ala Tyr Gln Leu Tyr Gly Pro Gly Pro Gly Val65 70 75 80Val Lys Ala Tyr Leu Pro Val Asn Glu Ser Phe Ala Phe Thr Ala Asp 85 90 95Leu Arg Ser Asn Thr Gly Gly Gln Gly Pro Gly Pro Gly Asn Ile Leu 100 105 110Ala Val Ser Phe Ala Pro Leu Val Gln Leu Ser Lys Asn Asp Asn Gly 115 120 125Thr Pro Asp Ser Val Gly Gly Pro Gly Pro Gly Gln Ser Asn Tyr Gln 130 135 140His Ile Thr Asn Phe Glu Trp Cys Ile Ser Ile Leu Val Glu Leu Thr145 150 155 160Arg Leu Glu Gly Gly Pro Gly Pro Gly Tyr Tyr Thr Val Phe Asp Arg 165 170 175Asp Asn Asn Arg Val Ser Phe Ala Asn Ala Val Val Leu Gly Pro Gly 180 185 190Pro Gly His Ser Gly Leu Val Thr Phe Gln Ala Phe Ile Asp Val Met 195 200 205Ser Arg Glu Thr Thr Asp Thr Asp Thr Ala Asp Gly Pro Gly Pro Gly 210 215 220Leu Asp Leu Ala Ala Leu Glu Asp Val Ser Ala Asn Cys Leu Thr Glu225 230 235 240Thr Leu Glu Asp Lys Asn Glu Gly Val Gly Pro Gly Pro Gly Val Leu 245 250 255Ser Phe Val Gly Gln Thr Arg Val Leu Met Ile Asn Gly Glu Glu Val 260 265 270Glu Glu Thr Glu Leu Met Gly Gly Pro Gly Pro Gly Glu Val Ser Gly 275 280 285Leu Glu Gln Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu Trp 290 295 300Thr Ser Ser Asn Val Gly Gly Pro Gly Pro Gly Met Thr Glu Gln Gln305 310 315 320Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser Ala Ile Gln Gly Asn 325 330 335Val Thr Ser Ile His Ser Leu Leu Asp Asp Tyr Lys Asp Asp Asp Asp 340 345 350Lys Gly Ser Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 355 360 365Ile Met Leu Ile Pro Ile Ala Val Gly Gly Ala Leu Ala Gly Leu Val 370 375 380Leu Ile Val Leu Ile Ala Tyr Leu Ile Gly Arg Lys Arg Cys Phe Cys385 390 395 40020404PRTArtificial SequenceMHC-I polypeptide for MHC-II presentation (sorting endosome target) 20Met Leu Leu Leu Pro Phe Gln Leu Leu Ala Val Leu Phe Pro Gly Gly1 5 10 15Asn Ser Glu Asp Lys Ile Thr Gln Tyr Glu Lys Ser Leu Tyr Tyr Cys 20 25 30Ser Phe Leu Glu Ala Leu Val Arg Asp Val Cys Ile Gly Pro Gly Pro 35 40 45Gly Cys Val Asp Cys Leu Asp Arg Thr Asn Thr Ala Gln Val Met Val 50 55 60Gly Lys Cys Ala Leu Ala Tyr Gln Leu Tyr Gly Pro Gly Pro Gly Val65 70 75 80Val Lys Ala Tyr Leu Pro Val Asn Glu Ser Phe Ala Phe Thr Ala Asp 85 90 95Leu Arg Ser Asn Thr Gly Gly Gln Gly Pro Gly Pro Gly Asn Ile Leu 100 105 110Ala Val Ser Phe Ala Pro Leu Val Gln Leu Ser Lys Asn Asp Asn Gly 115 120 125Thr Pro Asp Ser Val Gly Gly Pro Gly Pro Gly Gln Ser Asn Tyr Gln 130 135 140His Ile Thr Asn Phe Glu Trp Cys Ile Ser Ile Leu Val Glu Leu Thr145 150 155 160Arg Leu Glu Gly Gly Pro Gly Pro Gly Tyr Tyr Thr Val Phe Asp Arg 165 170 175Asp Asn Asn Arg Val Ser Phe Ala Asn Ala Val Val Leu Gly Pro Gly 180 185 190Pro Gly His Ser Gly Leu Val Thr Phe Gln Ala Phe Ile Asp Val Met 195 200 205Ser Arg Glu Thr Thr Asp Thr Asp Thr Ala Asp Gly Pro Gly Pro Gly 210 215 220Leu Asp Leu Ala Ala Leu Glu Asp Val Ser Ala Asn Cys Leu Thr Glu225 230 235 240Thr Leu Glu Asp Lys Asn Glu Gly Val Gly Pro Gly Pro Gly Val Leu 245 250 255Ser Phe Val Gly Gln Thr Arg Val Leu Met Ile Asn Gly Glu Glu Val 260 265 270Glu Glu Thr Glu Leu Met Gly Gly Pro Gly Pro Gly Glu Val Ser Gly 275 280 285Leu Glu Gln Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu Trp 290 295 300Thr Ser Ser Asn Val Gly Gly Pro Gly Pro Gly Met Thr Glu Gln Gln305 310 315 320Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser Ala Ile Gln Gly Asn 325 330 335Val Thr Ser Ile His Ser Leu Leu Asp Asp Tyr Lys Asp Asp Asp Asp 340 345 350Lys Gly Ser Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 355 360 365Ile Met Leu Ile Pro Ile Ala Val Gly Gly Ala Leu Ala Gly Leu Val 370 375

