Methods and compounds for neoantigen vaccines

Abstract

The present disclosure includes compounds and methods for treating a subject with a disease, such as cancer. The treatment method includes administering to the patient a therapeutically effective amount of one or more peptides corresponding to tumor neoantigens or administering to the patient a therapeutically effective amount of one or more oligonucleotides, each having a nucleic acid sequence encoding a peptide corresponding to a tumor neoantigen. Tumor neoantigens can be identified from patient-specific tumor mutations in the patient's tumor cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 166,697, filed March 26, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] Reference to Electronically Submitted Sequence Listing An official copy of the Sequence Listing has been submitted electronically via EFS-Web as an ASCII formatted sequence listing with the file name "91482-255WO-PCT_Sequence_Listing.txt" created on March 25, 2022 and having a size of 7,379 bytes, and is filed concurrently herewith. The Sequence Listing contained in this ASCII formatted document is a part of the present specification and is incorporated herein by reference in its entirety.

[0003] The present invention relates to novel proteins and methods for their preparation and use as therapeutic or prophylactic agents, for example for treating cancer. [Background technology]

[0004] Cancer is a group of diseases involving abnormal cell growth that can spread to various parts of the body. Hundreds of different types of cancer affect humans, with millions diagnosed and millions more diagnosed each year. The most common types of cancer include lung, breast, prostate, and colorectal cancer, among others. Treatments for cancer include surgery, radiation therapy, chemotherapy, immunotherapy, hormonal therapy, and stem cell replacement. Treatment options are invasive and can be associated with a variety of unwanted side effects.

[0005] Thus, although the scientific community has made advances in this field, there remains a need in the art for improved compounds and methods for the prevention and treatment of cancer. Summary of the Invention

[0006] There is a need for therapeutic strategies for the treatment of diseases, including cancer. Therapeutic cancer vaccines have been developed to actively drive antitumor immune responses. Unlike prophylactic vaccines, which are used prophylactically to treat infectious diseases, therapeutic vaccines are designed to treat established cancers by stimulating immune responses against specific tumor-associated antigens. The advantage of active immunotherapy is that it has the potential to provide long-lasting anticancer activity by engaging both the innate and adaptive arms of the immune response. Neoantigens are major histocompatibility complex (MHC)-presented peptides of novel sequences formed by somatic mutations and represent particularly promising immunotherapy targets because they appear exclusively on tumors, causing minimal off-target effects.

[0007] The present invention provides a method for treating cancer in a patient. The treatment method can include administering to the patient a therapeutically effective amount of one or more peptides corresponding to tumor neoantigens or administering to the patient a therapeutically effective amount of one or more oligonucleotides, each having a nucleic acid sequence encoding a peptide corresponding to a tumor neoantigen. Neoantigens can be identified from patient-specific tumor mutations in the patient's tumor cells.

[0008] According to various embodiments, the method can include: (a) obtaining tumor cells from a tumor resected from a patient; (b) detecting a plurality of patient-specific tumor mutations in the tumor cells by genomic analysis of tumor DNA and / or RNA and normal DNA and / or RNA from the patient; (c) identifying neoantigens derived from somatic mutations that exhibit specific binding to a human leukocyte antigen (HLA) protein or fragment thereof corresponding to the patient's genotype; and (d) administering to the patient a therapeutically effective amount of a peptide corresponding to the tumor neoantigen ("neoantigen peptide"). An adjuvant may be administered to the patient simultaneously with the neoantigen peptide.

[0009] According to other embodiments, the method may include: (a) obtaining tumor cells from a tumor resected from a patient; (b) detecting a plurality of patient-specific tumor mutations in the tumor cells by genomic analysis of tumor DNA and / or RNA and normal DNA and / or RNA from the patient; (c) identifying neoantigens derived from somatic mutations that exhibit specific binding to a human leukocyte antigen (HLA) protein or a fragment thereof corresponding to the patient's genotype; (d) designing a peptide based on the neoantigen; (e) generating a nucleic acid sequence encoding the peptide; and (f) administering to the patient a therapeutically effective amount of an oligonucleotide having a nucleic acid sequence encoding the peptide.

[0010] In certain embodiments, detecting multiple patient-specific tumor mutations can include genomic profiling by next-generation sequencing of a panel of targeted genes. In one embodiment, genomic profiling includes whole genome profiling, whole exome profiling, and / or transcriptome profiling.

[0011] In other embodiments, genomic analysis can include identifying multiple patient-specific tumor mutations in expressed genes by nucleic acid sequencing of tumor and normal samples from the patient, where the mutations are present in the genome of the patient's cancer cells but not present in normal cells from the subject.

[0012] In yet other embodiments, the plurality of patient-specific tumor mutations can include point mutations, splice site mutations, frameshift mutations, read-through mutations, gene fusion mutations, insertions, deletions, or combinations thereof; the plurality of patient-specific tumor mutations encodes at least one mutant polypeptide having a tumor-specific neoepitope that binds to an HLA protein or a fragment thereof with greater affinity than the wild-type polypeptide.

[0013] In some embodiments, the method may further comprise identifying the patient's MHC class 1 and 2 genotype. In one embodiment, identifying the patient's MHC class 1 and 2 genotype comprises analysis of whole exome sequencing (WES) and / or RNA sequencing from tumor and / or normal tissue.

[0014] In certain embodiments, identifying neoantigens can include (i) providing a library of peptide constructs, each peptide construct of the library comprising a peptide portion and an identifying nucleic acid portion that identifies the peptide portion, wherein at least one peptide portion of the peptide construct is capable of specifically binding to an HLA protein or a fragment thereof; (ii) contacting the HLA protein or a fragment thereof with the library of peptide constructs; (iii) separating at least one peptide construct comprising a peptide portion capable of specifically binding to an HLA protein or a fragment thereof from peptide constructs comprising a peptide portion that is not capable of specifically binding to an HLA protein or a fragment thereof; and (iv) sequencing all or a portion of the identifying nucleic acid portion of at least one peptide construct capable of specifically binding to an HLA protein or a fragment thereof.

[0015] In some embodiments, the library of peptide constructs can include variant peptides designed from the analysis of multiple patient-specific tumor mutations that predict the effect of each mutation on the corresponding protein and exclude silent mutations and mutations in non-coding regions. In one embodiment, the variant peptides include mutations that are predicted to affect the structure of the corresponding protein.

[0016] In other embodiments, the step of identifying neoantigens can include (i) generating a genetically encoded combinatorial library of polypeptides by phage display, ribosome display, mRNA display, bicistronic DNA display, P2A DNA display, CIS display, yeast display, or bacterial display, where the combinatorial library comprises polypeptides linked to corresponding nucleic acid molecules encoding the polypeptides; (ii) contacting the combinatorial library with an HLA protein or fragment thereof; (iii) isolating an HLA protein or fragment thereof that exhibits specific binding to the combinatorial library; and (iv) sequencing all or a portion of the nucleic acid molecules of the combinatorial library bound to the HLA protein or fragment thereof to identify neoantigens.

[0017] In one embodiment, a combinatorial library of polypeptides can include variant peptides designed from the analysis of multiple patient-specific tumor mutations that predict the effect of each mutation on the corresponding protein and exclude silent mutations and mutations in non-coding regions.

[0018] In certain embodiments, specific binding between a neoantigen and an HLA protein or fragment thereof is achieved by binding to at least one nucleic acid molecule comprising: (i) an MHC class II component comprising at least a portion of an MHC class II α chain and at least a portion of an MHC class II β chain, such that the MHC class II α chain and the MHC class II β chain form a peptide-binding groove; and a nucleotide sequence encoding a space holder molecule and a first processable linker. (ii) culturing a cell transformed with an MHC class II component, wherein the space holder molecule is linked to an MHC class II component by a processable linker, such that the space holder molecule binds in the peptide binding groove thereby preventing binding of any other peptides in the peptide binding groove; the culturing is performed to produce an MHC class II component; (ii) recovering the MHC class II component; (iii) releasing the space holder molecule from the peptide binding groove by processing the processable linker; (iv) incubating the MHC class II component in the presence of a neoantigen, wherein the incubation promotes binding of the neoantigen to the peptide binding groove; and (v) recovering the MHC class II component bound to the neoantigen.

[0019] In one embodiment, the spacer holder molecule can have the consensus sequence AAXAAAAAAAXAA (SEQ ID NO: 30). In another embodiment, the spacer holder molecule is selected from the group consisting of PVSKMRMATPLLMQA (SEQ ID NO: 25); AAMAAAAAAAMAA (SEQ ID NO: 26); AAMAAAAAAAAAA (SEQ ID NO: 27); AAFAAAAAAAAAA (SEQ ID NO: 28); and ASMSAASAASMAA (SEQ ID NO: 29).

[0020] In some embodiments, the processable linker is linked to the MHC class II α chain of the MHC class II component. In other embodiments, the step of recovering the neoantigen-bound MHC class II component comprises affinity chromatography with an antibody that recognizes the MHC class II component.

[0021] In certain embodiments, specific binding between a neoantigen and an HLA protein or fragment thereof is determined by phage display, where the HLA protein or fragment thereof is expressed on the surface of a phage, and the neoantigen is incubated with the phage to assay for specific binding.

[0022] In one embodiment, the method further comprises an in silico analysis to determine specific binding between the neoantigen and an MHC class I protein or a fragment thereof, the in silico analysis comprising using a computational algorithm to predict relative binding to the MHC I protein based on the peptide sequence of the neoantigen.

[0023] In another aspect, the present invention relates to a method of treating cancer in a patient, comprising obtaining tumor cells from a tumor resected from the patient; detecting a plurality of patient-specific tumor mutations in the tumor cells by genomic analysis of tumor DNA and / or RNA and normal DNA and / or RNA from the patient; identifying neoantigens derived from somatic mutations that exhibit specific binding to a human leukocyte antigen (HLA) protein or fragment thereof corresponding to the patient's genotype; generating a messenger ribonucleic acid (mRNA) polynucleotide comprising an open reading frame encoding one or more peptides based on the neoantigens; and administering a therapeutically effective amount of the mRNA polynucleotide to the patient.

[0024] In some embodiments, the mRNA polynucleotide comprises at least one chemical modification. In one embodiment, the chemical modification is selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine. In another embodiment, the chemical modification is at carbon 5 of uracil. In another embodiment, the chemical modification is N1-methylpseudouridine or N1-ethylpseudouridine.

[0025] In certain embodiments, at least 80% of the uracils in the open reading frame have a chemical modification. In other embodiments, the mRNA polynucleotide further encodes a 5' end cap. In one embodiment, the 5' end cap is 7mG(5')ppp(5')NlmpNp.

[0026] In other embodiments, the disclosed methods further comprise administering a therapeutically effective amount of a therapeutic population of tumor infiltrating lymphocytes (TILs). In some embodiments, the therapeutic population of TILs is administered simultaneously or sequentially with the peptide or mRNA polynucleotide. The therapeutic population of TILs is activated and / or educated by at least one neoantigen presented by the peptide or encoded by the mRNA polynucleotide.

[0027] In some embodiments, administering a peptide or mRNA polynucleotide with a therapeutic population of TILs enhances the immunogenic response and / or anti-tumor activity in the patient. In one embodiment, the increase in immunogenic response and / or anti-tumor activity in the patient is synergistic.

[0028] The foregoing features and elements are not exclusive and may be combined in various combinations unless expressly stated otherwise. These features and elements and their operation will become more apparent in light of the following description. It should be understood, however, that the following description is intended to be illustrative and non-limiting in nature.

[0029] The subject matter of the present disclosure is particularly pointed out and distinctly claimed in the concluding portion of this specification. A more complete understanding of the present disclosure, however, can be best obtained by reference to the detailed description and claims when considered in conjunction with the drawings, in which like numerals may refer to like elements. [Brief description of the drawings]

[0030] [Figure 1] We demonstrate how genomic tools can be used to generate personalized neoantigen vaccines. [Figure 2-1] An overview of approaches for the development of neoantigen peptide-based vaccines and methods of treating patients using neoantigen peptide-based vaccines is provided. [Figure 2-2] An overview of approaches for the development of neoantigen peptide-based vaccines and methods of treating patients using neoantigen peptide-based vaccines is provided. [Diagram 3] A step-by-step workflow is shown for the efficient synthesis and analysis of large libraries of DNA-barcoded peptides; the peptide-DNA library shown exhibits puromycin ("Puro") adapters that facilitate ligation of DNA to the encoded peptides. [Figure 4] Analysis of tumor exomes to identify candidate neoantigens, characterization of candidate neoantigens by MHC-peptide-DNA conjugate assays, and evaluation of T cell responses to neoantigens bound to HLA proteins in MHC-peptide-DNA conjugate assays are shown. [Diagram 5] 1 shows DNA conjugate assay library design for analysis of mutations identified from analysis of tumor exomes. [Figure 6] An embodiment of an MHC-peptide-DNA conjugate assay used to identify neoantigens that bind to MHC class II molecules is shown; in the control group ("non-cleaved"), the CLIP peptide is not removed from the HLA construct and candidate neoantigens bind non-specifically, whereas in the test group ("cleaved"), removal of the CLIP peptide opens up the recognition site in the HLA construct, allowing specific binding of the candidate neoantigens. [Figure 7] Identification of enriched peptides (i.e., neoantigens) from two human patients (i.e., TG0006 and TG00013) is shown; the number of mutations identified in each patient by exome analysis is shown under each patient ID; the human leukocyte antigen serotype of the HLA complex used in the binding assay is identified in the upper left corner of each graph. Enriched peptides are shown as larger data points. [Figure 8] Figures 8A and 8B show additional data sets identifying enriched peptides (i.e., neoantigens) from human patient TG00013; enriched peptides are displayed as data points at the top of each graph corresponding to peptides with log mean % reads of cleaved HLA complexes close to 0 (i.e., binding to cleaved HLA complexes close to 100%). [Figure 9] The amino acid sequences of the enriched peptides, the corresponding mutations of each peptide, and the human leukocyte antigen serotype of the HLA complex to which the peptides specifically bind for human patient TG00013 are shown. [Figure 10]A comparison of MHC-peptide-DNA conjugate assays to in silico predictions to identify neoantigens is shown; the IEDB consensus sequences used in the in silico analysis are obtained from the Immune Epitope Database and Analysis Resource (IEDB), a public resource maintained by the National Institute of Allergy and Infectious Diseases (NIAID). [Figure 11] We present information on several human patients in which tumor-infiltrating lymphocytes (TILs) were harvested and evaluated by generating peptides to activate and educate these TILs to enhance their recognition of neoantigens and antitumor activity. [Figure 12] 1 shows a non-limiting example of peptide vaccine production according to the disclosed methods. [Figure 13-1] Figure 1 shows the infiltration of tumor cell organoids by peripheral blood mononuclear cells (PBMCs) derived from tumor-bearing patients. PBMCs were administered alone or with DMSO (negative control), in combination with IL-2, or in combination with IL-2 and a peptide identified as a neoantigen. Images were taken 24 hours after subjecting the organoids to the various treatments. As shown in the bar graph, after only 24 hours, PBMCs combined with IL-2 and peptides showed significant infiltration of the organoids. [Figure 13-2] Figure 1 shows the infiltration of tumor cell organoids by peripheral blood mononuclear cells (PBMCs) derived from tumor-bearing patients. PBMCs were administered alone or with DMSO (negative control), in combination with IL-2, or in combination with IL-2 and a peptide identified as a neoantigen. Images were taken 24 hours after subjecting the organoids to the various treatments. As shown in the bar graph, after only 24 hours, PBMCs combined with IL-2 and peptides showed significant infiltration of the organoids. [Figure 13-3]Figure 1 shows the infiltration of tumor cell organoids by peripheral blood mononuclear cells (PBMCs) derived from tumor-bearing patients. PBMCs were administered alone or with DMSO (negative control), in combination with IL-2, or in combination with IL-2 and a peptide identified as a neoantigen. Images were taken 24 hours after subjecting the organoids to the various treatments. As shown in the bar graph, after only 24 hours, PBMCs combined with IL-2 and peptides showed significant infiltration of the organoids. [Figure 14] Figure 1 shows studies to test the safety and efficacy of the disclosed peptide vaccines. ORR: objective response rate; DOR: duration of any observed response; PFS: progression-free survival; OS: overall survival. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Unless specifically stated otherwise, it is to be understood that references to "a," "an," and / or "the" may include one or more than one, and references to a singular item may also include a plurality of items. Reference to an element by the indefinite articles "a," "an," and / or "the" does not exclude the possibility that more than one element is present, unless the context clearly requires that one and only one element is present. As used herein, the term "comprise," as well as conjugations or any other variations thereof, is used in an open-ended sense meaning that the items following the word are included but do not exclude items not specifically mentioned.