380Leu Ile Val Leu Ile Ala Tyr Leu Ile Gly Lys Lys His Cys Ser Tyr385 390 395 400Gln Asp Ile Leu212712DNAArtificial SequenceMHC-II nucleic acid for MHC-II presentation (lysosomal target) 21atgctgctgc tgcccttcca gttgctggct gtcctctttc ccggcggcaa ctccgaggca 60ctgcaggcct accacctgga tcctcaatgc tggggcgtga acgtgcagcc ctactccggg 120agccctgcca atgtcgcagt ttatacagct ttggtagagc ctcatggcag aatcatgggt 180ctcgatcttc cagacggcgg gcatctcacg ggccctggtc cgggtgatac cgatgaagct 240gaggatccag agaaaatgct tgccaacttt gagtccggaa agcataagta tcgacagaca 300gccatgttta ccgccaccat gccaccagcc gtcgagagac tggctagatc ctacctgagg 360agacctgctg tggtgtacgg cccaggcccg ggcaggcggt atctgttgca gaacacagct 420ctcgaagtct tcatggcaaa tcgcacttct gttatgttca actttcctga acaagccacg 480gtgaagaaag tggtatattc ccttcccagg gttggggtcg gcacaagcta tgggctcccc 540caagcagggc caggaccagg gaagattgag cccgatatga tgtcaatgga acatagcttc 600gaaacggcat cccatgacgg agaggctggc ccctccccag aagtgttgca gggccctgga 660cctggcaagg ttctgatggc atccacttcc tacttgccct cccaggtcac tgagatgttt 720aaccagggaa gggcatttgc agcagtcaga ttgccttttt gtggacacaa aaacatctgt 780agcctgacaa caatccagaa gattccacga ctgctcgtag gacctggacc tggcatgccc 840gcagccgagt tggcactcag cgccttcctc gtcctcgtct ttttgtgggt gcattctttg 900cgccgccttc tcgaatgctt ctatgtatct gtattctcta acgccgccat ccatgttgta 960caatactgtt tcggcctcgt gtactacggt cctggccccg gactcctcga gctcctgcac 1020tgcccacttg gtcactgtca tctgtgttcc gagccgatgt tcacgtttgt gtaccccaca 1080attttcccac tcagagagac tcctatggct gggcttcacc agcggcgaac gtccatcggc 1140ttcgtggcct attgtggccc aggcccaggc gcccattttc gccttgtgtc aaaggaaaag 1200atgccgtggg acagcatcaa actgaccttc gaggcaaccg gacctaggca catgagcttc 1260tatgtgcgca cccacaaggg ttctaccctc agccagtggt cactgggtaa tggaattcca 1320gtgggccctg gaccgggagg taggacacag caaatgctca ttcccgcttg gcaacaggtt 1380acacctatgg ctccagccgc cgccacattg acattcgagg gcatggccgg atctcagagg 1440ctgggcgact ggggaaagat gattccccac agcaaccatt acaactccgt tgggccaggc 1500cctggtgaac ttcagctcgt ccagttggag ggcgggggcg gcagcgggac atatagagtc 1560gggaacgccc aaccatcact cgccgattgt ctggacgccg gggatcttgc ccagcgcctc 1620agagagcatg gcgccgaagt gcccacagaa cctaaagagg gaccaggacc cggcgagctt 1680gaaaagttcc gcaagtctga ggagggtaag caacgggccg ccgcccccag tgccgccagc 1740tcaccagcag acgtccagag cctgaagaag gccatgtcct ctctgcagaa tgacagggac 1800cggctgctca aggaactgaa gaatctgggc cccggccctg ggggcaaaca cgaccgggat 1860ctgctgattg ggaccgctaa gcacggtctg aatcgaactg attactacat catgaatgga 1920cctcagctct catttctcga cgcatacaga aactacgccc agcataaacg aactgacaca 1980caggctcctg gcagtggacc cggccctgga attctcgagg tggacaaaag cggccctatt 2040actctcttgg tgcagggaca catggaggga gaggtctggg ggctgtctac tcatccatac 2100ctgcccatat gcgccaccgt tagtgatgac aaaactctca gaatttggga cttgtccccg 2160tccggcccag gtcctggtat gctcaccgct cggcttttgc tcccaaggct gctctgtctt 2220cagggcagga ccactagtta ttcaacagcc gcagtactgc ccaatccaat cccaaacccc 2280gagatttgtt ataataagtt gtttatcaat aacgagtggc acgacgccgt cggcccgggc 2340cctggggaag ttagtggtct ggaacagctg gaatctatca ttaatttcga gaaactgaca 2400gaatggactt ccagcaacgt cgggggacca ggtcctggaa tgacagagca gcagtggaat 2460tttgccggaa ttgaggccgc tgcatctgcc atccaaggaa atgtgacatc cattcactcc 2520ctcctcgatg attacaagga cgacgacgac aagggaagtg actacaaaga ccacgacggg 2580gactacaagg accacgacat tatgctgatc cccatcgccg tgggcggggc cctggccggc 2640ctcgtgctga tcgtccttat cgcctacctc atcggcagga agaggagtca cgcaggctac 2700cagactatct ag 2712222697DNAArtificial SequenceMHC-II nucleic acid for MHC-II presentation (recycling endosome target) 22atgctgctgc tgcccttcca gttgctggct gtcctctttc ccggcggcaa ctccgaggca 60ctgcaggcct accacctgga tcctcaatgc tggggcgtga acgtgcagcc ctactccggg 120agccctgcca atgtcgcagt ttatacagct ttggtagagc ctcatggcag aatcatgggt 180ctcgatcttc cagacggcgg gcatctcacg ggccctggtc cgggtgatac cgatgaagct 