[0032] "Neoepitope" is understood in the art to refer to an epitope that appears or develops in a subject following exposure to a particular event or the occurrence of a particular event (e.g., the onset or progression of a particular disease, disorder, or condition, e.g., infection, cancer, cancer stage, etc.). As used herein, a neoepitope is one whose presence and / or level correlates with exposure to the event or the occurrence of the event. In some embodiments, a neoepitope is one that triggers an immune response against cells that express it (e.g., at a reasonable level). In some embodiments, a neoepitope is one that triggers an immune response that kills or otherwise destroys cells that express it (e.g., at a reasonable level). In some embodiments, the relevant event that triggers the neoepitope is or includes a somatic mutation in a cell. In some embodiments, the neoepitope was not expressed in non-cancer cells to a level and / or in a manner that triggers and / or supports an immune response (e.g., an immune response sufficient to target cancer cells that express the neoepitope). In some embodiments, the neoepitope is a neoantigen.

[0033] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0034] The term "peptide construct" as used herein refers to a peptide of any length attached to an identification oligonucleotide. The attachment may be via an intervening linker, and the attachment may be covalent or non-covalent. The identification oligonucleotide may be a message that is translated to form the peptide portion of the construct, or any other sequence that is known and can be used to identify the attached peptide by sequencing. A "peptide construct set" refers to a pool of peptide constructs generated from a custom-designed set of oligonucleotides. The set may contain as little as one copy per species of peptide construct, but typically contains many copies of each peptide construct.

[0035] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, and those that are later modified. Non-natural amino acids are not encoded by the genetic code, and can, but do not necessarily, have the same basic structure as naturally occurring amino acids. "Amino acid analog" refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha carbon attached to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs can have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimetics" refers to a compound that has a structure that is different from the general chemical structure of an amino acid, but functions similarly to a naturally occurring amino acid.

[0036] Amino acids can be represented either by the three letter symbols recommended by IUPAC or by the one-letter symbols, the IUAPC letter code being: G=glycine; A=alanine; L=leucine; M=methionine; F=phenylalanine; W=tryptophan; K=lysine; Q=glutamine; E=glutamic acid; S=serine; P=proline; V=valine; I=isoleucine; C=cysteine; Y=tyrosine; H=histidine; R=arginine; N=asparagine; D=aspartic acid; T=threonine.

[0037] The terms "homologous" and "similar" refer to the relationship between proteins that have a "common evolutionary origin", including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species. Such proteins (and the genes that encode them) have sequence homology as reflected by their sequence similarity, whether in terms of percent similarity or in terms of the presence of specific residues or motifs as conserved positions. In certain embodiments, two peptide sequences are "substantially homologous or similar" if at least about 80%, or at least about 90%, or at least about 95% of the amino acids match over a defined length of the amino acid sequence.

[0038] The term "variant" applies to both amino acid and nucleic acid sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for any given protein. For example, the codons GCA, GCC, GCG and GCU all code for the amino acid alanine. Variants can include individual substitutions, deletions or additions to nucleic acid, peptide, polypeptide or protein sequences that change, add or delete a single amino acid or a small percentage of amino acids in the coded sequence.

[0039] A "functionally conservative variant" is a variant in which a given amino acid residue in a protein or enzyme is altered without changing the overall conformation and function of the polypeptide, including but not limited to replacing the amino acid with one having similar properties (e.g., polarity, hydrogen bond potential, acidic, basic, hydrophobic, aromatic, etc.). Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic basic amino acids and can be interchangeable. Similarly, the hydrophobic amino acid isoleucine can be replaced with leucine, methionine or valine. Such alterations are expected to have little or no effect on the apparent molecular weight or isoelectric point of the protein or polypeptide.

[0040] Since amino acids other than those designated as conserved may vary within a protein, the percent similarity of protein or amino acid sequence between any two proteins of similar function may vary, for example, from 70% to 99% as determined according to an alignment scheme. "Variant" also includes polypeptides having at least 60% amino acid identity, preferably at least 75%, most preferably at least 85%, even more preferably at least 90%, and even more preferably at least 95% amino acid identity as determined by BLAST or FASTA algorithms, and having the same or substantially similar properties or functions as the native or parent protein to which it is compared. Certain variants are "gain-of-function" variants, meaning polypeptide variants in which the modification of at least one given amino acid residue in a protein or enzyme improves a particular function of the polypeptide, including but not limited to the activity of the protein. The modification of the amino acid residue may be the replacement of an amino acid with one having similar properties.

[0041] The term "binding" as used herein refers to an attractive interaction between two molecules that results in a stable association of the molecules close to each other. Molecular binding can be classified into the following types: non-covalent, reversible covalent, and irreversible covalent. Molecules that can participate in molecular binding include proteins, nucleic acids, carbohydrates, lipids, and small organic molecules, such as pharmaceutical compounds. For example, proteins that form stable complexes with other molecules are often referred to as receptors, while their binding partners are called ligands. Nucleic acids can also form stable complexes with themselves or other nucleic acids, such as DNA-protein complexes, DNA-DNA complexes, DNA-RNA complexes, and protein-protein complexes.

[0042] As used herein, the term "specific binding" refers to the specificity of a binder, e.g., a protein or antibody, to preferentially bind to a target, e.g., a polypeptide antigen, receptor, or antibody. When referring to a binding partner, e.g., a protein, nucleic acid, antibody, or other affinity capture agent, "specific binding" can include a binding reaction of two or more binding partners with high affinity and / or complementarity to ensure selective hybridization under the specified assay conditions. Typically, specific binding will be at least three times the standard deviation of the background signal. Thus, under the specified conditions, the binding partner binds to its specific target molecule and does not bind in any significant amount to other molecules present in the sample. The recognition by the binder or antibody of a specific target in the presence of other potential interfering substances is what is unique about such binding. Preferably, a binder, antibody or antibody fragment, peptide, or fusion peptide that is specific to or specifically binds to a target binds to the target with a higher affinity than it binds to other non-target substances. Also, preferably, the binder, antibody or antibody fragment, peptide, or fusion peptide that is specific for or specifically binds to a target avoids binding to non-target substances, such as a significant percentage of non-target substances present in a test sample. The binding affinity of an antibody to a target antigen, antigenic fragment, peptide, or fusion peptide that contains a cognate epitope can be easily determined using any of a number of methods available in the art, including, but not limited to, enzyme-linked immunosorbent assay (ELISA). In some embodiments, the binder, antibody or antibody fragment, peptide, or fusion peptide of the present disclosure avoids binding to more than about 90% of non-target substances, although higher percentages are clearly contemplated and preferred. For example, the binder, antibody, or antibody fragment, or peptide of the present disclosure avoids binding to about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, and about 99% or more of non-target substances.In other embodiments, the binders, antibodies, or antibody fragments, or peptides of the present disclosure avoid binding to more than about 10%, 20%, 30%, 40%, 50%, 60%, or 70%, or more than about 75%, or more than about 80%, or more than about 85% of non-target material.

[0043] The terms "target", "target molecule" and "targeting agent" are used interchangeably herein and refer to a protein, toxin, enzyme, pathogen, cell, or biomarker that is incubated with a library to identify peptides that exhibit specific binding to the target. A target or marker can be any molecular structure that is produced by a cell, expressed within a cell, accessible on the cell surface, or secreted by a cell. A marker can be any protein, carbohydrate, fat, nucleic acid, catalytic site, or any target of these, such as an enzyme, glycoprotein, cell membrane, virus, cell, organ, organelle, or any mono- or multi-molecular structure, or any other such structure, whether alone or in combination, currently known or yet to be disclosed. A target may also be called a marker, and the terms are used interchangeably. A target can be represented by a sequence of amino acids, or the sequence of one or more strands of nucleic acid from which it can be derived. For example, a target can be represented by a protein sequence. Alternatively, a target can be represented by a nucleic acid sequence, a protein or peptide encoded by a nucleic acid sequence, or a fragment thereof.

[0044] Examples of such nucleic acids include both single-stranded and double-stranded nucleic acid sequences, including miRNA, tRNA, siRNA, mRNA, cDNA, or genomic DNA sequences that contain complementary sequences. The concept of a marker is not limited to the exact nucleic acid sequence or protein sequence product that it can represent. Rather, a marker encompasses all molecules that can be detected by methods that evaluate the expression of the marker. Examples of molecules encompassed by a marker include point mutations, silent mutations, deletions, frameshift mutations, translocations, alternative splicing derivatives, differentially methylated sequences, differentially modified protein sequences, truncations, soluble forms of cell membrane-associated markers, and any other mutations that result in a product that can be identified as a marker. The term "target" further encompasses the product (i.e., protein) of a gene or its gene alleles whose expression or activity is directly or indirectly related to a particular phenotype or cell state, or physiological characteristic.

[0045] The terms "capture agent" and "capture group" as used herein refer to any moiety that allows the capture of a target molecule or peptide construct through binding or linkage to an affinity group or domain on the target molecule or an affinity tag of the peptide construct. The bond between the capture agent and its affinity tag can be covalent and / or non-covalent. Capture agents include, for example, members of a binding pair that selectively bind to an affinity tag on a fusion peptide, chemical bonds added by recombinant techniques or other mechanisms, cofactors for enzymes, and the like. Capture agents can be associated with peptide constructs using conventional techniques, including hybridization, cross-linking (e.g., covalent immobilization with furocoumarins such as psoralens), ligation, attachment via chemically reactive groups, introduction by post-translational modification, and the like.

[0046] "Sequence determination", "sequencing" and the like include determining information about the nucleotide base sequence of a nucleic acid. Such information may include identification or determination of partial sequence information and complete sequence information of the nucleic acid. Sequence information may be determined with various degrees of statistical certainty or reliability. In one embodiment, the term includes the determination of the identity and order of multiple consecutive nucleotides in a nucleic acid. "High-throughput sequencing" or "next-generation sequencing" includes the determination of sequences using methods that essentially determine many (typically thousands to billions) of nucleic acid sequences in parallel, i.e., DNA templates are prepared for sequencing in a bulk process rather than one at a time, and many sequences are read out, preferably in parallel, or using an ultra-high throughput serial process that may itself be parallelized. Such methods include, but are not limited to, pyrosequencing (e.g., as commercialized by 454 Life Sciences, Inc., Branford, CT); sequencing by ligation (e.g., SOLiD™ technology, as commercialized by Life Technologies, Inc., Carlsbad, CA); sequencing by synthesis using modified nucleotides (e.g., TruSeq™ and HiSeq™ technologies by Illumina, San Diego, CA; HeliScope™ by Helicos Biosciences Corporation, Cambridge, MA; and PacBio RS by Pacific Biosciences of California, Inc., Menlo Park, CA), sequencing by ion detection technology (e.g., Ion Torrent™ technology, Life Technologies, Carlsbad, CA); DNA nanoball sequencing (Complete Genomics, Inc., Mountain View, CA); nanopore-based sequencing technology (e.g., Oxford Nanopore Technologies, LTD, Oxford, UK), and such highly parallel sequencing methods.

[0047] The term "exome" is used according to its art-understood meaning to refer to the set of exon sequences found within a particular genome. The term "mutation" as used herein refers to a permanent change in the DNA sequence that constitutes a gene. In some embodiments, mutations range in size from a single DNA building block (DNA base) to large segments of a chromosome. In some embodiments, mutations can include missense mutations, frameshift mutations, duplications, insertions, nonsense mutations, deletions, and repeat expansions. In some embodiments, a missense mutation is a change in a single DNA base pair that results in the replacement of one amino acid with another in the protein produced by the gene. In some embodiments, a nonsense mutation is also a change in a single DNA base pair. However, instead of replacing one amino acid with another, the altered DNA sequence signals the cell to stop building the protein prematurely. In some embodiments, an insertion changes the number of DNA bases in a gene by adding a piece of DNA. In some embodiments, a deletion changes the number of DNA bases by removing a piece of DNA. In some embodiments, a small deletion may remove one or a few base pairs in a gene, while a larger deletion may remove the entire gene or several adjacent genes. In some embodiments, a duplication consists of a piece of DNA that is abnormally copied one or more times. In some embodiments, a frameshift mutation occurs when the addition or loss of a DNA base changes the reading frame of a gene. A reading frame consists of a group of three bases, each of which codes for one amino acid. In some embodiments, a frameshift mutation shifts the grouping of these bases to change the code for the amino acid. In some embodiments, an insertion, deletion, and duplication can all be frameshift mutations. In some embodiments, a repeat expansion is another type of mutation. In some embodiments, a nucleotide repeat is a short DNA sequence that is repeated many times in a row. For example, a trinucleotide repeat is composed of a three base pair sequence, and a tetranucleotide repeat is composed of a four base pair sequence. In some embodiments, a repeat expansion is a mutation that increases the number of times a short DNA sequence is repeated.

[0048] As used herein, "small molecule" refers to a molecule less than 5 kilodaltons, more typically less than 1 kilodalton. As used herein, "small molecule" includes peptides. "Affinity tag" is given its ordinary meaning in the art. An affinity tag is any biological or chemical material that can be easily attached to a target biological or chemical material. An affinity tag may be attached to a target biological or chemical molecule by any suitable method. For example, in some embodiments, an affinity tag may be attached to a target molecule using genetic methods. For example, a nucleic acid sequence encoding an affinity tag may be inserted near a sequence encoding a biological molecule; the sequence may be located anywhere within, adjacent to, or near the nucleic acid, such as within, adjacent to, or near, to allow the affinity tag to be expressed with the biological molecule. In other embodiments, an affinity tag may also be attached to a target biological or chemical molecule after the molecule is produced (e.g., expressed or synthesized). As an example, an affinity tag such as biotin may be chemically linked, e.g., covalently, to a target protein or peptide to facilitate binding of the target to streptavidin.

[0049] Affinity tags include, for example, metal binding tags, such as histidine tags, GST (in glutathione / GST binding), streptavidin (in biotin / streptavidin binding). Other affinity tags include Myc or Max in Myc / Max pair, or polyamino acids, such as polyhistidine. At various places in this specification, specific affinity tags are described in conjunction with binding interactions. The molecule with which the affinity tag interacts (i.e. binds), which may be a known biological or chemical binding partner, is the "recognition entity". It should be understood that the present invention, in any embodiment using affinity tags, encompasses a series of individual embodiments, each of which encompasses any of the selections of affinity tags described herein.

[0050] A "recognition entity" can be any chemical or biological material that can bind to an affinity tag. Recognition entities can be, for example, small molecules such as maltose (which binds to MBP, or maltose binding protein), glutathione, NTA / Ni 2+ The affinity tag / recognition entity interaction may be, for example, polyhistidine / NTA / Ni, biotin (which can bind to streptavidin), or an antibody. The affinity tag / recognition entity interaction may facilitate attachment of the target molecule, for example, to another biological or chemical material, or to a substrate (e.g., a nitrocellulose membrane or other immobilized substrate). An example of an affinity tag / recognition entity interaction is polyhistidine / NTA / Ni 2+ , glutathione S-transferase / glutathione, maltose binding protein / maltose, streptavidin / biotin, biotin / streptavidin, antigen (or antigen fragment) / antibody (or antibody fragment), and the like.

[0051] The term "ribosome display" refers to a reaction system that can produce a ternary complex of mRNA, ribosomes, and the corresponding protein of interest. Ribosome display can be used to screen cell surface receptors, antibodies, and their fragments for target antigen or ligand binding. The steps of making the reaction system can include: 1) generating a DNA library and transcribing the library into an RNA library; 2) purifying the RNA and translating it in vitro by a cell-free protein synthesis system; 3) binding the ribosome complex of the translation reaction to the target antigen or ligand; 4) selecting the bound ribosome complex; and 5) isolating the RNA from the complex and reverse transcribing the transcript into cDNA, which can be amplified, sequenced, and / or further modified.