240gaggatccag agaaaatgct tgccaacttt gagtccggaa agcataagta tcgacagaca 300gccatgttta ccgccaccat gccaccagcc gtcgagagac tggctagatc ctacctgagg 360agacctgctg tggtgtacgg cccaggcccg ggcaggcggt atctgttgca gaacacagct 420ctcgaagtct tcatggcaaa tcgcacttct gttatgttca actttcctga acaagccacg 480gtgaagaaag tggtatattc ccttcccagg gttggggtcg gcacaagcta tgggctcccc 540caagcagggc caggaccagg gaagattgag cccgatatga tgtcaatgga acatagcttc 600gaaacggcat cccatgacgg agaggctggc ccctccccag aagtgttgca gggccctgga 660cctggcaagg ttctgatggc atccacttcc tacttgccct cccaggtcac tgagatgttt 720aaccagggaa gggcatttgc agcagtcaga ttgccttttt gtggacacaa aaacatctgt 780agcctgacaa caatccagaa gattccacga ctgctcgtag gacctggacc tggcatgccc 840gcagccgagt tggcactcag cgccttcctc gtcctcgtct ttttgtgggt gcattctttg 900cgccgccttc tcgaatgctt ctatgtatct gtattctcta acgccgccat ccatgttgta 960caatactgtt tcggcctcgt gtactacggt cctggccccg gactcctcga gctcctgcac 1020tgcccacttg gtcactgtca tctgtgttcc gagccgatgt tcacgtttgt gtaccccaca 1080attttcccac tcagagagac tcctatggct gggcttcacc agcggcgaac gtccatcggc 1140ttcgtggcct attgtggccc aggcccaggc gcccattttc gccttgtgtc aaaggaaaag 1200atgccgtggg acagcatcaa actgaccttc gaggcaaccg gacctaggca catgagcttc 1260tatgtgcgca cccacaaggg ttctaccctc agccagtggt cactgggtaa tggaattcca 1320gtgggccctg gaccgggagg taggacacag caaatgctca ttcccgcttg gcaacaggtt 1380acacctatgg ctccagccgc cgccacattg acattcgagg gcatggccgg atctcagagg 1440ctgggcgact ggggaaagat gattccccac agcaaccatt acaactccgt tgggccaggc 1500cctggtgaac ttcagctcgt ccagttggag ggcgggggcg gcagcgggac atatagagtc 1560gggaacgccc aaccatcact cgccgattgt ctggacgccg gggatcttgc ccagcgcctc 1620agagagcatg gcgccgaagt gcccacagaa cctaaagagg gaccaggacc cggcgagctt 1680gaaaagttcc gcaagtctga ggagggtaag caacgggccg ccgcccccag tgccgccagc 1740tcaccagcag acgtccagag cctgaagaag gccatgtcct ctctgcagaa tgacagggac 1800cggctgctca aggaactgaa gaatctgggc cccggccctg ggggcaaaca cgaccgggat 1860ctgctgattg ggaccgctaa gcacggtctg aatcgaactg attactacat catgaatgga 1920cctcagctct catttctcga cgcatacaga aactacgccc agcataaacg aactgacaca 1980caggctcctg gcagtggacc cggccctgga attctcgagg tggacaaaag cggccctatt 2040actctcttgg tgcagggaca catggaggga gaggtctggg ggctgtctac tcatccatac 2100ctgcccatat gcgccaccgt tagtgatgac aaaactctca gaatttggga cttgtccccg 2160tccggcccag gtcctggtat gctcaccgct cggcttttgc tcccaaggct gctctgtctt 2220cagggcagga ccactagtta ttcaacagcc gcagtactgc ccaatccaat cccaaacccc 2280gagatttgtt ataataagtt gtttatcaat aacgagtggc acgacgccgt cggcccgggc 2340cctggggaag ttagtggtct ggaacagctg gaatctatca ttaatttcga gaaactgaca 2400gaatggactt ccagcaacgt cgggggacca ggtcctggaa tgacagagca gcagtggaat 2460tttgccggaa ttgaggccgc tgcatctgcc atccaaggaa atgtgacatc cattcactcc 2520ctcctcgatg attacaagga cgacgacgac aagggaagtg actacaaaga ccacgacggg 2580gactacaagg accacgacat tatgctgatc cccatcgccg tgggcggggc cctggccggc 2640ctcgtgctga tcgtccttat cgcctacctc atcggcagga aacgctgttt ctgttaa 2697232709DNAArtificial SequenceMHC-II nucleic acid for MHC-II presentation (sorting endosome target) 23atgctgctgc tgcccttcca gttgctggct gtcctctttc ccggcggcaa ctccgaggca 60ctgcaggcct accacctgga tcctcaatgc tggggcgtga acgtgcagcc ctactccggg 120agccctgcca atgtcgcagt ttatacagct ttggtagagc ctcatggcag aatcatgggt 180ctcgatcttc cagacggcgg gcatctcacg ggccctggtc cgggtgatac cgatgaagct 240gaggatccag agaaaatgct tgccaacttt gagtccggaa agcataagta tcgacagaca 300gccatgttta ccgccaccat gccaccagcc gtcgagagac tggctagatc ctacctgagg 360agacctgctg tggtgtacgg cccaggcccg ggcaggcggt atctgttgca gaacacagct 420ctcgaagtct tcatggcaaa tcgcacttct gttatgttca actttcctga acaagccacg 480gtgaagaaag tggtatattc ccttcccagg gttggggtcg gcacaagcta tgggctcccc 540caagcagggc caggaccagg gaagattgag cccgatatga tgtcaatgga acatagcttc 600gaaacggcat cccatgacgg agaggctggc ccctccccag aagtgttgca gggccctgga 660cctggcaagg ttctgatggc atccacttcc tacttgccct cccaggtcac tgagatgttt 720aaccagggaa gggcatttgc agcagtcaga ttgccttttt gtggacacaa aaacatctgt 780agcctgacaa caatccagaa gattccacga ctgctcgtag gacctggacc tggcatgccc 840gcagccgagt tggcactcag cgccttcctc gtcctcgtct ttttgtgggt gcattctttg 900cgccgccttc tcgaatgctt ctatgtatct gtattctcta acgccgccat ccatgttgta 960caatactgtt tcggcctcgt gtactacggt cctggccccg gactcctcga gctcctgcac 1020tgcccacttg gtcactgtca tctgtgttcc gagccgatgt tcacgtttgt gtaccccaca 1080attttcccac tcagagagac tcctatggct gggcttcacc agcggcgaac gtccatcggc 1140ttcgtggcct attgtggccc aggcccaggc gcccattttc gccttgtgtc aaaggaaaag 1200atgccgtggg acagcatcaa actgaccttc gaggcaaccg gacctaggca catgagcttc 1260tatgtgcgca cccacaaggg ttctaccctc agccagtggt cactgggtaa tggaattcca 1320gtgggccctg gaccgggagg taggacacag caaatgctca ttcccgcttg gcaacaggtt 1380acacctatgg ctccagccgc cgccacattg acattcgagg gcatggccgg atctcagagg 1440ctgggcgact ggggaaagat gattccccac agcaaccatt acaactccgt tgggccaggc 1500cctggtgaac ttcagctcgt ccagttggag ggcgggggcg gcagcgggac atatagagtc 1560gggaacgccc aaccatcact cgccgattgt ctggacgccg gggatcttgc ccagcgcctc 1620agagagcatg gcgccgaagt gcccacagaa cctaaagagg gaccaggacc cggcgagctt 1680gaaaagttcc gcaagtctga ggagggtaag caacgggccg ccgcccccag tgccgccagc 1740tcaccagcag acgtccagag cctgaagaag gccatgtcct ctctgcagaa tgacagggac 1800cggctgctca aggaactgaa gaatctgggc cccggccctg ggggcaaaca cgaccgggat 1860ctgctgattg ggaccgctaa gcacggtctg aatcgaactg attactacat catgaatgga 1920cctcagctct catttctcga cgcatacaga aactacgccc agcataaacg aactgacaca 1980caggctcctg gcagtggacc cggccctgga attctcgagg tggacaaaag cggccctatt 2040actctcttgg tgcagggaca catggaggga gaggtctggg ggctgtctac tcatccatac 2100ctgcccatat gcgccaccgt tagtgatgac aaaactctca gaatttggga cttgtccccg 2160tccggcccag gtcctggtat gctcaccgct cggcttttgc tcccaaggct gctctgtctt 2220cagggcagga ccactagtta ttcaacagcc gcagtactgc ccaatccaat cccaaacccc 2280gagatttgtt ataataagtt gtttatcaat aacgagtggc acgacgccgt cggcccgggc 2340cctggggaag ttagtggtct ggaacagctg gaatctatca ttaatttcga gaaactgaca 2400gaatggactt ccagcaacgt cgggggacca ggtcctggaa tgacagagca gcagtggaat 2460tttgccggaa ttgaggccgc tgcatctgcc atccaaggaa atgtgacatc cattcactcc 2520ctcctcgatg attacaagga cgacgacgac aagggaagtg actacaaaga ccacgacggg 2580gactacaagg accacgacat tatgctgatc cccatcgccg tgggcggggc cctggccggc 2640ctcgtgctga tcgtccttat cgcctacctc atcggcaaga agcactgctc atatcaggac 2700atcctgtga 270924903PRTArtificial SequenceMHC-II polypeptide for MHC-II presentation (lysosomal target) 24Met Leu Leu Leu Pro Phe Gln Leu Leu Ala Val Leu Phe Pro Gly Gly1 5 10 15Asn Ser Glu Ala Leu Gln Ala Tyr His Leu Asp Pro Gln Cys Trp Gly 20 25 30Val Asn Val Gln Pro Tyr Ser Gly Ser Pro Ala Asn Val Ala Val Tyr 35 40 45Thr Ala Leu Val Glu Pro His Gly Arg Ile Met Gly Leu Asp Leu Pro 50 55 60Asp Gly Gly His Leu Thr Gly Pro Gly Pro Gly Asp Thr Asp Glu Ala65 70 75 80Glu Asp Pro Glu Lys Met Leu Ala Asn Phe Glu Ser Gly Lys His Lys 85 90 95Tyr Arg Gln Thr Ala Met Phe Thr Ala Thr Met Pro Pro Ala Val Glu 100 105 110Arg Leu Ala Arg Ser Tyr Leu Arg Arg Pro Ala Val Val Tyr Gly Pro 115 120 125Gly Pro Gly Arg Arg Tyr Leu Leu Gln Asn Thr Ala Leu Glu Val Phe 130 135 140Met Ala Asn Arg Thr Ser Val Met Phe Asn Phe Pro Glu Gln Ala Thr145 150 155 160Val Lys Lys Val Val Tyr Ser Leu Pro Arg Val Gly Val Gly Thr Ser 165 170 175Tyr Gly Leu Pro Gln Ala Gly Pro Gly Pro Gly Lys Ile Glu Pro Asp 180 185 190Met Met Ser Met Glu His Ser Phe Glu Thr Ala Ser His Asp Gly Glu 195 200 205Ala Gly Pro Ser Pro Glu Val Leu Gln Gly Pro Gly Pro Gly Lys Val 210 215 220Leu Met Ala Ser Thr Ser Tyr Leu Pro Ser Gln Val Thr Glu Met Phe225 230 235 240Asn Gln Gly Arg Ala Phe Ala Ala Val Arg Leu Pro Phe Cys Gly His 245 250 255Lys Asn Ile Cys Ser Leu Thr Thr Ile Gln Lys Ile Pro