[0052] In some embodiments, the pharmaceutical composition comprises one or more peptides or oligonucleotides described herein together with a pharma- ceutically acceptable carrier, diluent or excipient. In preparing pharmaceutical compositions comprising peptides or oligonucleotides described in the teachings herein, various vehicles, vectors, excipients, and routes of administration may be used. Pharmaceutical compositions generally comprise a pharma- ceutically acceptable carrier and a pharmacologically (or therapeutically) effective amount of a peptide or oligonucleotide. In various embodiments, pharmaceutical compositions comprising one or more peptides may comprise an adjuvant or other agent for inducing or enhancing the immune response of a patient. In other embodiments, pharmaceutical compositions comprising one or more oligonucleotides may be delivered without an adjuvant.

[0053] The pharmaceutical compositions described herein can be administered by any means that allows the active agent to reach the site of action of the agent in the subject's body.The dose administered varies depending on factors such as pharmacological characteristics; mode and route of administration; age, health and weight of recipient subject; nature and degree of symptoms; concurrent treatment; and frequency of treatment.

[0054] As used herein, the terms "administration" and "administering" of an agent to a subject include any route of introducing or delivering an agent to a subject to perform its intended function. Administration can be performed by any suitable route, including intravenous, intramuscular, intraperitoneal, inhalation, intranasal, or subcutaneous. Administration includes self-administration and administration by another person.

[0055] The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent or combination of agents described herein sufficient to affect the intended application, including but not limited to disease treatment. The therapeutically effective amount may vary depending on the intended application (in vitro or in vivo), the subject and disease state to be treated (e.g., the subject's weight, age, and sex), the severity of the disease state, or the mode of administration. The term also applies to a dose that will induce a specific response in target cells. The specific dose will vary depending on the particular agent selected, the subsequent administration regimen, whether the agent is administered in combination with other agents, the timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is delivered.

[0056] The terms "treatment", "treating", "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, with respect to completely or partially preventing the disease or its symptoms, and / or may be therapeutic, with respect to a partial or complete cure for the disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses any treatment of disease in a mammal, particularly in a human, including: (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., arresting its onset or progression; and (c) relieving the disease, i.e., causing regression of the disease and / or relieving one or more disease symptoms. "Treatment" is also meant to encompass the delivery of an agent that provides a pharmacological effect even in the absence of disease or symptoms. For example, "treatment" encompasses the delivery of a composition that can induce an immune response or confer immunity in the absence of a disease state, e.g., in the case of a vaccine.

[0057] The term "patient" or "subject" as used herein refers to any living organism to which the provided compositions are administered or may be administered, for example, for experimental, diagnostic, preventive, cosmetic, and / or therapeutic purposes. For example, the subject can refer to a human or a non-human animal. In some aspects, the subject refers to any vertebrate, including, but not limited to, humans and other primates (e.g., chimpanzees and other ape and monkey species), livestock (e.g., cows, sheep, pigs, goats, and horses), domestic mammals (e.g., dogs and cats), laboratory animals (e.g., rodents such as mice, rats, and guinea pigs), and birds (e.g., domestic, wild, and game birds such as chickens, turkeys, and other poultry birds, ducks, geese, etc.). In some embodiments, the subject is a mammal. In further embodiments, the subject is a human.

[0058] In some embodiments, the patient suffers from or is susceptible to one or more disorders or conditions. In some embodiments, the patient shows one or more symptoms of a disease, disorder or condition. In some embodiments, the patient has been diagnosed with one or more diseases, disorders or conditions. In some embodiments, the disorder or condition is or includes cancer, or the presence of one or more tumors.

[0059] Detection of tumor mutations and / or neoepitopes Cancers may be screened to detect mutations and / or neoepitopes described herein (e.g., to detect neoantigen identity and / or neoepitope nature, level and / or frequency) using any of a variety of known techniques. In some embodiments, a particular mutation or neoepitope, or its expression, is detected at the nucleic acid level (e.g., in DNA or RNA). One of skill in the art will appreciate that a mutation or neoepitope, or its expression, may be detected in a sample containing DNA or RNA from a cancer cell. Furthermore, one of skill in the art will appreciate that a sample containing DNA or RNA from a cancer cell may include, but is not limited to, circulating tumor DNA (ctDNA), cell-free DNA (cfDNA), cells, tissues, or organs. In some embodiments, a mutation or neoepitope, or its expression, is detected at the protein level (e.g., in a sample containing a polypeptide from a cancer cell (which sample may be or may include a polypeptide complex or other higher order structure, including, but not limited to, a cell, tissue, or organ)).

[0060] In some specific embodiments, detection involves nucleic acid sequencing. In some embodiments, detection involves whole exome sequencing. In some embodiments, detection involves immunoassays. In some embodiments, detection involves the use of microarrays. In some embodiments, detection involves massively parallel exome sequencing. In some embodiments, detection involves genome sequencing. In some embodiments, detection involves RNA sequencing. In some embodiments, detection involves standard DNA or RNA sequencing. In some embodiments, detection involves mass spectrometry.

[0061] In some embodiments, detection includes next-generation sequencing (DNA and / or RNA). In some embodiments, detection includes genome sequencing, genome resequencing, targeted sequencing panels, transcriptome profiling (RNA-Seq), DNA-protein interaction (ChIP-sequencing), and / or epigenomic characterization. In some embodiments, resequencing of the patient's genome may be utilized, for example, to detect genomic mutations.

[0062] In some embodiments, detection involves using techniques such as ELISA, Western blotting, immunoassays, mass spectrometry, microarray analysis, and the like. In some embodiments, detection involves next generation sequencing (DNA and / or RNA). In some embodiments, detection involves next generation sequencing of targeted gene panels (e.g., ASHION® Tumor / Normal Exome-RNA Test (GEMEXTRA®, MSK-IMPACT, or FOUNDATIONONE®). In some embodiments, detection involves genomic profiling.

[0063] In some embodiments, detection includes genomic profiling using the GEMEXTRA® test. The GEMEXTRA® test is a comprehensive exome and transcriptome profiling assay, both of which provide information for cancer patient care and enable future research of the disease. Over 19,000 genes are assayed via hybridization capture and clinical depth sequencing for the identification of somatic point mutations, small and large insertions and deletions, and structural rearrangements. Microsatellite instability (MSI) and tumor mutation burden (TMB) are also measured to provide information for the application of immuno-oncology therapy. To ensure the somatic origin of the identified variants, both germline and tumor exomes are sequenced and compared. The entire transcriptome is also sequenced to allow detection of gene fusions and alternative splicing events from the patient's RNA. GEMEXTRA® is applicable to both solid and hematological cancers.

[0064] In some embodiments, detecting comprises genomic profiling using Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) (Cheng DT, Mitchell TN, Zehir A, et al. Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT): A Hybridization Capture-Based Next-Generation Sequencing Clinical Assay for Solid Tumor Molecular Oncology. J Mol Diagn. 2015;17(3):251-264; and Ross DS, Zehir A, Cheng DT, et al. Next-Generation Assessment of Human Epidermal Growth Factor Receptor 2 (ERBB2) Amplification Status: Clinical Validation in the Context of a Hybrid Capture-Based, Comprehensive Solid Tumor Genomic Profiling Assay. J Mol Diagn. 2015;17(3):251-264). Diagn. 2017;19(2):244-254). MSK-IMPACT is a comprehensive molecular profiling assay that encompasses hybridization capture and deep sequencing of all exons and selected introns of multiple oncogenes and tumor suppressor genes, allowing detection of point mutations, small and large insertions or deletions, and rearrangements. MSK-IMPACT also captures intergenic and intronic single nucleotide polymorphisms (e.g., tiling probes) that are interspersed throughout the genome and aid in accurate assessment of genome-wide copy number. In some embodiments, the probes may target megabases.

[0065] In some embodiments, detection includes genomic profiling using FOUNDATIONONE® CDX™ ("FlCDx") assay. FlCDx assay is a next-generation sequencing-based in vitro diagnostic device for the detection of genomic signatures including substitution, insertion, and deletion alterations (indels) and copy number alterations (CNAs) in 324 genes and selected gene rearrangements, as well as microsatellite instability (MSI) and tumor mutation burden (TMB) using DNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor tissue specimens. FlCDx has been approved by the US Food and Drug Administration (FDA) for several tumor indications, including NSCLC, melanoma, breast cancer, colorectal cancer, and ovarian cancer.

[0066] The FlCDx assay uses a single DNA extraction method from routine FFPE biopsy or surgical resection specimens (50-1000ng of which will undergo whole-genome shotgun library construction and hybridization-based capture of all coding exons from 309 cancer-related genes), one promoter region, one non-coding (ncRNA), and selected intronic regions from 34 commonly rearranged genes (21 of which also contain coding exons). In total, the assay detects alterations in a total of 324 genes. Using the ILLUMINA® HiSeq4000 platform, hybrid capture selected libraries are sequenced to high uniform depth (targeting a median coverage of >500× and >99% of exons at coverage of >100×). The sequence data is then processed using a customized analysis pipeline designed to detect all classes of genomic alterations, including base substitutions, indels, copy number alterations (amplifications and homozygous gene deletions), and selected genomic rearrangements (e.g. gene fusions).In addition, genomic signatures are reported, including microsatellite instability (MSI) and tumor mutation burden (TMB).

[0067] In some embodiments, detection may include sequencing of exon and / or intron sequences from at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more genes (e.g., oncogenes and / or tumor suppressor genes). For example, literature reports indicate that deep sequencing of all exons and selected introns of 468 oncogenes and tumor suppressor genes has been achieved using MSK-IMPACT.

[0068] Alternatively, or in addition, in some embodiments, detection may include sequencing of intergenic and / or intronic single nucleotide polymorphisms. For example, literature reports indicate that deep sequencing of >1000 intergenic and intronic single nucleotide polymorphisms has been achieved using MSK-IMPACT.

[0069] In some embodiments, detection comprises sequencing of splice variants.Cancer cells can adapt and evolve by developing mechanisms to escape regulation by microenvironment.Therefore, the diversity and plasticity provided by alternative splicing provides the opportunity for cancer cells to generate protein isoforms suitable for tumor growth and / or spread (David CJ and Manley J L. Genes Dev. 2010;24(21):2343-2364). Genome-wide approaches have revealed that large-scale alternative splicing occurs during tumorigenesis (Venables JP et al. Nat Struct Mol Biol. 2009; 16(6):670-676), and genomic portraits of alternative splicing patterns have proven useful for tumor classification (Venables J P. Bioessays. 2006; 28(4):378-386; Skoteim RI et al. Int J Biochem Cell Biol. 2007; 39(7-8):1432-1449; Omenn GS et al. Dis Markers. 2010; 28(4):241-251).

[0070] Reports of aberrant splicing events and changes in the ratio of alternatively spliced ​​transcripts in different cancers have been noted (Rajan P et al., Nat Rev Urol. 2009; 6(8): 454-460). These events result in novel transcripts not observed in normal cellular counterparts. Nearly all areas of tumor biology have been reported to be affected by alternative splicing, including metabolism, apoptosis, cell cycle control, invasion, metastasis, and angiogenesis (Venables J P., Bioessays. 2006; 28(4): 378-386; Ghigna C et al., Curr Genomics. 2008; 9(8): 556-570).

[0071] One of the earliest examples of alternative splice variants with opposing apoptotic effects is BCL-x. BCL-x pre-mRNA can be alternatively spliced ​​to generate two splice variants, the anti-apoptotic Bcl-xL (long form) and the pro-apoptotic Bcl-xS (short form) (Boise LH et al., Cell. 1993;74(4):597-608). A high Bc1-xL / Bc1-xS ratio that promotes tumor cell survival can be found in several cancer types, including human lymphoma, breast cancer, and human hepatocellular carcinoma (Minn AJ et al., J Biol Chem. 1996; 271(11):6306-6312.44-46; Olopade OI et al., Cancer J Sci Am. 1997; 3(4):230-237; Takehara T et al., Hepatology. 2001; 34(1):55-61). Another example of an apoptosis-related gene that undergoes alternative splicing in cancer cells is the Fas receptor gene.

[0072] The Fas receptor, expressed on the cell surface of many cell types, is activated by Fas ligand produced by cytotoxic T cells, which initiates a death signaling cascade leading to apoptosis of cells expressing the Fas receptor (Bouillet P et al., Nat Rev Immunol. 2009; 9(7):514-519). There are at least three short mRNA variants of Fas that shed the encoded transmembrane domain, and the resulting translated protein variants are presumably secreted by cancer cells to serve as decoy receptors for Fas ligand, thus allowing the cancer cells to escape apoptosis (Cheng J et al., Science. 1994; 263(5154):1759-1762; Cascino I et al., Journal of immunology. 1995; 154(6):2706-2713).

[0073] Alternative splicing of the H-Ras oncogene occurs within a previously unknown spliced ​​exon (designated IDX) caused by an intronic mutation within the H-Ras gene (Cohen JB et al., Cell. 1989;58(3):461-472). This mutation at the IDX splice site results in an H-Ras mRNA variant that is more resistant to the nonsense-mediated mRNA decay (NMD) process and is consequently overexpressed in cancer (Barbier J et al., Mol Cell Biol. 2007;27(20):7315-7333). Alternative splicing also plays a role in promoting invasive and metastatic behavior in cancer. CD44 was one of the first genes whose splice variants were specifically associated with metastasis, and variants containing exons 4-7 and 6-7 (v4-7 and v6-7, respectively) were shown to be expressed in metastatic pancreatic cancer cell lines but not in the corresponding parental tumors (Gunthert U et al. Cell. 1991;65(1):13-24).

[0074] Mutation calling software tools In certain embodiments, the Pegasus Human Research Pipeline at TGen. https: / / github.com / tgen / pegasusPipe is used. Sequences are aligned to the HG19 or HG38 human genome. In one embodiment, somatic mutations are called using three separate mutation callers: Seurat, Mutect, and Strelka. All mutations found in more than one of the callers were selected and used to generate peptides. The rationale is that each caller has its own strengths and weaknesses. By indexing mutations called by multiple callers, the risk of identifying false positives is reduced. The callers are available at Mutect: https: / / software.broadinstitute.org / cancer / cga / mutect (Broad Institute Software); Seurat: https: / / github.com / tgen / seurat (TGen Caller); and Strelka: https: / / github.com / Illumina / strelka (GNU General Public License). Best practices are followed for DNA and RNA workflows, as well as CLIA-certified somatic cell calls that are unaltered to generate peptides as candidate neoantigens for further analysis.

[0075] In some embodiments, a single or multiple mutation callers may be utilized to call genomic variants. Callers that may be used for sequencing tumor and normal tissue samples include: Strelka, Strelka2, VarDict, VarScan2, qSNP, Shimmer, RADIA, SOAPsnv, SomaticSniper, FaSD-somatic, Samtools, JointSNVMix, Virmid, SNVSniffer, Seurat, CaVEMan, MuTect, MuTect2, LoFreq, EBCall, deepSNV, LoLoPicker, MuSE , MutationSeq, SomaticSeq, SnooPer, FreeBayes, HapMuC, SPLINTER, Pisces, DeepSNVMiner, smCounter, DeepVariant, Cake, Tnscope, DNAscope, NeoMutate, Maftools, scABA, Sanivar, Sarek, BATCAVE, SomaticNet, CoVaCS, RegTools, xAtlas, R2D2, SiNPle, Bambino, and exactSNP. Sequencing callers that may be used for tumor-only samples include: Platypus, LumosVar, LumosVar2, SNVMix2, SNVer, OutLyzer, ISOWN, SomVarIUS, SiNVICT, FreeBayes, SNPiR, eSNV-dectect, RNAIndel, VaDir, and Clairvoyante.