Arg Leu Leu 260 265 270Val Gly Pro Gly Pro Gly Met Pro Ala Ala Glu Leu Ala Leu Ser Ala 275 280 285Phe Leu Val Leu Val Phe Leu Trp Val His Ser Leu Arg Arg Leu Leu 290 295 300Glu Cys Phe Tyr Val Ser Val Phe Ser Asn Ala Ala Ile His Val Val305 310 315 320Gln Tyr Cys Phe Gly Leu Val Tyr Tyr Gly Pro Gly Pro Gly Leu Leu 325 330 335Glu Leu Leu His Cys Pro Leu Gly His Cys His Leu Cys Ser Glu Pro 340 345 350Met Phe Thr Phe Val Tyr Pro Thr Ile Phe Pro Leu Arg Glu Thr Pro 355 360 365Met Ala Gly Leu His Gln Arg Arg Thr Ser Ile Gly Phe Val Ala Tyr 370 375 380Cys Gly Pro Gly Pro Gly Ala His Phe Arg Leu Val Ser Lys Glu Lys385 390 395 400Met Pro Trp Asp Ser Ile Lys Leu Thr Phe Glu Ala Thr Gly Pro Arg 405 410 415His Met Ser Phe Tyr Val Arg Thr His Lys Gly Ser Thr Leu Ser Gln 420 425 430Trp Ser Leu Gly Asn Gly Ile Pro Val Gly Pro Gly Pro Gly Gly Arg 435 440 445Thr Gln Gln Met Leu Ile Pro Ala Trp Gln Gln Val Thr Pro Met Ala 450 455 460Pro Ala Ala Ala Thr Leu Thr Phe Glu Gly Met Ala Gly Ser Gln Arg465 470 475 480Leu Gly Asp Trp Gly Lys Met Ile Pro His Ser Asn His Tyr Asn Ser 485 490 495Val Gly Pro Gly Pro Gly Glu Leu Gln Leu Val Gln Leu Glu Gly Gly 500 505 510Gly Gly Ser Gly Thr Tyr Arg Val Gly Asn Ala Gln Pro Ser Leu Ala 515 520 525Asp Cys Leu Asp Ala Gly Asp Leu Ala Gln Arg Leu Arg Glu His Gly 530 535 540Ala Glu Val Pro Thr Glu Pro Lys Glu Gly Pro Gly Pro Gly Glu Leu545 550 555 560Glu Lys Phe Arg Lys Ser Glu Glu Gly Lys Gln Arg Ala Ala Ala Pro 565 570 575Ser Ala Ala Ser Ser Pro Ala Asp Val Gln Ser Leu Lys Lys Ala Met 580 585 590Ser Ser Leu Gln Asn Asp Arg Asp Arg Leu Leu Lys Glu Leu Lys Asn 595 600 605Leu Gly Pro Gly Pro Gly Gly Lys His Asp Arg Asp Leu Leu Ile Gly 610 615 620Thr Ala Lys His Gly Leu Asn Arg Thr Asp Tyr Tyr Ile Met Asn Gly625 630 635 640Pro Gln Leu Ser Phe Leu Asp Ala Tyr Arg Asn Tyr Ala Gln His Lys 645 650 655Arg Thr Asp Thr Gln Ala Pro Gly Ser Gly Pro Gly Pro Gly Ile Leu 660 665 670Glu Val Asp Lys Ser Gly Pro Ile Thr Leu Leu Val Gln Gly His Met 675 680 685Glu Gly Glu Val Trp Gly Leu Ser Thr His Pro Tyr Leu Pro Ile Cys 690 695 700Ala Thr Val Ser Asp Asp Lys Thr Leu Arg Ile Trp Asp Leu Ser Pro705 710 715 720Ser Gly Pro Gly Pro Gly Met Leu Thr Ala Arg Leu Leu Leu Pro Arg 725 730 735Leu Leu Cys Leu Gln Gly Arg Thr Thr Ser Tyr Ser Thr Ala Ala Val 740 745 750Leu Pro Asn Pro Ile Pro Asn Pro Glu Ile Cys Tyr Asn Lys Leu Phe 755 760 765Ile Asn Asn Glu Trp His Asp Ala Val Gly Pro Gly Pro Gly Glu Val 770 775 780Ser Gly Leu Glu Gln Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr785 790 795 800Glu Trp Thr Ser Ser Asn Val Gly Gly Pro Gly Pro Gly Met Thr Glu 805 810 815Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser Ala Ile Gln 820 825 830Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Asp Tyr Lys Asp Asp 835 840 845Asp Asp Lys Gly Ser Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp 850 855 860His Asp Ile Met Leu Ile Pro Ile Ala Val Gly Gly Ala Leu Ala Gly865 870 875 880Leu Val Leu Ile Val Leu Ile Ala Tyr Leu Ile Gly Arg Lys Arg Ser 885 890 895His Ala Gly Tyr Gln Thr Ile 90025898PRTArtificial SequenceMHC-II polypeptide for MHC-II presentation (recycling endosome target) 25Met Leu Leu Leu Pro Phe Gln Leu Leu Ala Val Leu Phe Pro Gly Gly1 5 10 15Asn Ser Glu Ala Leu Gln Ala Tyr His Leu Asp Pro Gln Cys Trp Gly 20 25 30Val Asn Val Gln Pro Tyr Ser Gly Ser Pro Ala Asn Val Ala Val Tyr 35 40 45Thr Ala Leu Val Glu Pro His Gly Arg Ile Met Gly Leu Asp Leu Pro 50 55 60Asp Gly Gly His Leu Thr Gly Pro Gly Pro Gly Asp Thr Asp Glu Ala65 70 75 80Glu Asp Pro Glu Lys Met Leu Ala Asn Phe Glu Ser Gly Lys His Lys 85 90 95Tyr Arg Gln Thr Ala Met Phe Thr Ala Thr Met Pro Pro Ala Val Glu 100 105 110Arg Leu Ala Arg Ser Tyr Leu Arg Arg Pro Ala Val Val Tyr Gly Pro 115 120

125Gly Pro Gly Arg Arg Tyr Leu Leu Gln Asn Thr Ala Leu Glu Val Phe 130 135 140Met Ala Asn Arg Thr Ser Val Met Phe Asn Phe Pro Glu Gln Ala Thr145 150 155 160Val Lys Lys Val Val Tyr Ser Leu Pro Arg Val Gly Val Gly Thr Ser 165 170 175Tyr Gly Leu Pro Gln Ala Gly Pro Gly Pro Gly Lys Ile Glu Pro Asp 180 185 190Met Met Ser Met Glu His Ser Phe Glu Thr Ala Ser His Asp Gly Glu 195 200 205Ala Gly Pro Ser Pro Glu Val Leu Gln Gly Pro Gly Pro Gly Lys Val 210 215 220Leu Met Ala Ser Thr Ser Tyr Leu Pro Ser Gln Val Thr Glu Met Phe225 230 235 240Asn Gln Gly Arg Ala Phe Ala Ala Val Arg Leu Pro Phe Cys Gly His 245 250 255Lys Asn Ile Cys Ser Leu Thr Thr Ile Gln Lys Ile Pro Arg Leu Leu 260 265 270Val Gly Pro Gly Pro Gly Met Pro Ala Ala Glu Leu Ala Leu Ser Ala 275 280 285Phe Leu Val Leu Val Phe Leu Trp Val His Ser Leu Arg Arg Leu Leu 290 295 300Glu Cys Phe Tyr Val Ser Val Phe Ser Asn Ala Ala Ile His Val Val305 310 315 320Gln Tyr Cys Phe Gly Leu Val Tyr Tyr Gly Pro Gly Pro Gly Leu Leu 325 330 335Glu Leu Leu His Cys Pro Leu Gly His Cys His Leu Cys Ser Glu Pro 340 345 350Met Phe Thr Phe Val Tyr Pro Thr Ile Phe Pro Leu Arg Glu Thr Pro 355 360 365Met Ala Gly Leu His Gln Arg Arg Thr Ser Ile Gly Phe Val Ala Tyr 370 375 380Cys Gly Pro Gly Pro Gly Ala His Phe Arg Leu Val Ser Lys Glu Lys385 390 395 400Met Pro Trp Asp Ser Ile Lys Leu Thr Phe Glu Ala Thr Gly Pro Arg 405 410 415His Met Ser Phe Tyr Val Arg Thr His Lys Gly Ser Thr Leu Ser Gln 420 425 430Trp Ser Leu Gly Asn Gly Ile Pro Val Gly Pro Gly Pro Gly Gly Arg 435 440 445Thr Gln Gln Met Leu Ile Pro Ala Trp Gln Gln Val Thr Pro Met Ala 450 455 460Pro Ala Ala Ala Thr Leu Thr Phe Glu Gly Met Ala Gly Ser Gln Arg465 470 475 480Leu Gly Asp Trp Gly Lys Met Ile Pro His Ser Asn His Tyr Asn Ser 485 490 495Val Gly Pro Gly Pro Gly Glu Leu Gln Leu Val Gln Leu Glu Gly Gly 500 505 510Gly Gly Ser Gly Thr Tyr Arg Val Gly Asn Ala Gln Pro Ser Leu Ala 515 520 525Asp Cys Leu Asp Ala Gly Asp Leu Ala Gln Arg Leu Arg Glu His Gly 530 535 540Ala Glu Val Pro Thr Glu Pro Lys Glu Gly Pro Gly Pro Gly Glu Leu545 550 555 560Glu Lys Phe Arg Lys Ser Glu Glu Gly Lys Gln Arg Ala Ala Ala Pro 565 570 575Ser Ala Ala Ser Ser Pro Ala Asp Val Gln Ser Leu Lys Lys Ala Met 580 585 590Ser Ser Leu Gln Asn Asp Arg Asp Arg Leu Leu Lys Glu Leu Lys Asn 595 600 605Leu Gly Pro Gly Pro Gly Gly Lys His Asp Arg Asp Leu Leu Ile Gly 610 615 620Thr Ala Lys His Gly Leu Asn Arg Thr Asp Tyr Tyr Ile Met Asn Gly625 630 635 640Pro Gln Leu Ser Phe Leu Asp Ala Tyr Arg Asn Tyr Ala Gln His Lys 645 650 655Arg Thr Asp Thr Gln Ala Pro Gly Ser Gly Pro Gly Pro Gly Ile Leu 660 665 670Glu Val Asp Lys Ser Gly Pro Ile Thr Leu Leu Val Gln Gly His Met 675 680 685Glu Gly Glu Val Trp Gly Leu Ser Thr His Pro Tyr Leu Pro Ile Cys 690 695 700Ala Thr Val Ser Asp Asp Lys Thr Leu Arg Ile Trp Asp Leu Ser Pro705 710 715 720Ser Gly Pro Gly Pro Gly Met Leu Thr Ala Arg Leu Leu Leu Pro Arg 725 730 735Leu Leu Cys Leu Gln Gly Arg Thr Thr Ser Tyr Ser Thr Ala Ala Val 740 745 750Leu Pro Asn Pro Ile Pro Asn Pro Glu Ile Cys Tyr Asn Lys Leu Phe 755 760 765Ile Asn Asn Glu Trp His Asp Ala Val Gly Pro Gly Pro Gly Glu Val 770 775 780Ser Gly Leu Glu Gln Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr785 790 795 800Glu Trp Thr Ser Ser Asn Val Gly Gly Pro Gly Pro Gly Met Thr Glu 805 810 815Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser Ala Ile Gln 820 825 830Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Asp Tyr Lys Asp Asp 835 840 845Asp Asp Lys Gly Ser Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp 850 855 860His Asp Ile Met Leu Ile Pro Ile Ala Val Gly Gly Ala Leu Ala Gly865 870 875 880Leu Val Leu Ile Val Leu Ile Ala Tyr Leu Ile Gly Arg Lys Arg Cys 885 890 895Phe Cys26902PRTArtificial SequenceMHC-II polypeptide for MHC-II presentation (sorting endosome target) 26Met Leu Leu Leu Pro Phe Gln Leu Leu Ala Val Leu Phe Pro Gly Gly1 5 10 15Asn Ser Glu Ala Leu Gln Ala Tyr His Leu Asp Pro Gln Cys Trp Gly 20 25 30Val Asn Val Gln Pro Tyr Ser Gly Ser Pro Ala Asn Val Ala Val Tyr 35 40 45Thr Ala Leu Val Glu Pro His Gly Arg