[0076] In certain embodiments, a single or multiple mutation callers that recognize splice variants may be utilized to call genomic variants. Callers that may be used for sequencing tumor and normal tissue samples include: RegTools, ASGAL, MATS (rMATS), SUPPA (SUPPA2), Leafcutter, MAJIQ, JunctionSeq, findAS, Cufflinks / Cuffdiff, IsoformSwitchAnalyzeR, ALEXA-seq, MISO, SplicingCompass, Flux Capacitor, JuncBASE, SpliceR, FineSplice, ARH-seq, Spladder, DEXSeq, edgeR, Limma, DiffSplice, dSpliceType, SpliceDetector, HISAT2, STAR, Subread, Subjunct, PennDiff, DSGseq, AltAnalyze, Splicing Express, SpliceTrap, PSGInfer, FDM, IsoEM2, MADS+, Spanki, SpliceMap, CASPER, AVISPA, PASA, SpliceSeq, MATT, Aspli, IPSA, SANJUAN, VAST-Tools, SpliceV, Asprofile, DESeq2, Yanagi, ABLas, IRIS, and AS-Quant.

[0077] HLA typing In some embodiments, whole exome sequencing (WES) and / or RNA sequencing from tumor and / or normal tissue may be used to identify MHC class 1 and 2 genotypes for individual patients.

[0078] Single or multiple HLA callers can be used to identify the HLA type. HLA callers that can be used for DNA sequencing include: BWAkit, PHLAT, OptiType, HLAMiner, HISAT, HISAT2, xHLA, HLA-Vbseq, GATK HLA Caller, PolySolver, HLAReporter, Athlates, HLAseq, HLAssign, SOAP-HLA, STC-SEq, HapLogic, Gyper, HLAtyphon, HLA-LA, HLA-PRG, MHC-PRG, Prohlatype, GraphTyper, ALPHLARD, Omixon, SNP2HLA, HLAscan, NeoEpitopePred, and Assign. HLA callers that can be used for RNA sequencing include: BWAkit, seq2HLA, PHLAT, HLAProfiler, OptiType, HLAMiner, HLAForest, arcasHLA, HISAT, HISAT2, HLAseq, HLAssign, STC-Seq, HLATyphon, ALPHLARD, Omixon, and HLAscan.

[0079] In some embodiments, whole exome sequencing data may be analyzed for HLA typing using Polysolver (Shukla et al., 2015). Polysolver is an algorithm for inferring alleles of the three major MHC class I (HLA-A, HLA-B, HLA-C) genes. For example, a patient's HLA class I alleles may be inferred from whole exome sequencing data using Polysolver. In other variations, in the absence of whole exome sequencing data, a patient's HLA class I alleles may be inferred from transcriptome sequencing data by alternative HLA typing algorithms.

[0080] In one embodiment, HLA typing is performed in silico using one or more techniques, such as OptiType, run on a computing device. See Szolek et al., OptiType: precision HLA typing from next-generation sequencing data, Bioinformatics. 2014-12-01; 30(23), incorporated herein in its entirety for all purposes. Various other in silico techniques may also be used. See Major et al., HLA typing from 1000 genomes whole genome and whole exome Ilumina data, PLoS One. 2013-11-06; 8(11):e78410; Wittig et al., Development of a high-resolution NGS-based HLA-typing and analysis pipeline, Nucl. Acids Res. (2015).

[0081] Generation of peptides from tumor mutations In some embodiments, mutation data is obtained as described above in the section entitled "Detection of Tumor Mutations and / or Neoepitopes". Mutation calls may be converted to mutant protein sequences using a single or multiple tools, including but not limited to Varcode, customProDB, and pyGeno. The tools will generate peptide sequences for downstream in vitro and in silico HLA binding assays. In addition, in some embodiments, peptides will be tested to ensure that each peptide is unique to the human proteome. Peptides may be aligned to several different protein databases from NCBI, including non-redundant protein sequences, reference proteins, model organisms, UniProtKB / Swiss-Prot, patented protein sequences, protein data bank proteins, metagenomic proteins, and transcriptome shotgun assembly proteins. Using the databases listed above, rBlast may be utilized to run protein BLAST to identify proteins with 100% identity to the human proteome.

[0082] In certain embodiments, Varcode software is utilized in python to predict the impact of genome variant data.Varcode generates wild type and mutant protein sequences from mutations and / or neoepitopes with desired peptide size.In certain embodiments, the desired peptide size is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids.In one embodiment, the desired peptide size is 15 amino acids.

[0083] Varcode dependency is Pyensembl, which is a python access point to Ensembl reference genome data for generating peptides. In some embodiments, Varcode predicts the effect of each mutation on the protein. Silent mutations and mutations in non-coding regions on the genome are excluded. Varcode is then used to generate mutations throughout the peptide sequence. The variant peptides (i.e., candidate neoantigens) containing mutations generated by Varcode are further analyzed by in silico methods and / or biochemical assays (e.g., peptide-DNA conjugate assays) to identify which peptides bind to patient-specific MHC class I and II proteins, which are ultimately recognized by the patient's tumor infiltrating lymphocytes (TILs).

[0084] Analysis of neoantigens by peptide-DNA conjugate approach The peptide-DNA conjugate platform is suitable for rapid screening of large populations of diverse molecules for candidate neoantigens. In some embodiments, the present invention provides the following strategies for identifying neoantigens. According to various embodiments, the peptide-DNA conjugate method includes a method for pooled, highly parallel expression of proteins, each associated with a nucleic acid barcode. In some embodiments, the peptide-DNA conjugate method includes a method for pooled, highly parallel in vitro expression of proteins, each covalently linked to a DNA barcode via a puromycin-containing linkage. In other embodiments, the peptide-DNA conjugate method includes phage display, mRNA display, or other methods.

[0085] The peptide-DNA conjugate approach is a unique technology that can rapidly generate large numbers of potential neoantigens as candidate binders for patient-specific HLA proteins. The peptide-DNA conjugate approach is a method to generate peptide libraries (10-50 amino acids long) in which each peptide is conjugated to a unique DNA tag that can be used to monitor peptide abundance after binding experiments. Importantly, the peptide sequences can be used in large multiplexed binding assays with at least 100,000 unique programmable peptides.

[0086] Binding assays for any molecular target can be used to screen large libraries of diverse peptide-DNA conjugates. Biological structures (e.g., patient-specific HLA proteins or fragments thereof corresponding to the patient's genotype) can be mixed with diverse peptides, separated from non-binding peptides, and then queried for "sticky" ones. A particular library can be an intelligently designed library based on prior knowledge of the target (e.g., from exome sequencing and analysis).

[0087] The peptide-DNA conjugate platform is described in more detail in U.S. Pat. No. 9,958,454; U.S. Pat. No. 10,288,608; and U.S. Patent Application Publication No. 2016 / 0025726, the contents of which are incorporated herein by reference.

[0088] In some embodiments, the peptide-DNA conjugate method is used to identify neoantigens. In certain aspects, the identification of neoantigens includes the steps of: (i) providing a library of peptide constructs, each peptide construct of the library comprising a peptide portion and an identifying nucleic acid portion that identifies the peptide portion, and at least one peptide portion of the peptide construct is capable of specifically binding to an HLA protein or a fragment thereof; (ii) contacting the HLA protein or a fragment thereof with the library of peptide constructs; (iii) separating at least one peptide construct comprising a peptide portion that is capable of specifically binding to an HLA protein or a fragment thereof from a peptide construct comprising a peptide portion that is not capable of specifically binding to an HLA protein or a fragment thereof; and (iv) sequencing all or a portion of the identifying nucleic acid portion of at least one peptide construct that is capable of specifically binding to an HLA protein or a fragment thereof. In some aspects, the identifying nucleic acid portion of each peptide construct comprises a polynucleotide sequence that encodes the peptide portion of the peptide construct, or a complement thereof.

[0089] Identification of neoantigens by phage display, ribosome display, mRNA display, yeast display, bacterial display, and related technologies In some embodiments, the polypeptide of interest is genetically encoded to facilitate identification of neoantigens. An example of a genetically encoded polypeptide library is an mRNA display library. Another example is a replicable genetic display package (rgdp) library, such as a phage display library. In one embodiment, the polypeptide of interest is genetically encoded as a phage display library. In these embodiments, the nucleic acid may be comprised by a phage genome. In these embodiments, the polypeptide may be comprised by a phage coat.

[0090] In some embodiments, the present invention may be used to generate a genetically encoded combinatorial library of polypeptides generated by translating several nucleic acids into corresponding polypeptides and linking molecules of the molecular scaffold to the polypeptides. The genetically encoded combinatorial library of polypeptides can be generated by phage display, yeast display, ribosome display, bacterial display, or mRNA display.

[0091] Phage display Techniques and methodologies for performing phage display can be found in WO2009 / 098450.

[0092] Various phage display systems have been developed over the years, utilizing different phage vectors (M13 filamentous phage, lambda, T4, and T7 phage) and different phage coat proteins for covalent fusion. M13 filamentous phage is the most commonly used. The advantage of filamentous phage display systems is the lysogenic life cycle of this phage and the availability of M13 phagemid vectors (Webster, 1006; Hufton et al., 1999). Lysogenic phages integrate their DNA into the genome of the host cell, are replicated along with the bacterial cell, and do not require bacterial cell lysis to form phage particles. Instead, phage particles are shed from the bacterial surface without inducing cell death (Webster, 1996). Furthermore, the development of M13 phagemid vectors allows for excellent workability. Phagemids are plasmids that contain the origin of replication and packaging signals of a filamentous phage together with a gene encoding a phage coat protein coupled with a plasmid replication origin and a DNA insert (Webster, 1996; Armstrong et al., 1996). Phage propagation requires that the phagemid-infected bacterial cell be "superinfected" with a so-called helper phage, which provides all other phage components essential for the formation of viable phage virions. In addition to excellent workability, the use of phagemid vector systems allows monovalent display of recombinant proteins (up to one recombinant protein per phage virion), since the helper phage contributes non-recombinant phage coat proteins (Armstrong et al., 1996). Various M13 vector systems are available for phage display via different coat proteins (Smith and Petrenko, 1997; Barbas, 1993). The major coat protein pVIII and the minor coat protein pIII are most frequently used for display purposes (Armstrong et al., 1996; Rodi and Makowski, 1999). The N-termini of both proteins are exposed on the phage surface, so that foreign DNA sequences are inserted upstream of the genes encoding the coat proteins.The development of phage vectors for C-terminal fusion to the M13 minor coat protein pVI, whose C-terminus is exposed on the surface of the phage, has been an important step toward the development of cDNA phage display libraries ( Hufton et al., 1999 ; Jespers et al., 1995 ).

[0093] More recently, display methods for lytic phage systems, namely lambda, T4 and T7 phages, have also been developed. For the formation and shedding of recombinant lytic phage virions containing recombinant coat proteins, bacterial cells need to be lysed during phage propagation (Russel, 1991). Furthermore, there are no plasmid vectors available for lytic phages, making DNA isolation and experimental approaches more laborious compared to working with plasmids (Sambrook et al., 1989). As the life cycles of both lytic and lysogenic phages employ different phage assembly strategies, both approaches allow the display of different proteins (Hufton et al., 1999). Because the virion proteins of M13 filamentous phage (and therefore also recombinant phage coat proteins) are embedded in the bacterial membrane prior to phage virion assembly, this process imposes constraints on the proteins that can be displayed on the surface of the phage; for efficient display, the cDNA products must be able to cross the bacterial membrane, allowing the formation of viable infectious virions (Webster, 1996; Russell, 1991; Rodi et al., 2002). On the other hand, in virion production of lytic phage, recombinant proteins are formed and retained within the cytosol of the bacterial cell prior to and during virion assembly, so the spectrum of recombinant proteins that can be displayed by lytic phage is less constrained (Hufton et al., 1999; Russell, 1991; Krumpe et al., 2006).

[0094] Phage display has been described, for example, in Ladner et al., U.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; WO 92 / 18619; WO 91 / 17271; WO 92 / 20791; WO 92 / 15679; WO 93 / 01288; WO 92 / 01047; WO 92 / 09690; WO 90 / 02809; de Haard et al. (1999) J. Biol. Chem 274:18218-30; Hoogenboom et al. (1998) Immunotechnology 4:1-20; Hoogenboom et al. (2000) Immunol Today 2:371-8; Fuchs et al. (1991) Bio / Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) Proc Natl Acad Sci USA 89:3576-3580; Garrard et al. (1991) Bio / Technology 9:1373-1377; Rebar et al. (1996) Methods Enzymol. 267:129-49; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982.

[0095] Ribosome display In certain embodiments, the methods described herein include the use of ribosome display to identify neoantigens. Ribosome display can be used to perform in vitro protein synthesis of a protein of interest (e.g., a candidate neoantigen). Ribosome display can also be used to perform in vitro protein evolution to generate proteins with desired properties, e.g., Fab fragments that bind to a specific target molecule. Ribosome display is a well-known technique useful for generating libraries. This entirely in vitro method has been shown to be extremely useful in the past. 16 This allows for libraries with a diversity of members. The process produces translated proteins associated with RNA precursors that can be used as complexes to select proteins with desired properties (e.g., bind to immobilized target molecules). Subsequently, RNA-protein complexes that exhibit desired properties, e.g., high binding affinity, can be reverse transcribed into cDNA and the sequences amplified via PCR. This process also provides for repeated cycles of repeated or recursive protein expression. In addition, nucleic acid mutations can be efficiently introduced into the selected nucleic acid library in subsequent cycles, leading to successive DNA diversification and thus protein evolution. The end result is a nucleic acid sequence that can be used to generate a protein of interest (e.g., a patient-specific HLA protein, and ultimately a neoantigen that specifically binds to the patient's TILs).

[0096] In the method provided herein, a protein of interest is displayed on the surface of a ribosome where the protein of interest will be translated. Briefly, a library of RNA molecules is translated in an in vitro translation system to the 3' end of the RNA molecule so that the ribosome does not fall off. This is achieved by not incorporating a functional stop codon into the RNA template. Usually, the stop codon is recognized by a release factor that triggers the release of the nascent polypeptide from the ribosome. In ribosome display, the peptide emerges from the ribosome but does not fall off freely from the complex. This allows, for example, the nascent polypeptide to associate with another polypeptide to form a functional dimer. In some cases, there is an additional folding step in an oxidizing environment (important for forming disulfide bonds).

[0097] The entire complex of the folded protein of interest, ribosomes, and RNA, which is stable for several days, can then be screened for the presence or absence of desired properties, such as specific binding by the translated protein of interest to a binding counter ligand. The RNA encoding the selected protein of interest can be reverse transcribed into single-stranded cDNA, which can be converted into double-stranded DNA and amplified by PCR to provide the coding sequence of the selected protein (e.g., neoantigen). The reverse transcription reaction can be performed on the mRNA that is associated within the ribosome display complex, or the mRNA can be isolated from the ribosome display complex and then used in the reverse transcription step. Suitable methods for disrupting / dissociating the ribosome complex are known in the art and include EDTA treatment and / or phenol extraction.

[0098] Generally, a nucleic acid (DNA) construct for ribosome display contains a promoter (T7, SP6, or T3), a translation initiation signal, e.g., a Shine-Dalgarno (prokaryotes) or Kozak (eukaryotes) sequence, a start codon, and a coding sequence for a protein of interest (e.g., the V H or V LThe nucleic acid sequence of the present invention contains a nucleic acid sequence encoding one or more detection tags (e.g., a histidine tag). One or more nucleic acid sequences encoding one or more detection tags may be included to provide for the production of a protein further comprising one or more detection tags (e.g., a histidine tag). To allow the complete nascent protein to be displayed and folded into its active conformation, a spacer domain of at least 23-30 amino acids in length can be added to the C-terminus to allow the protein to fully exit the ribosome. The spacer can provide a known sequence for the design of primers for RT-PCR recovery of the DNA sequence.

[0099] To remove the stop codon from DNA, a 3' primer lacking the stop codon can be used during PCR construction. The construct designed for bacterial-based display system can incorporate sequences containing stem-loop structures at the 5' and 3' ends of DNA to stabilize mRNA against degradation by RNase activity in bacterial cell-free system.

[0100] The mRNA translation system used in the methods described herein may be any suitable available system. Prokaryotic or eukaryotic translation systems may be used, for example, E. coli crude lysate (e.g., commercially available from Promega Corp., Madison, Wis.; Agilent Technologies, Santa Clara, Calif.; GE Healthcare Biosciences, Pittsburgh, Pa.; Life Technologies, Carlsbad, Calif.), reconstituted ribosome systems, such as PURE (see, for example, Shimizu et al., Nat. Biotechnol., 19:751-755 (2001)), or cell-free protein synthesis systems as described below.

[0101] The PURE system can include approximately 32 individually purified components used in in vitro protein biosynthesis (e.g., initiation, elongation, and termination). In some embodiments, the components include initiation factors (e.g., IF1, IF2, IF3), elongation factors (e.g., EF-G, EF-Tu, EF-Ts), release factors (e.g., RF1, RF3), termination factors (e.g., RRF), 20 aminoacyl-tRNA synthetases, methionyl-tRNA transformylase, T7 RNA polymerase, ribosomes, 46 tRNAs, NTPs, creatine phosphate, 10-formyl-5,6,7,8-tetrahydrofolate, 20 amino acids, creatine kinase, myokinase, nucleoside diphosphate kinase, and pyrophosphatase.

[0102] Ribosome display has been successfully used to generate antibody fragments with high affinity to target.Detailed description of ribosome display can be found, for example, in Hanes, J.Proc.Natl.Acad.Sci.USA, 95:14130-14135 (1998); Schaffitzel et al., J.Immunological Methods, 231:119-135 (1999); He et al., J.Immunological Methods, 231:105-117 (1999); Roberts RW, Current Opinion in Chemical Biology, 3:268-273 (1999).

[0103] Other display methods for in vitro selection Other in vitro library display methods, including but not limited to, mRNA display, bicistronic display, P2A display, and CIS display (cis-acting display), can be used in the methods described herein.

[0104] mRNA display In mRNA display, each member of the RNA library is directly attached to the protein of interest that it encodes by a stable covalent bond to puromycin, an antibiotic that can mimic the aminoacyl terminus of tRNA (see, e.g., Robert RW and Szostak JW, Proc. Natl. Acad. Sci. USA, 94:12297-12302 (1997)). Puromycin is an aminonucleoside antibiotic active in both prokaryotes and eukaryotes, derived from Streptomyces alboniger. Protein synthesis is inhibited by premature chain termination during translation, which occurs in the ribosome. One part of the molecule serves as an analogue of the 3' end of tyrosyl-tRNA, one part of its structure mimics a molecule of adenosine, and another part mimics a molecule of tyrosine. It enters the A site and transfers to the growing chain, causing the formation of a puromycinated nascent chain and the release of a premature chain. The 3' position contains an amide bond instead of the standard ester bond of tRNA, making the molecule fairly resistant to hydrolysis and halting progression along the ribosome.

[0105] Other puromycin analog inhibitors of protein synthesis include O-demethylpuromycin, O-propargyl-puromycin, 9-{3'-deoxy-3'-[(4-methyl-L-phenylalanyl)amino]-β-D-ribofuranosyl}-6-(N,N'-dimethylamino)purine [L-(4-Me)-Phe-PANS], and 6-dimethylamino-9-[3-(p-azido-L-beta-phenylalanylamino)-3-deoxy-beta-ribofuranosyl]purine.

[0106] Members of the RNA library can be ligated to puromycin via a linker, such as, but not limited to, a polynucleotide or a chemical linker, for example, polyethylene glycol (Fukuda et al., Nucleic Acid Research, 34(19):e127 (2006)). In some embodiments, a polynucleotide linker comprising RNA is linked to puromycin at the 3' end. In other embodiments, a PEG linker is linked to puromycin.

[0107] As puromycin, which is linked to the 3' end of an RNA molecule, enters the ribosome, it establishes a covalent bond to the nascent protein (encoded by the RNA molecule) as a result of peptidyl transferase activity in the ribosome, and a stable amide bond is formed between the protein and the O-methyl tyrosine moiety of puromycin.

[0108] The RNA library of RNA-puromycin fusions can be subjected to in vitro translation to generate RNA-puromycin-protein complexes (e.g., RNA-puromycin-neoantigen complexes) as described herein. In some embodiments, the RNA-puromycin fusions contain RNA molecules encoding candidate neoantigens identified by exome analysis of patient-derived tumor and wild-type cells.

[0109] Affinity selection can be performed on the mRNA-displayed protein library to screen for the presence or absence of proteins with desired properties, such as specific binding to a binding counter ligand. The mRNA display can be performed in solution or on a solid support. The selected, mRNA-displayed proteins of interest can be purified by standard methods known in the art, such as affinity chromatography. The mRNA can be cloned, PCR amplified, and / or sequenced to determine the coding sequence of the selected proteins of interest. In one embodiment, the selected mRNA display library contains a population of neoantigens that bind to patient-specific HLA proteins or fragments thereof, and ultimately to the patient's TILs.

[0110] In some embodiments, the members of the library of nucleic acid members are linked to puromycin, and each member encodes a predicted protein of interest that has a primary amino acid sequence that is different from other proteins encoded by other nucleic acid members. The mRNA display system can contain a population or mixture of different complexes, such that each complex has a different mRNA linked to puromycin, a ribosome, and a predicted protein of interest.

[0111] Biscistronic DNA display Bicistronic DNA display can be utilized for in vitro selection of proteins of interest (see, e.g., Sumida et al., Nucleic Acid Research, 37(22):e147 (2009)). The method is based on forming complexes of in vitro translated proteins of interest with their encoding DNA, which is used to determine the sequence of the protein of interest. Typically, a DNA template is generated that contains multiple ORFs that can be linked to the proteins they encode. Furthermore, the coupled transcription / translation reaction is compartmentalized in a water-in-oil emulsion (e.g., micelles).

[0112] In some cases, when multiple ORFs are used, they can be separated at the ribosome binding side. In some embodiments, the coding sequence of the protein of interest is fused to the coding sequence of streptavidin. In some embodiments, the DNA template is biotinylated via a linker. The linker can be cleavable.

[0113] During in vitro transcription / translation, the protein can be expressed in the micelles. In one non-limiting exemplary embodiment, the DNA template contains the coding sequence of a candidate neoantigen, and the candidate neoantigen is expressed in the micelles. In an embodiment, the candidate neoantigen forms and associates with the DNA encoding the candidate neoantigen via a linkage, such as a streptavidin-biotin linkage. The DNA display protein of interest, such as the candidate neoantigen, can be recovered from the emulsion and screened by affinity selection. The DNA template of the selected DNA display protein of interest can be cleaved from the complex by a method such as UV irradiation, and then PCR amplified, cloned, and / or sequenced.

[0114] In some embodiments, the methods provided herein include generating a library of nucleic acid members, each member encoding a prospective protein of interest (i.e., a candidate neoantigen) that has a different primary amino acid than other proteins encoded by other nucleic acid members. In some embodiments, the library includes DNA members, and the method includes transcribing the library into RNA and translating the RNA in a cell-free protein synthesis system to generate a bicistronic DNA display system in a water-in-oil emulsion. In some embodiments, the bicistronic DNA display system contains a population of proteins of interest that are selected for specific binding to a binding pair ligand. In one embodiment, the bicistronic DNA display system contains a population of candidate neoantigens, each candidate neoantigen associated with a DNA molecule that encodes the candidate neoantigen.

[0115] P2A DNA display In P2A DNA display, which utilizes the cis activity of endonuclease P2A, a fusion protein of P2A and a protein of interest is bound to the same DNA molecule from which it is expressed (see, for example, Reiersen et al., Nucleic Acids Research, 33(1):e10). The DNA template may contain a coding sequence for P2A, a promoter, and a coding sequence encoding a protein of interest fused to a gene at an origin of replication. The coding sequence for P2A can be obtained and a gene fusion with a protein of interest can be constructed by standard methods known to those skilled in the art.

[0116] In some embodiments, P2A DNA display is used to select a protein of interest. A protein of interest can be selected by generating a library of fusion proteins, where each member of the library comprises a fusion protein between P2A and a protein of interest, and selecting the protein of interest based on a desired property, e.g., specific binding to a binding pair ligand. The library can be constructed from a library of nucleic acid members, each member comprising a DNA template encoding a different protein of interest genetically fused to the coding sequence of P2A (i.e., each protein of interest has a different primary amino acid sequence from other proteins encoded by other members of the nucleic acid library).

[0117] In some embodiments, the P2A DNA display library can be screened by affinity selection strategies, for example, in solution or on a solid support. The selected proteins of interest can be purified, for example, by affinity chromatography, and the complexed DNA can be PCR amplified, cloned, and / or sequenced.

[0118] CIS Display Similar to P2A DNA display, CIS display involves a DNA-based approach to directly link in vitro transcribed / translated proteins to the DNA molecules that encode them (see, e.g., Odegrip et al., Proc. Natl. Acad. Sci. USA, 101(9):2806-2810). This method uses the DNA replication initiator protein, RepA, to non-covalently link the DNA molecule on which it is expressed if the DNA molecule has a CIS element. DNA molecules can be made to encode proteins of interest (e.g., candidate neoantigens) in addition to RepA.

[0119] In some embodiments, CIS display is used to select a protein of interest. A protein of interest can be selected by generating a DNA library of fusion proteins, each member of which contains a fusion protein between RepA and a protein of interest, and selecting the protein of interest based on a desired property, e.g., specific binding to a binding pair ligand. The library can be constructed from a DNA library, in which each member of the library contains a DNA template that encodes a different protein of interest genetically fused to the coding sequence of RepA (i.e., each protein of interest has a different primary amino acid sequence from other proteins encoded by other members of the nucleic acid library).

[0120] In some embodiments, members of the DNA library contain a coding sequence for a protein of interest, a coding sequence for RepA, a CIS element, an origin of replication and a promoter, and the coding sequence for the protein of interest is genetically fused to the coding sequence for RepA. The CIS element can be genetically linked to the RepA coding sequence.

[0121] In some embodiments, the library contains fusion proteins between RepA and a protein of interest, and DNA molecules encoding the fusion proteins. RepA DNA display libraries can be screened, for example, by affinity selection strategies in solution or on a solid support. The selected proteins of interest can be purified, for example, by affinity chromatography, and the complexed DNA can be PCR amplified, cloned, and / or sequenced.

[0122] Cell-free Protein Synthesis (CFPS) Cell-free protein synthesis systems can be used to express biologically active proteins of interest (i.e., neoantigens) as described herein. Cell extracts have been developed that support the synthesis of proteins in vitro from purified mRNA transcripts or from mRNA transcribed from DNA during an in vitro synthesis reaction.

[0123] The CFPS of a polypeptide in a reaction mix includes bacterial extracts and / or defined reagents. The reaction mix includes at least ATP or an energy source; templates for the production of macromolecules, such as DNA, mRNA, etc.; amino acids necessary for polypeptide synthesis, and such cofactors, enzymes and other reagents, such as ribosomes, tRNA, polymerases, transcription factors, aminoacyl synthetases, elongation factors, initiation factors, etc. In one embodiment of the present invention, the energy source is a homeostatic energy source. Also included may be enzymes that catalyze the regeneration of ATP from high-energy phosphate bonds, such as acetate kinase, creatine kinase, etc. Such enzymes may be present in the extract used for translation or may be added to the reaction mix. Such synthesis reaction systems are well known in the art and described in the literature.

[0124] The template for cell-free protein synthesis can be either mRNA or DNA. The template can include the sequence for any particular gene of interest and can code for a full-length polypeptide or any length of fragment thereof. The nucleic acid that serves as protein synthesis template can be derived from natural sources as needed, or can be synthetic or recombinant. For example, DNA can be recombinant DNA, such as plasmid, virus, etc.

[0125] As used herein, the term "reaction mix" refers to a reaction mixture capable of catalyzing the synthesis of a polypeptide from a nucleic acid template. The reaction mixture includes an extract from a bacterial cell, for example, an E. coli S30 extract. S30 extracts are well known in the art and are described, for example, in Lesley, SA et al. (1991), J. Biol. Chem. 266, 2632-8. Synthesis can be carried out under either aerobic or anaerobic conditions.

[0126] In some embodiments, the bacterial extract is dried. The dried bacterial extract can be reconstituted in Milli-Q water (e.g., reverse osmosis water) at 110% of the original solids, as determined by measuring the percent solids of the starting material. In one embodiment, an accurately weighed aliquot of the dried extract, corresponding to 110% of the original solids of 10 mL of extract, is added to 10 mL of Milli-Q water in a glass beaker equipped with a stir bar on a magnetic stirrer. The resulting mixture is stirred until the powder is dissolved. Once dissolved, the material is transferred to a 15 mL Falcon tube and stored at -80C unless used immediately.

[0127] The volume percentage of extractables in the reaction mix varies, with extractables typically being at least about 10% of the total volume, more typically at least about 20%, and in some cases at least about 50%, or at least about 60% of the total volume, and typically providing additional benefit if provided at up to about 75%.

[0128] A typical system includes a nucleic acid template that encodes a protein of interest. The nucleic acid template can be an RNA molecule (e.g., mRNA) or a nucleic acid (e.g., RNA, DNA) that encodes an mRNA, and can be in any form (e.g., linear, circular, supercoiled, single-stranded, double-stranded, etc.). The nucleic acid template directs the production of the desired protein.

[0129] To maintain the template, the cells used to generate the extract can be selected for the reduction, substantial reduction or elimination of the activity of harmful enzymes, or enzymes with modified activity. Bacterial cells with modified nuclease or phosphatase activity (e.g., having at least one mutant phosphatase or nuclease gene or a combination thereof) can be used to synthesize cell extracts to increase synthesis efficiency. For example, the E. coli strain used to make the S30 extract for CFPS can be deficient in RNase E or RNase A (e.g., by mutation).

[0130] In a typical CFPS reaction, a gene encoding a protein of interest is expressed in a transcription buffer, resulting in mRNA that is translated into the protein of interest in a CFPS extract and a translation buffer. The transcription buffer, cell-free extract, and translation buffer can be added separately, or two or more of these solutions can be combined prior to their addition, or added simultaneously.

[0131] To synthesize a protein of interest in vitro, the CFPS extract at some point contains an mRNA molecule that codes for the protein of interest. In some CFPS systems, the mRNA is added exogenously after it has been purified from a natural source or synthetically prepared in vitro from cloned DNA using RNA polymerases such as RNA polymerase II, SP6 RNA polymerase, T3 RNA polymerase, T7 RNA polymerase, RNA polymerase III and / or phage-derived RNA polymerase. In other systems, the mRNA is generated in vitro from a template DNA, and both transcription and translation occur in this type of CFPS reaction. In some embodiments, the transcription and translation system is coupled or includes complementary transcription and translation systems that perform both RNA and protein synthesis in the same reaction. In such in vitro transcription and translation systems, the CFPS extract contains all the components (exogenous or endogenous) required for both transcription (to generate mRNA) and translation (to synthesize protein) in a single system.

[0132] The cell-free protein synthesis reaction mixture includes the following components: a template nucleic acid, such as a DNA or PCR fragment, comprising at least one promoter and, optionally, a gene of interest operably linked to one or more other regulatory sequences (e.g., a cloning or expression vector containing the gene of interest); an RNA polymerase that recognizes the promoter to which the gene of interest is operably linked, and, optionally, one or more transcription factors directed to the optional regulatory sequences to which the template nucleic acid is operably linked; ribonucleotide triphosphates (rNTPs); optionally, other transcription factors and their cofactors; ribosomes; transfer RNA (tRNA); other or optional translation factors (e.g., translation initiation factors, elongation factors, and termination factors) and their cofactors; one or more energy sources (e.g., ATP, GTP); optionally, one or more energy regenerating components (e.g., PEP / pyruvate kinase, AP / acetate kinase, or creatine phosphate / creatine kinase); optionally, yield and / or efficiency enhancing factors (e.g., nucleases, nuclease inhibitors, protein stabilizers, chaperones) and their cofactors; and; optionally, a solubilizing agent. The reaction mix further includes amino acids and other materials specifically required for protein synthesis, including salts (e.g., potassium, magnesium, ammonium, and manganese salts of acetate, glutamate, or sulfate), polymeric compounds (e.g., polyethylene glycol, dextran, diethylaminoethyl dextran, quaternary aminoethyl and aminoethyl dextran, etc.), cyclic AMP, inhibitors of proteins or nucleases, inhibitors or regulators of protein synthesis, oxidation / reduction regulators (e.g., DTT, ascorbic acid, glutathione, and / or their oxides), non-denaturing detergents (e.g., Triton X-100), buffer components, spermine, spermidine, putrescine, etc. The components of the CFPS reaction are described in more detail in U.S. Pat. Nos. 7,338,789 and 7,351,563, and U.S. Patent Application Publication No. 2010 / 0184135, the disclosures of which are incorporated by reference in their entireties for all purposes.