Ile Met Gly Leu Asp Leu Pro 50 55 60Asp Gly Gly His Leu Thr Gly Pro Gly Pro Gly Asp Thr Asp Glu Ala65 70 75 80Glu Asp Pro Glu Lys Met Leu Ala Asn Phe Glu Ser Gly Lys His Lys 85 90 95Tyr Arg Gln Thr Ala Met Phe Thr Ala Thr Met Pro Pro Ala Val Glu 100 105 110Arg Leu Ala Arg Ser Tyr Leu Arg Arg Pro Ala Val Val Tyr Gly Pro 115 120 125Gly Pro Gly Arg Arg Tyr Leu Leu Gln Asn Thr Ala Leu Glu Val Phe 130 135 140Met Ala Asn Arg Thr Ser Val Met Phe Asn Phe Pro Glu Gln Ala Thr145 150 155 160Val Lys Lys Val Val Tyr Ser Leu Pro Arg Val Gly Val Gly Thr Ser 165 170 175Tyr Gly Leu Pro Gln Ala Gly Pro Gly Pro Gly Lys Ile Glu Pro Asp 180 185 190Met Met Ser Met Glu His Ser Phe Glu Thr Ala Ser His Asp Gly Glu 195 200 205Ala Gly Pro Ser Pro Glu Val Leu Gln Gly Pro Gly Pro Gly Lys Val 210 215 220Leu Met Ala Ser Thr Ser Tyr Leu Pro Ser Gln Val Thr Glu Met Phe225 230 235 240Asn Gln Gly Arg Ala Phe Ala Ala Val Arg Leu Pro Phe Cys Gly His 245 250 255Lys Asn Ile Cys Ser Leu Thr Thr Ile Gln Lys Ile Pro Arg Leu Leu 260 265 270Val Gly Pro Gly Pro Gly Met Pro Ala Ala Glu Leu Ala Leu Ser Ala 275 280 285Phe Leu Val Leu Val Phe Leu Trp Val His Ser Leu Arg Arg Leu Leu 290 295 300Glu Cys Phe Tyr Val Ser Val Phe Ser Asn Ala Ala Ile His Val Val305 310 315 320Gln Tyr Cys Phe Gly Leu Val Tyr Tyr Gly Pro Gly Pro Gly Leu Leu 325 330 335Glu Leu Leu His Cys Pro Leu Gly His Cys His Leu Cys Ser Glu Pro 340 345 350Met Phe Thr Phe Val Tyr Pro Thr Ile Phe Pro Leu Arg Glu Thr Pro 355 360 365Met Ala Gly Leu His Gln Arg Arg Thr Ser Ile Gly Phe Val Ala Tyr 370 375 380Cys Gly Pro Gly Pro Gly Ala His Phe Arg Leu Val Ser Lys Glu Lys385 390 395 400Met Pro Trp Asp Ser Ile Lys Leu Thr Phe Glu Ala Thr Gly Pro Arg 405 410 415His Met Ser Phe Tyr Val Arg Thr His Lys Gly Ser Thr Leu Ser Gln 420 425 430Trp Ser Leu Gly Asn Gly Ile Pro Val Gly Pro Gly Pro Gly Gly Arg 435 440 445Thr Gln Gln Met Leu Ile Pro Ala Trp Gln Gln Val Thr Pro Met Ala 450 455 460Pro Ala Ala Ala Thr Leu Thr Phe Glu Gly Met Ala Gly Ser Gln Arg465 470 475 480Leu Gly Asp Trp Gly Lys Met Ile Pro His Ser Asn His Tyr Asn Ser 485 490 495Val Gly Pro Gly Pro Gly Glu Leu Gln Leu Val Gln Leu Glu Gly Gly 500 505 510Gly Gly Ser Gly Thr Tyr Arg Val Gly Asn Ala Gln Pro Ser Leu Ala 515 520 525Asp Cys Leu Asp Ala Gly Asp Leu Ala Gln Arg Leu Arg Glu His Gly 530 535 540Ala Glu Val Pro Thr Glu Pro Lys Glu Gly Pro Gly Pro Gly Glu Leu545 550 555 560Glu Lys Phe Arg Lys Ser Glu Glu Gly Lys Gln Arg Ala Ala Ala Pro 565 570 575Ser Ala Ala Ser Ser Pro Ala Asp Val Gln Ser Leu Lys Lys Ala Met 580 585 590Ser Ser Leu Gln Asn Asp Arg Asp Arg Leu Leu Lys Glu Leu Lys Asn 595 600 605Leu Gly Pro Gly Pro Gly Gly Lys His Asp Arg Asp Leu Leu Ile Gly 610 615 620Thr Ala Lys His Gly Leu Asn Arg Thr Asp Tyr Tyr Ile Met Asn Gly625 630 635 640Pro Gln Leu Ser Phe Leu Asp Ala Tyr Arg Asn Tyr Ala Gln His Lys 645 650 655Arg Thr Asp Thr Gln Ala Pro Gly Ser Gly Pro Gly Pro Gly Ile Leu 660 665 670Glu Val Asp Lys Ser Gly Pro Ile Thr Leu Leu Val Gln Gly His Met 675 680 685Glu Gly Glu Val Trp Gly Leu Ser Thr His Pro Tyr Leu Pro Ile Cys 690 695 700Ala Thr Val Ser Asp Asp Lys Thr Leu Arg Ile Trp Asp Leu Ser Pro705 710 715 720Ser Gly Pro Gly Pro Gly Met Leu Thr Ala Arg Leu Leu Leu Pro Arg 725 730 735Leu Leu Cys Leu Gln Gly Arg Thr Thr Ser Tyr Ser Thr Ala Ala Val 740 745 750Leu Pro Asn Pro Ile Pro Asn Pro Glu Ile Cys Tyr Asn Lys Leu Phe 755 760 765Ile Asn Asn Glu Trp His Asp Ala Val Gly Pro Gly Pro Gly Glu Val 770 775 780Ser Gly Leu Glu Gln Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr785 790 795 800Glu Trp Thr Ser Ser Asn Val Gly Gly Pro Gly Pro Gly Met Thr Glu 805 810 815Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser Ala Ile Gln 820 825 830Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Asp Tyr Lys Asp Asp 835 840 845Asp Asp Lys Gly Ser Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp 850 855 860His Asp Ile Met Leu Ile Pro Ile Ala Val Gly Gly Ala Leu Ala Gly865 870 875 880Leu Val Leu Ile Val Leu Ile Ala Tyr Leu Ile Gly Lys Lys His Cys 885 890 895Ser Tyr Gln Asp Ile Leu 900