[0133] Depending on the particular enzymes present in the extract, for example, one or more of the many known nuclease inhibitors, polymerase inhibitors, or phosphatase inhibitors can be selected and used advantageously to improve the efficiency of synthesis.

[0134] Protein and nucleic acid synthesis typically requires an energy source. Energy is required for the initiation of transcription to generate mRNA (e.g., when a DNA template is used, and high-energy phosphate, e.g., in the form of GTP, is used to initiate translation). Each subsequent step of one codon (three nucleotides; one amino acid) by the ribosome requires further hydrolysis of GTP to GDP. Typically, ATP is also required. For an amino acid to be polymerized during protein synthesis, the amino acid must first be activated. Thus, a significant amount of energy from high-energy phosphate bonds is required to allow protein and / or nucleic acid synthesis to proceed.

[0135] Energy sources are chemical substrates that can be enzymatically processed to provide energy for achieving the desired chemical reaction. Energy sources that allow the release of energy for synthesis by cleavage of high-energy phosphate bonds, such as those found in nucleoside triphosphates, e.g., ATP, are commonly used. Any source that can be converted into high-energy phosphate bonds is particularly suitable. ATP, GTP, and other triphosphates can usually be considered as equivalent energy sources to support protein synthesis.

[0136] To provide energy for the synthesis reactions, the system may include additional energy sources such as glucose, pyruvate, phosphoenolpyruvate (PEP), carbamoyl phosphate, acetyl phosphate, creatine phosphate, phosphopyruvate, glyceraldehyde-3-phosphate, 3-phosphoglycerate and glucose-6-phosphate, which can generate or regenerate high-energy triphosphate compounds such as ATP, GTP, other NTPs.

[0137] If sufficient energy is not initially present in the synthesis system, an additional energy source is preferably supplemented. Energy sources may also be added or supplemented during an in vitro synthesis reaction.

[0138] In some embodiments, the cell-free protein synthesis reaction comprises NTPs, E. coli tRNA, amino acids, Mg 2+ Acetate, Mg 2+ Glutamate, K + Acetate, K + Glutamate, folinic acid, Tris pH 8.2, DTT, pyruvate kinase, T7 RNA polymerase, disulfide isomerase, phosphoenolpyruvate (PEP), NAD, CoA, Na + This is performed using the PANOx-SP system, which contains oxalate, putrescine, spermidine, and S30 extract.

[0139] In some cases, cell-free synthesis reactions do not require the addition of a common secondary energy source, but use the simultaneous activation of oxidative phosphorylation and protein synthesis. In some cases, CFPS is carried out in reactions such as the Cytomim system. The Cytomim system is defined as a reaction condition carried out in the absence of polyethylene glycol at an optimized magnesium concentration. This system does not accumulate phosphate, which is known to inhibit protein synthesis.

[0140] The presence of an active oxidative phosphorylation pathway can be tested using inhibitors that specifically inhibit steps in the pathway, such as electron transport chain inhibitors. Examples of inhibitors of the oxidative phosphorylation pathway include toxins such as cyanide, carbon monoxide, azide, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and 2,4-dinitrophenol, antibiotics such as oligomycin, insecticides such as rotenone, and competitive inhibitors of succinate dehydrogenase, such as malonate and oxaloacetate.

[0141] In some embodiments, the cell-free protein synthesis reaction comprises NTPs, E. coli tRNA, amino acids, Mg 2+ Acetate, Mg 2+ Glutamate, K + Acetate, K + Glutamate, folinic acid, Tris pH 8.2, DTT, pyruvate kinase, T7 RNA polymerase, disulfide isomerase, sodium pyruvate, NAD, CoA, Na + The method is carried out using a Cytomim system that includes oxalate, putrescine, spermidine, and S30 extract. In some embodiments, the energy substrates of the Cytomim system are pyruvate, glutamate, and / or glucose. In some embodiments of the system, nucleoside triphosphates (NTPs) are replaced with nucleoside monophosphates (NMPs).

[0142] The cell extract can be treated with iodoacetamide to inactivate enzymes that may reduce disulfide bonds and impair proper protein folding. In some embodiments, the cell extract contains exogenous protein chaperones. The protein chaperones can be expressed by the bacterial strain used to make the cell-free extract, or the protein chaperones can be added to the cell extract. Non-limiting examples of exogenous protein chaperones include disulfide bond isomerases (PDIs), such as, but not limited to, E. coli DsbC, and peptidyl prolyl cis-trans isomerases (PPIases), such as, but not limited to, FkpA. In some embodiments, the extract contains both PDIs and PPIases, such as, but not limited to, both DsbC and FkpA. Glutathione disulfide (GSSG) and / or glutathione (GSH) can also be added to the extract in a ratio that promotes proper protein folding and prevents the formation of abnormal protein disulfides.

[0143] In some embodiments, the CFPS reactants contain inverted membrane vesicles for carrying out oxidative phosphorylation. These vesicles may be formed during the high pressure homogenization step of the preparation of the cell extraction processes described herein and remain in the extract used in the reaction mix.

[0144] Methods for preparing cell extracts are described, for example, in Zawada, J. "Preparation and Testing of E. coli S30 In Vitro Transcription Translation Extracts," in Douthwaite, JA and Jackson, RH (eds.), Ribosomal display and Related Technologies: Methods and Protocols, Methods in Molecular Biology, vol. 805, pp. 31-41 (Humana Press, 2012); Jewett et al., Molecular Systems Biology: 4, 1-10 (2008); Shin J. and Norieaux V., J. Biol. Eng., 4:8 (2010). Briefly, bacterial cultures are grown, harvested, suspended in an appropriate buffer (e.g., S30 buffer) and homogenized to lyse the cells.

[0145] The cell-free extract can be thawed to room temperature before use in the CFPS reaction. When synthesizing proteins with disulfide bonds, the extract can be incubated with 50 μM iodoacetamide for 30 minutes. In some embodiments, the CFPS reaction comprises about 8 mM magnesium glutamate, about 10 mM ammonium glutamate, about 130 mM potassium glutamate, about 35 mM sodium pyruvate, about 1.2 mM AMP, about 0.86 mM each of GMP, UMP, and CMP, about 2 mM amino acids (about 1 mM for tyrosine), about 4 mM sodium oxalate, about 0.5 mM putrescine, about 1.5 mM spermidine, about 16.7 mM potassium phosphate, about 100 mM T7 RNA polymerase, about 2-10 μg / mL plasmid DNA template, about 1-10 μM E. coli DsbC, and an extract treated with about 30% (v / v) iodoacetamide with a total concentration of about 2 mM oxidized (GSSG) glutathione. Optionally, the cell-free extract may contain 1 mM reduced (GSH).

[0146] Lysate generation The methods and systems described herein can use cell lysates for in vitro translation of a target protein of interest. For convenience, the organism used as a source of lysate can be referred to as the source organism or host cell. The host cell can be a bacterial cell, a yeast cell, a mammalian cell or a plant cell, or any other cell type capable of synthesizing a protein. The lysate contains components capable of translating messenger ribonucleic acid (mRNA) encoding the desired protein, and optionally components capable of transcribing DNA encoding the desired protein. Such components include, for example, a DNA-directed RNA polymerase (RNA polymerase), any transcriptional activators required to initiate transcription of the DNA encoding the desired protein, transfer ribonucleic acid (tRNA), aminoacyl-tRNA synthetase, 70S ribosomes, N 10 -Formyltetrahydrofolate, formylmethionine-tRNAf MetThese include synthases, peptidyl transferases, initiation factors such as IF-1, IF-2, and IF-3, elongation factors such as EF-Tu, EF-Ts, and EF-G, and release factors such as RF-1, RF-2, and RF-3.

[0147] In an embodiment, the bacterial cells from which the lysate is derived are used. Bacterial lysates from any strain of bacteria can be used in the method of the present invention. Bacterial lysates can be obtained as follows. Selected bacteria are grown to logarithmic phase under any of a number of growth media and growth conditions for growing a particular bacterium, which are well known in the art and easily optimized by practitioners. For example, the natural environment for synthesis utilizes cell lysates from bacterial cells grown in a medium containing glucose and phosphate, where glucose is present at a concentration of at least about 0.25% (weight / volume), more usually at least about 1%; and usually not more than about 4%, more usually not more than about 2%. An example of such a medium is 2YTPG medium, but those skilled in the art will understand that many culture media can be suitable for this purpose, since many media suitable for growing bacteria such as E. coli, both with defined and undefined nutrient sources, have been published. The overnight harvested cells can be lysed by suspending the cell pellet in an appropriate cell suspension buffer and disrupting the suspended cells by sonication, crushing the suspended cells in a French press, continuous flow high pressure homogenization, or any other method known in the art that is useful for efficient cell lysis. The cell lysate is then centrifuged or filtered to remove large DNA fragments and cell debris.

[0148] The bacterial strains used to make cell lysates generally have reduced nuclease and / or phosphatase activity to increase cell-free synthesis efficiency.For example, the bacterial strains used to make cell-free extracts may have mutations in the genes encoding the nucleases RNase E and RNase A.The strains may also have mutations to stabilize components of cell synthesis reactions, such as deletion of genes such as tnaA, speA, sdaA or gshA, which prevent the degradation of the amino acids tryptophan, arginine, serine and cysteine, respectively, in cell-free synthesis reactions.In addition, the strains may have mutations to stabilize protein products of cell-free synthesis, such as knockout of proteases ompT or lonP.

[0149] DNA expression cassette containing no stop codon In some embodiments, the nucleic acid template used in the ribosome display reaction system comprises a DNA expression cassette that can express RNA that codes for a protein of interest, and the RNA lacks an operable stop codon.In order to remove the stop codon from the protein coding region in the expression cassette, PCR primers that lack stop codons can be used to amplify the coding region of the protein of interest, resulting in the amplification of the entire coding region from the translation start to the sequence that codes for the C-terminal amino acid.

[0150] MHC binding assay using neoantigen libraries The major histocompatibility complex (MHC) is a collection of genes that code for glycoproteins called MHC proteins. The main function of MHC proteins in vivo is to present antigens in a form that can be recognized by the TCR. MHC proteins bind to antigens in the form of antigenic peptides to form MHC-peptide complexes.

[0151] MHC proteins, also known as human leukocyte antigens (HLA) in the H-2 region of humans and mice, are divided into two categories: class I and class II MHC proteins. These proteins are composed of clusters of highly polymorphic genes. Specifically, human HLA-A, HLA-B, and HLA-C are known as class I MHC molecules, while human HLA-DP, HLA-DQ, and HLA-DR are known as class II MHC molecules. HLA loci include HLA-DP, HLA-DN, HLA-DM, HLA-DO, HLA-DQ, HLA-DR, HLA-A, HLA-B, and HLA-C. Each of these loci contains different alleles in the human population. The different subtypes encoded by these allelic variants are intended to be within the scope of the present invention.

[0152] MHC class II proteins are heterodimeric integral membrane proteins that contain one α chain and one β chain in non-covalent association. The α chain has two extracellular domains (α1 and α2), as well as transmembrane (TM) and cytoplasmic (CYT) domains. The β chain contains two extracellular domains (β1 and β2), as well as TM and CYT domains. MHC class I proteins are integral membrane proteins that contain a glycoprotein heavy chain that has three extracellular domains (i.e., α1, α2, and α3) as well as TM and CYT domains. The heavy chain is a glycoprotein that encodes β2-microglobulin (β 2m ), which is non-covalently associated with a soluble subunit called the ribosomal protein.

[0153] In some embodiments, the binding assays of the invention (e.g., MHC-peptide-DNA conjugate assays) employ the use of space holder molecules linked to MHC class II components, e.g., β-chains, by processable linkers. The space holder molecules of the invention can be any peptide that can bind to the peptide binding groove of an MHC protein such that binding of any other peptides in the peptide binding groove is prevented. Preferably, the space holder molecules bind in the peptide binding groove with moderate affinity, more preferably with low affinity, at about neutral pH. In one embodiment, the length of the space holder molecule ranges from about 5 to about 40 amino acid residues, more preferably from about 6 to about 30 amino acid residues, 8 to about 20 amino acid residues, and even more preferably from about 12 to 15 amino acid residues. In a further embodiment, the space holder molecule is about 13 amino acid residues. Examples of suitable space holder molecules include, but are not limited to, (in single letter amino acid code) PVSKMRMATPLLMQA (SEQ ID NO:25), also known as CLIP; AAMAAAAAAAMAA (SEQ ID NO:26); AAMAAAAAAAAAA (SEQ ID NO:27); AAFAAAAAAAAAA (SEQ ID NO:28); ASMSAASAASMAA (SEQ ID NO:29), and functional equivalents thereof. In one embodiment, the space holder molecule has the consensus sequence AAXAAAAAAAXAA (SEQ ID NO:30), where X is any amino acid. The ability to maintain the space holder molecule in the binding groove of the MHC class II molecule prevents the formation of "empty" molecules. The formation of "empty" MHC class II molecules is a major limiting factor, as these "empty" molecules tend to aggregate, making the isolation of functional MHC class II components difficult.

[0154] The space holder molecules of the present invention can be covalently linked to MHC molecules by linkers having amino acid sequences that contain target sites for enzymes that can cleave proteins. Such linkers are referred to herein as "processable linkers". Examples of processable linkers of the present invention include linkers that contain target sites for enzymes such as collagenases, metalloproteases, serine proteases, cysteine ​​proteases, kallikreins, thrombin, and plasminogen activators. Preferred processable linkers of the present invention include linkers that have a thrombin cleavage site.

[0155] Suitable linkers useful in the present invention can also be designed using various methods. For example, using X-ray crystallographic data of MHC protein, linkers of suitable length and charge can be designed so that the linker does not interfere with the binding of space holder molecules to the peptide-binding groove of MHC class II components. Such methods are included in the present invention.

[0156] The length of the linker of the present invention is preferably sufficiently short (i.e., sufficiently small in size) so that the linker does not substantially inhibit binding between the space holder molecule and the MHC class II component. The length of the linker of the present invention can range from about 1 amino acid residue to about 40 amino acid residues, more preferably from about 5 amino acid residues to about 30 amino acid residues, and even more preferably from about 8 amino acid residues to about 20 amino acid residues.

[0157] Cleavage of the linker promotes the release of the space holder molecule, thereby freeing the peptide-binding groove.The MHC class II component can then be incubated with a library of candidate neoantigens (e.g., a peptide-DNA conjugate library of peptides designed from exome analysis of tumor and wild-type cells) to promote binding of the antigenic peptide molecule to the MHC class II component.After a sufficient time to allow binding, which can be easily determined by one skilled in the art, the MHC class II component bound to the antigenic peptide molecule is recovered.

[0158] In certain embodiments, the steps of cleaving the linker and incubating with the antigenic peptide molecule are repeated using different antigenic peptide molecules. The advantage of repeating these steps is that it allows the formation of multiple MHC class II components that recognize several antigenic epitopes. Furthermore, these steps can be performed using MHC class II molecules constructed with various allelic types of MHC class II genes. Thus, this feature of the assay allows the generation of several MHC class II components specific to the genotypes of multiple different MHC alleles.

[0159] Alternatively, the binding assays of the invention (e.g., MHC-peptide-DNA conjugate assays) utilize a phage display system (see, e.g., Hammer J et al., Promiscuous and allele-specific anchors in HLA-DR-binding peptides, Cell (1993) 74(1):197-203). MHC II components are expressed in a phage display. Phage expressing the MHC II components are then incubated with a library of candidate neoantigens (e.g., a peptide-DNA conjugate library of peptides designed from exome analysis of tumor and wild-type cells) to promote binding of the antigenic peptide molecules to the MHC class II components. After a sufficient time to allow binding, which can be readily determined by one of skill in the art, the MHC class II components bound to the antigenic peptide molecules are recovered.

[0160] In certain embodiments, in silico analysis is used to determine the specific binding between neoantigens and MHC class I proteins or fragments thereof. In silico analysis involves applying computational algorithms to predict relative binding to MHC I proteins based on the peptide sequence of the neoantigen. Tools such as netMHCpan are used to predict peptide / neoantigen binding to MHC proteins (see Jurtz et al., NetMHCpan-4.0: Improved Peptide-MHC Class I Interaction Predictions Integrating Eluted Ligand and Peptide Binding Affinity Data, J Immunol (2017) 199(9):3360-3368). The input to this analysis is the peptide sequence and MHC allele of interest, and the output is the predicted binding affinity.

[0161] In certain embodiments, neoantigens that demonstrate binding to MHC class I or MHC class II molecules in MHC binding assays or in silico are pooled to form a peptide mixture, which peptide mixture, or oligonucleotides encoding peptides from the peptide mixture, can then be administered directly to a patient.

[0162] Vaccine administration Administration of peptides or oligonucleotides according to the methods provided herein may be performed by any suitable method for administering peptides or oligonucleotides, including, but not limited to, by injection, transfusion, subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intrathecal, intravenous or intralymphatic injection, or intraperitoneally. In some embodiments, the neoantigen or oligonucleotide is administered into the cavity formed by resection of tumor tissue (i.e., intracavitary delivery) or directly into the tumor prior to resection (i.e., intratumoral delivery). In one embodiment, the peptide or oligonucleotide is administered by intravenous injection.

[0163] In one embodiment, the present invention includes a method of treating cancer with one or more neoantigen peptides or one or more oligonucleotides each having a nucleic acid sequence encoding a neoantigen peptide, wherein the patient has been pretreated with non-myeloablative chemotherapy prior to neoantigen or oligonucleotide treatment according to the present invention.

[0164] Methods of Treating Cancer The cancer treated by the disclosed compositions and methods can be any solid tumor. The cancer can also be metastatic and / or recurrent. Non-limiting examples of cancer include acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, cervical cancer, glioma, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, ovarian cancer, peritoneal, omental, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, soft tissue cancer, testicular cancer, thyroid cancer, ureteral cancer, bladder cancer, and gastrointestinal cancer, e.g., esophageal cancer, gastric cancer, These include cancer of the pancreas, stomach, small intestine, gastrointestinal carcinoid tumors, oral cavity, colorectal, and hepatobiliary cancers.

[0165] Triple-negative breast cancer (TNBC), which accounts for about 15% of all breast cancers, is a highly aggressive type of tumor that lacks estrogen receptor (ER), progesterone receptor (PR), and ERBB2 (HER2) gene amplification. TNBC does not respond to hormonal therapy, such as tamoxifen or aromatase inhibitors, or to therapy that targets the HER2 receptor, such as Herceptin (trastuzumab). As there are limited targets available for TNBC, there is currently a strong interest in finding new targets and thus personalized medicines that can treat this type of breast cancer. Thus, in some embodiments, the cancer is triple-negative breast cancer (TNBC).

[0166] Cancer Combination Therapy The disclosed compositions and methods can be used in combination with other cancer immunotherapies. There are two different types of immunotherapies: passive immunotherapy uses components of the immune system to direct targeted cytotoxic activity against cancer cells without necessarily initiating an immune response in the patient, whereas active immunotherapy actively induces an endogenous immune response. Passive strategies include the use of monoclonal antibodies (mAbs), which are produced by B cells in response to specific antigens. The development of hybridoma technology in the 1970s and the identification of tumor-specific antigens allowed for the pharmaceutical development of mAbs that can specifically target tumor cells for destruction by the immune system. So far, mAbs have been the most successful story for immunotherapy, and the top three best-selling anti-cancer drugs in 2012 were mAbs. Among them is Rituximab (Rituxan, Genentech), which binds to the CD20 protein, which is highly expressed on the surface of B-cell malignancies such as non-Hodgkin's lymphoma (NHL). Rituximab has been approved by the FDA for the treatment of NHL and chronic lymphocytic leukemia (CLL) in combination with chemotherapy. Another important mAb is trastuzumab (Herceptin, Genentech), which has revolutionized the treatment of HER2 (human epidermal growth factor receptor 2)-positive breast cancer by targeting the expression of HER2.

[0167] To generate optimal "killer" CD8 T cell responses, T cell receptor activation and costimulation are also required, which can be provided by ligation of tumor necrosis factor receptor family members, including OX40 (CD134) and 4-1BB (CD137). OX40 is of particular interest because treatment with activating (agonist) anti-OX40 mAb increases T cell differentiation and cytolytic function, leading to enhanced antitumor immunity against a variety of tumors.

[0168] A large number of anti-cancer drugs are available for combination with the present methods and compositions. The following is a non-exhaustive list of anti-cancer (antineoplastic) drugs that may be used with or without irradiation: Acivicin; Aclarubicin; Acodazole hydrochloride; AcrQnine; Adzelesin; Aldesleukin; Altretamine; Ambomycin; Amethanethrone acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene hydrochloride; Bisnafide dimesylate; Bizelesin; Bleomycin sulfate; Brequinar sodium; Bropirimine; Busulfan; Cacti cyclophosphamide;cyclophosphamide mesylate ... Hydrochloride);Elsamitrucin;Enloplatin;Enpromate;Epipropizine;Epirubicin hydrochloride;Elbrozole;Esorubicin hydrochloride;Estramustine;Estramustine sodium phosphate;Etanidazole;Iodized poppy oil ethyl ester I 131;Etoposide;Etoposide phosphate;Etoprine;Fadrozole hydrochloride;Fazarabine;Fenretinide;Floxuridine;Fludarabine phosphate;Fluorouracil;Flurocitabine;Fosquidone;Fostriecin sodium;Gemcitabine;Gemcitabine hydrochloride;Au198;Hydroxyurea;Idarubicin hydrochloride;Ifosfamide;Irmofosine;Iproplatin;Irinotecan hydrochloride;Lanreotide acetate;Letrozole;Leuprolide acetate;Liarozole hydrochloride;Lometrexol sodium;Lomustine;Losoxantrone hydrochloride;Masoprocol;Maytansine;Mechlorethamine hydrochloride;Megestrol acetate;Melengestrol acetate;Melphalan;Menogaril;Mercaptopurine;Methotrexate;Methotrexate sodium;Metoprine;Meturedepa;Mitindomide;Mitocalcine;Mitochromine;Mitogillin;Mitomarcin;Mitomycin;Mitosper;Mitotane;Mitoxantrone hydrochloride;Mycophenolic acid;Nocodazole;Nogalamycin;Ormaplatin;Oxisuran;Paclitaxel;Pegaspargase;Periomycin;Pentamustine;Peplomycin sulfate; Perfosfamide;Pipobroman;Piposulfan;Piroxantrone hydrochloride;Plicamycin;Promestane;Porfimer sodium;Porfiromycin;Prednimustine;Procarbazine hydrochloride;Puromycin;Puromycin hydrochloride;Pyrazofurin;Riboprine;Rogletimide;Safmgol;Safingol hydrochloride;Semustine;Simtrazene;Sparfosate sodium;Sparsomycin;Spirogermanium hydrochloride;Spiromustine;Spiroplatin;Streptonigrin;Streptozocin;Strontium Sr89 chloride;Sulofenur;Talisomycin;Taxanes;Taxoids;Tecogalan sodium Sodium);Tegafur;Teroxantrone Hydrochloride;Temoporfin;Teniposide;Teroxylon;Testolactone;Thiamiprine;Thioguanine;Thiotepa;Tiazofurin;Tirapazamine;Topotecan Hydrochloride;Toremifene Citrate;Trestorone Acetate;Triciribine Phosphate;Trimetrexate;Trimetrexate Glucuronate;Triptorelin;Tubulozole Hydrochloride;Uracil Mustard;Uredep;Vapreotide;Verteporfin;Vinblastine Sulfate;Vincristine Sulfate;Vindesine;Vindesine Sulfate;Vinepidine Sulfate;Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride. ;

[0169] The disclosed compositions and methods can be used in combination with one or more immune checkpoint inhibitors.Immune checkpoint inhibitor refers to a compound that inhibits proteins in checkpoint signaling pathways.Proteins in checkpoint signaling pathways include, for example, CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, PD-L1, PD-L2, TIM-3, TIGIT, Lair1, CD244, HAVCR2, CD200, CD200R1, CD200R2, CD200R4, LILRB4, PILRA, ICOSL, 4-1BB, or VISTA.Immune checkpoint inhibitors are known in the art.

[0170] For example, an immune checkpoint inhibitor can be a small molecule, such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid, or other organic or inorganic molecule.

[0171] Alternatively, the immune checkpoint inhibitor is an antibody or its fragment.For example, the antibody or its fragment is specific to the protein in checkpoint signaling pathway such as CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, PD-L1, PD-L2, TIM-3, TIGIT, Lair1, CD244, HAVCR2, CD200, CD200R1, CD200R2, CD200R4, LILRB4, PILRA, ICOSL, 4-1BB or VISTA.

[0172] Examples of anti-immune checkpoint antibodies include, for example, ipiliumab (anti-CTLA-4), pembrolizumab (anti-PD-L1), nivolumab (anti-PD-L1), atezolizumab (anti-PD-L1), and durvalumab (anti-PD-L1).

[0173] The present invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as figures, are hereby incorporated by reference in their entirety for all purposes. EXAMPLES

[0174] Example 1. Overview of neoantigen vaccine The inventors have developed a differentiated peptide vaccine approach (FIGS. 1 and 2) that applies novel peptide:MHC screening technology (i.e., "peptide-DNA conjugate assay" or "PepSeq") or related methodologies (e.g., ribosome display) to predict personalized immunogenic neoantigens from genomic analysis. Peptides corresponding to the patient's neoantigens are designed, and these peptides are then administered to the patient. One or more oligonucleotides can be manufactured having nucleic acid sequences that encode the peptides corresponding to the neoantigens. The oligonucleotides can be administered to the patient.

[0175] The peptide-DNA conjugate assay provides a method for the efficient synthesis and analysis of large libraries of DNA-barcoded peptides (see Figure 3). Example 2. Methods for the identification of neoantigens from human tumors The following method allows the identification of neoantigens from human tumors.

[0176] Freshly resected tumor specimens from human patients with a variety of malignancies are split into two samples each. The first sample is used to extract DNA and RNA, allowing for whole exome sequencing and RNAseq. Together with a germline sample (e.g., blood or cheek swab), this allows for the identification of somatic variants and quantification of the expression of associated proteins.

[0177] Following analysis of the sequencing data, the tumor's genomic information is used to design a library of overlapping peptides covering the candidate mutations and their wild-type counterparts. DNA-encoded libraries of these peptides are synthesized in a highly parallelized fashion, for example using peptide-DNA conjugate or phage display platforms, and assayed in parallel against a panel of commonly expressed human MHC class II proteins. In parallel, an in silico model of MHC class I binding is applied to identify a second subset of candidate neoantigens. The resulting data allows prioritization of a subset of putative class I or II restricted neoantigens for downstream attention.

[0178] Using candidate neoantigens identified by peptide-DNA conjugate assays or phage display platforms and / or in silico prediction approaches, two parallel approaches to enrich for neoantigens are pursued. In the first approach, selective expansion of neoantigen-reactive T cells is achieved by culturing TILs (free of tumor cells) isolated from tumors with peptides representing the final candidate (i.e., preferred) neoantigen sequences in the presence of cytokines.

[0179] Furthermore, fluorescently labeled peptide:MHC probes corresponding to the final candidate sequences (and possibly in combination with cell exhaustion markers) are used to isolate neoantigen-reactive T cells by fluorescence-activated cell sorting. In either case, in vitro activation assays, possibly including the use of in vitro derived tumor organoids, are used to measure the activity of the cell products against autologous tumor cells.

[0180] Example 3. Alternative methods for identification of neoantigens from human tumors Freshly resected tumor specimens from human patients are used to extract DNA and RNA, allowing for whole exome sequencing and RNAseq. Together with germline samples (blood or cheek swabs), this allows for the identification of somatic variants and quantification of expression of associated proteins.

[0181] Following analysis of the sequencing data, the tumor's genomic information is used to design a library of overlapping peptides covering the candidate mutations and their wild-type counterparts. DNA-encoded libraries of these peptides are synthesized in a highly parallel manner using a peptide-DNA conjugate platform and assayed in parallel against patient-specific MHC class II (DR, DP, and DQ) variants. This process allows for rapid and reliable identification of candidate neoantigen peptides:MHC by selectivity and affinity. In parallel, in silico analysis can be performed on the peptide library applying computational algorithms that predict relative binding to MHC class I proteins based on the peptide sequence of the neoantigen. As a result, a small set of targeted neoantigens is selected among a typically large catalog of peptide-altered somatic tumor variants.

[0182] Example 4. Collection of samples from human patients and mice To test the efficacy of neo-TIL therapy, several human patients were enrolled in the clinical trial. The patients had been diagnosed with lung cancer, colorectal cancer, melanoma, breast cancer, and colon cancer. In addition, a BALB / c mouse model of colon cancer was used to harvest tumor-infiltrating lymphocytes (TILs).

[0183] Tumor biopsies were taken from each human patient and mouse. The numbers of isolated tumor cells, peripheral blood mononuclear cells, and TILs were determined for each human patient. In addition, the number of MHC alleles and mutations were identified for each human patient and BALB / c mouse model of colon cancer (see Table 1).

[0184] [Table 1]

[0185] Example 5. Exome sequencing, peptide library design, and binding assays using human samples Whole-exome sequencing of tumor exomes and exomes from normal cells was used to design a peptide-DNA conjugate library, as outlined in Figures 4 and 5. The peptides in the peptide-DNA conjugate library overlapped in sequence and contained wild-type ("W") and mutated ("M") residues (see Figure 5).

[0186] Binding assays were performed for human patients TG00006 and TG00013 using the methods outlined in Day et al., J Clin Invest. 2003;112(6):831-842, as shown in Figure 6. The binding assays identified peptides with high affinity for the specific human leukocyte antigen serotypes present in each patient (see the large data points in the upper left corner of each graph shown in Figure 7). Additional analysis of binding assays using peptide libraries for human patient TG00013 is shown in Figures 8A and 8B.

[0187] Nucleic acids encoding peptides with high affinity for HLA complexes were isolated using peptide constructs identified using peptide-DNA conjugate assays. These nucleic acids were sequenced and the corresponding amino acid sequences are shown in Figure 9 for human patient TG00013. Comparison of MHC-peptide-DNA conjugate results with in silico predictions can be used to identify and confirm neoantigens and / or neoepitopes, as shown in Figure 10.

[0188] Several additional human patients with various forms of cancer, including lung cancer, melanoma, pancreatic cancer, colorectal cancer, gastric adenocarcinoma, uveal melanoma, basal cell carcinoma, Merkel cell carcinoma, and cholangiocarcinoma, were evaluated by harvesting tumor infiltrating lymphocytes (TILs) and generating peptides to activate and educate these TILs to enhance recognition of neoantigens and antitumor activity (see FIG. 11). A non-limiting example of a process for producing a peptide vaccine is outlined in FIG. 12.

[0189] Example 6. Activation of peripheral blood mononuclear cells by peptides Peripheral blood mononuclear cells (PBMCs) are any peripheral blood cell with a round nucleus. These cells consist of lymphocytes (T cells, B cells, NK cells) and monocytes, whereas erythrocytes and platelets have no nucleus and granulocytes (neutrophils, basophils, and eosinophils) have segmented nuclei. In humans, lymphocytes make up the majority of the PBMC population, followed by monocytes, with dendritic cells making up only a small percentage.

[0190] PBMCs can be extracted from whole blood using FICOLL®, a hydrophilic polysaccharide that separates the layers of blood, and gradient centrifugation, which separates the blood into a top layer of plasma, followed by a layer of PBMCs, and a bottom fraction of polymorphonuclear cells (such as neutrophils and eosinophils) and red blood cells.

[0191] To test the efficiency of the peptide vaccines generated as disclosed herein, immune infiltration assays were performed on patient-derived tumor organoids. Briefly, tumor cells isolated from the original specimen were cultured in ultra-low affinity (ULA) plates to form 3D structures (organoids). Organoids are known to recapitulate the phenotypic characteristics of the original tissue, including antigenic potential. After co-culture of PBMCs with tumor organoids from the same patient, the level of PBMC infiltration into the organoids is quantified by z-stacking imaging.