Claims

1-123. (canceled)

124. A composition comprising: (a) an immunostimulatory cytokine; (b) a means for macrophage suppression; and (c) an adenovirus, wherein the adenovirus comprises a nucleic acid encoding a heterologous antigen.

125. The composition of claim 124, wherein the means for macrophage suppression is RP 182.

126. The composition of claim 125, wherein the immunostimulatory cytokine is ALT-803.

127. The composition of claim 126, wherein the adenovirus comprises an E1 gene region deletion.

128. The composition of claim 127, wherein the adenovirus comprises an E2b gene region deletion.

129. The composition of claim 128, wherein the heterologous antigen is a neoepitope.

130. The composition of claim 128, wherein the adenovirus further comprises a sequence that encodes a co-stimulatory molecule selected from the group consisting of CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, OX40L, 4-1BBL, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1, and LFA3.

131. The composition of claim 128, wherein the adenovirus further comprises a sequence that encodes a protein that interferes with checkpoint inhibition, and wherein the protein that interferes is an antagonist of CTLA-4, PD-1, TIM1 receptor, 2B4, or CD160.

132. A composition comprising: (d) a first adenovirus, wherein the first adenovirus comprises a nucleic acid encoding an immunostimulatory cytokine; (e) a means for macrophage suppression; and (f) a second adenovirus, wherein the second adenovirus comprises a nucleic acid encoding a heterologous antigen.

133. The composition of claim 132, wherein the means for macrophage suppression is RP 182.

134. The composition of claim 133, wherein the immunostimulatory cytokine is ALT-803.

135. The composition of claim 134, wherein the first and second adenoviruses each comprise an E1 gene region deletion.

136. The composition of claim 135, wherein the first and second adenoviruses each comprise an E2b gene region deletion.

137. The composition of claim 136, wherein the heterologous antigen is a neoepitope.

138. The composition of claim 136, wherein the second adenovirus further comprises a sequence that encodes a co-stimulatory molecule selected from the group consisting of CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, OX40L, 4-1BBL, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1, and LFA3.

139. The composition of claim 136, wherein the second adenovirus further comprises a sequence that encodes a protein that interferes with checkpoint inhibition, and wherein the protein that interferes is an antagonist of CTLA-4, PD-1, TIM1 receptor, 2B4, or CD160.

140. A composition comprising: (g) a first adenovirus, wherein the first adenovirus comprises a nucleic acid encoding an immunostimulatory cytokine; (h) a second adenovirus, wherein the second adenovirus comprises a nucleic acid encoding a means for macrophage suppression; and (i) a third adenovirus, wherein the second adenovirus comprises a nucleic acid encoding a heterologous antigen.

141. The composition of claim 140, wherein the means for macrophage suppression is RP 182.

142. The composition of claim 141, wherein the immunostimulatory cytokine is ALT-803.

143. The composition of claim 142, wherein the first, second, and third adenoviruses each comprise an E1 gene region deletion and an E2b gene region deletion.