[0192] PBMCs were stained with Invitrogen's CELLTRACKER™ CM-DiI, a red fluorescent dye suitable for monitoring cell migration or location over several generations, to facilitate quantification of PBMC infiltration into organoids.The following treatments were administered to organoids: 1) PBMCs; 2) PBMCs+IL-2; 3) PBMCs+IL-2+peptide vaccine ("PEP"); and 4) PBMCs+DMSO (vehicle).PBMC infiltration into organoids was quantified 24 hours and 48 hours after administration.As shown in the bar graph in Figure 13, after only 24 hours, PBMCs combined with IL-2 and peptides showed significant infiltration of organoids.

[0193] Example 7. Peptide vaccine studies A study to evaluate the safety and efficacy of peptide vaccines generated as disclosed herein is outlined in Figure 14. The study will be conducted in patients with advanced or metastatic cancer with the main inclusion and exclusion criteria listed in Figure 14. The study has the following objectives: · To evaluate the safety of administering peptide vaccines; To observe antitumor activity as assessed by objective response rate (ORR) and clinical benefit rate (CBR); To assess the duration of any observed response (DOR); To evaluate progression-free survival (PFS) and overall survival (OS) of all enrolled patients; · To characterize vaccine-induced immune responses by assessing several criteria, including antigen-specific CDS+ and CD4+ T cell responses in peripheral blood before, during, and after vaccine treatment; To evaluate exploratory biomarkers in peripheral blood and tumor biopsies, such as programmed death-ligand 1 (PD-L1) expression, abundance and phenotype of tumor-infiltrating lymphocytes (TIL) and myeloid cells, somatic mutational burden, and neoantigen burden.

[0194] The duration of treatment with the peptide vaccine is approximately 12 weeks. During the vaccination treatment period, safety, functionality, and immunological evaluations are performed. Safety and functionality evaluations are performed 30 days (± 7 days) after the last administration.

[0195] While the invention has been described in relation to particular embodiments thereof, it will be understood that further modifications are possible, and this application is intended to cover any variations, uses, or adaptations of the invention in accordance with the principles of the invention in general, including such departures from the present disclosure as are within known or customary practice in the art to which the invention pertains, and as are applicable to the essential features described above.

Claims

1. 1. A pharmaceutical composition for use in a method of treating cancer in a patient, comprising: comprising one or more peptides corresponding to neoantigens; The method comprises: obtaining tumor cells from a tumor resected from a patient; Detecting a plurality of patient-specific tumor mutations in tumor cells by genomic analysis of tumor DNA and / or RNA and normal DNA and / or RNA from the patient; identifying neoantigens resulting from somatic mutations that exhibit specific binding to a human leukocyte antigen (HLA) protein or fragment thereof corresponding to the patient's genotype, said neoantigens corresponding to said one or more peptides; administering to the patient a therapeutically effective amount of the one or more peptides; The method includes:

2. 2. The pharmaceutical composition of claim 1, wherein the step of detecting multiple patient-specific tumor mutations comprises genomic profiling by next-generation sequencing of a panel of targeted genes.

3. The pharmaceutical composition of claim 2 , wherein the genomic profiling comprises whole genome profiling, whole exome profiling, and / or transcriptome profiling.

4. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein the genomic analysis comprises identifying multiple patient-specific tumor mutations in expressed genes by nucleic acid sequencing of tumor and normal samples from the patient, wherein the mutations are present in the genome of the patient's cancer cells but not present in normal cells from the subject.

5. The plurality of patient-specific tumor mutations include point mutations, splice site mutations, frameshift mutations, read-through mutations, gene fusion mutations, insertions, deletions, or combinations thereof; the plurality of patient-specific tumor mutations encodes at least one mutant polypeptide having a tumor-specific neoepitope that binds to an HLA protein or a fragment thereof with greater affinity than a wild-type polypeptide; The pharmaceutical composition according to any one of claims 1 to 4.

6. A pharmaceutical composition described in any one of claims 1 to 5, wherein the method further comprises a step of identifying the patient's major histocompatibility complex (MHC) class 1 and 2 genotype.

7. The pharmaceutical composition of claim 6, wherein the step of identifying the patient's MHC class 1 and 2 genotype comprises analysis of whole exome sequencing (WES) and / or RNA sequencing from tumor and / or normal tissue.

8. The step of identifying neoantigens comprises: (i) providing a library of peptide constructs, each peptide construct of the library comprises a peptide portion and an identifying nucleic acid portion that identifies the peptide portion; At least one peptide moiety of the peptide construct is capable of specifically binding to an HLA protein or a fragment thereof; (ii) contacting the HLA protein or a fragment thereof with a library of peptide constructs; (iii) separating at least one peptide construct comprising a peptide portion capable of specifically binding to an HLA protein or a fragment thereof from peptide constructs comprising a peptide portion not capable of specifically binding to an HLA protein or a fragment thereof; (iv) sequencing all or a portion of an identified nucleic acid portion of at least one peptide construct capable of specifically binding to an HLA protein or a fragment thereof; The pharmaceutical composition according to any one of claims 1 to 7, comprising:

9. The pharmaceutical composition of claim 8, wherein the library of peptide constructs comprises variant peptides designed from the analysis of multiple patient-specific tumor mutations that predict the effect of each mutation on the corresponding protein and exclude silent mutations and mutations in non-coding regions.

10. The pharmaceutical composition of claim 9 , wherein the variant peptide contains a mutation predicted to affect the structure of the corresponding protein.

11. The step of identifying neoantigens comprises: (i) generating a genetically encoded combinatorial library of polypeptides by phage display, ribosome display, mRNA display, bicistronic DNA display, P2A DNA display, CIS display, yeast display, or bacterial display, wherein the combinatorial library comprises polypeptides linked to corresponding nucleic acid molecules that encode the polypeptides; (ii) contacting the combinatorial library with an HLA protein or fragment thereof; (iii) isolating HLA proteins or fragments thereof that exhibit specific binding to the combinatorial library; (iv) sequencing all or a portion of the nucleic acid molecules of the combinatorial library bound to the HLA protein or fragment thereof to identify neoantigens; The pharmaceutical composition according to any one of claims 1 to 7, comprising:

12. The pharmaceutical composition of claim 11, wherein the combinatorial library of polypeptides comprises variant peptides designed from the analysis of multiple patient-specific tumor mutations that predict the effect of each mutation on the corresponding protein and exclude silent mutations and mutations in non-coding regions.

13. The pharmaceutical composition of claim 12 , wherein the variant peptide contains a mutation predicted to affect the structure of the corresponding protein.

14. The pharmaceutical composition of any one of claims 1 to 13, wherein the method further comprises administering to the patient an adjuvant simultaneously with a therapeutically effective amount of one or more peptides.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the step of administering one or more peptides to the patient is intravenous, intraperitoneal, subcutaneous, intramuscular, or intratumoral.

16. Specific binding between a neoantigen and an HLA protein or a fragment thereof (i) an MHC class II component comprising at least a portion of an MHC class II α chain and at least a portion of an MHC class II β chain, such that the MHC class II α chain and the MHC class II β chain form a peptide binding groove; and Space holder molecule and first processable linker Culturing cells transformed with at least one nucleic acid molecule comprising a nucleotide sequence encoding: wherein the space holder molecule is linked to an MHC class II component by a processable linker, and the space holder molecule binds in the peptide binding groove thereby preventing binding of any other peptide in the peptide binding groove; and the culturing is performed to produce an MHC class II component; (ii) recovering the MHC class II components; (iii) releasing the space holder molecule from the peptide binding groove by processing the processable linker; (iv) incubating the MHC class II component in the presence of the neoantigen, where the incubation promotes binding of the neoantigen to the peptide binding groove; (v) recovering the MHC class II components bound to the neoantigens; The pharmaceutical composition according to any one of claims 1 to 13, wherein the composition is determined by

17. 17. The pharmaceutical composition of claim 16, wherein the space holder molecule has the consensus sequence AAXAAAAAAAXAA (SEQ ID NO: 30).

18. 18. The pharmaceutical composition of claim 17, wherein the space holder molecule is selected from the group consisting of PVSKMRMATPLLMQA (sequence number 25); AAMAAAAAAAAMAA (sequence number 26); AAMAAAAAAAAAAAAAA (sequence number 27); AAFAAAAAAAAAAAAA (sequence number 28); and ASMSAASAASMAA (sequence number 29).

19. The pharmaceutical composition according to any one of claims 16 to 18, wherein the processable linker is linked to the MHC class II α chain of the MHC class II component.

20. The pharmaceutical composition according to any one of claims 17 to 19, wherein the step of recovering the MHC class II component bound by the neoantigen comprises affinity chromatography with an antibody that recognizes the MHC class II component.

21. The pharmaceutical composition according to any one of claims 1 to 13, wherein the specific binding between the neoantigen and the HLA protein or a fragment thereof is determined by phage display, the HLA protein or a fragment thereof is expressed on the surface of a phage, and the neoantigen is incubated with the phage to assay for specific binding.

22. The pharmaceutical composition of any one of claims 1 to 21, wherein the method further comprises an in silico analysis to determine specific binding between the neoantigen and an MHC class I protein or a fragment thereof, the in silico analysis comprising a step of using a computational algorithm to predict relative binding to the MHC I protein based on the peptide sequence of the neoantigen.

23. 1. A pharmaceutical composition for use in a method of treating a patient having a disease, comprising: an oligonucleotide having a nucleic acid sequence encoding a peptide corresponding to a neoantigen; The method comprises: obtaining tumor cells from a tumor resected from a patient; Detecting a plurality of patient-specific tumor mutations in tumor cells by genomic analysis of tumor DNA and / or RNA and normal DNA and / or RNA from the patient; identifying neoantigens resulting from somatic mutations that exhibit specific binding to a human leukocyte antigen (HLA) protein or fragment thereof corresponding to the patient's genotype, said neoantigens corresponding to the peptide encoded by said oligonucleotide; administering to the patient a therapeutically effective amount of an oligonucleotide having a nucleic acid sequence encoding the peptide; 13. A pharmaceutical composition comprising:

24. 24. The pharmaceutical composition of claim 23, wherein the step of detecting multiple patient-specific tumor mutations comprises genomic profiling by next-generation sequencing of a panel of targeted genes.

25. 25. The pharmaceutical composition of claim 24, wherein the genomic profiling comprises whole genome profiling, whole exome profiling, and / or transcriptome profiling.

26. 26. The pharmaceutical composition of any one of claims 23 to 25, wherein the genomic analysis comprises identifying a plurality of patient-specific tumor mutations in expressed genes by nucleic acid sequencing of tumor and normal samples from the patient, wherein the mutations are present in the genome of the patient's cancer cells but not present in normal cells from the subject.

27. The plurality of patient-specific tumor mutations include point mutations, splice site mutations, frameshift mutations, read-through mutations, gene fusion mutations, insertions, deletions, or combinations thereof; the plurality of patient-specific tumor mutations encodes at least one mutant polypeptide having a tumor-specific neoepitope that binds to an HLA protein or a fragment thereof with greater affinity than a wild-type polypeptide; The pharmaceutical composition according to any one of claims 23 to 26.

28. The pharmaceutical composition of any one of claims 23 to 27, wherein the method further comprises identifying the patient's major histocompatibility complex (MHC) class 1 and 2 genotype.

29. 29. The pharmaceutical composition of claim 28, wherein the step of identifying the patient's MHC class 1 and 2 genotype comprises whole exome sequencing (WES) and / or RNA sequencing analysis from tumor and / or normal tissue.

30. The step of identifying neoantigens comprises: (i) providing a library of peptide constructs, each peptide construct of the library comprises a peptide portion and an identifying nucleic acid portion that identifies the peptide portion; At least one peptide moiety of the peptide construct is capable of specifically binding to an HLA protein or a fragment thereof; (ii) contacting the HLA protein or a fragment thereof with a library of peptide constructs; (iii) separating at least one peptide construct comprising a peptide portion capable of specifically binding to an HLA protein or a fragment thereof from peptide constructs comprising a peptide portion not capable of specifically binding to an HLA protein or a fragment thereof; (iv) sequencing all or a portion of an identified nucleic acid portion of at least one peptide construct capable of specifically binding to an HLA protein or a fragment thereof; The pharmaceutical composition according to any one of claims 23 to 29, comprising:

31. The pharmaceutical composition of claim 30, wherein the library of peptide constructs comprises variant peptides designed from the analysis of multiple patient-specific tumor mutations that predict the effect of each mutation on the corresponding protein and exclude silent mutations and mutations in non-coding regions.

32. 32. The pharmaceutical composition of claim 31 , wherein the variant peptide contains a mutation predicted to affect the structure of the corresponding protein.

33. 1. A pharmaceutical composition for use in a method of treating cancer in a patient, comprising: a messenger ribonucleic acid (mRNA) polynucleotide comprising an open reading frame encoding one or more peptides corresponding to a neoantigen; The method comprises: obtaining tumor cells from a tumor resected from a patient; Multiple patient-specific tumor mutations in tumor cells are identified using tumor DNA from the patient and / or detecting by genomic analysis of the RNA and normal DNA and / or RNA; identifying neoantigens resulting from somatic mutations that exhibit specific binding to a human leukocyte antigen (HLA) protein or fragment thereof corresponding to the patient's genotype, said neoantigens corresponding to one or more peptides encoded by an open reading frame contained in said mRNA polynucleotide; administering to the patient a therapeutically effective amount of an mRNA polynucleotide; 13. A pharmaceutical composition comprising:

34. 34. The pharmaceutical composition of claim 33, wherein the mRNA polynucleotide comprises at least one chemical modification.

35. 35. The pharmaceutical composition of claim 34, wherein the chemical modification is selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine.

36. 36. The pharmaceutical composition of claim 35, wherein the chemical modification is at carbon 5 of uracil.

37. 36. The pharmaceutical composition of claim 35, wherein the chemical modification is N1-methylpseudouridine or N1-ethylpseudouridine.

38. The pharmaceutical composition of any one of claims 33 to 37, wherein at least 80% of the uracils in the open reading frame have a chemical modification.

39. The pharmaceutical composition of any one of claims 33 to 38, wherein the mRNA polynucleotide further encodes a 5'-end cap.

40. 40. The pharmaceutical composition of claim 39, wherein the 5' end cap is 7mG(5')ppp(5')NlmpNp.

41. The pharmaceutical composition of any one of claims 33 to 40, wherein the step of detecting a plurality of patient-specific tumor mutations comprises genomic profiling by next generation sequencing of a panel of targeted genes.

42. 42. The pharmaceutical composition of claim 41, wherein the genomic profiling comprises whole genome profiling, whole exome profiling, and / or transcriptome profiling.

43. 43. The pharmaceutical composition of any one of claims 33 to 42, wherein the genomic analysis comprises identifying a plurality of patient-specific tumor mutations in expressed genes by nucleic acid sequencing of tumor and normal samples from the patient, wherein the mutations are present in the genome of the patient's cancer cells but not present in normal cells from the subject.

44. The plurality of patient-specific tumor mutations include point mutations, splice site mutations, frameshift mutations, read-through mutations, gene fusion mutations, insertions, deletions, or combinations thereof; the plurality of patient-specific tumor mutations encodes at least one mutant polypeptide having a tumor-specific neoepitope that binds to an HLA protein or a fragment thereof with greater affinity than a wild-type polypeptide; The pharmaceutical composition according to any one of claims 33 to 43.

45. The pharmaceutical composition of any one of claims 33 to 44, wherein the method further comprises identifying the patient's major histocompatibility complex (MHC) class 1 and 2 genotype.

46. 46. ​​The pharmaceutical composition of claim 45, wherein the step of identifying the patient's MHC class 1 and 2 genotype comprises whole exome sequencing (WES) and / or RNA sequencing analysis from tumor and / or normal tissue.

47. The step of identifying neoantigens comprises: (i) providing a library of peptide constructs, each peptide construct of the library comprises a peptide portion and an identifying nucleic acid portion that identifies the peptide portion; At least one peptide moiety of the peptide construct is capable of specifically binding to an HLA protein or a fragment thereof; (ii) contacting the HLA protein or a fragment thereof with a library of peptide constructs; (iii) separating at least one peptide construct comprising a peptide portion capable of specifically binding to an HLA protein or a fragment thereof from peptide constructs comprising a peptide portion not capable of specifically binding to an HLA protein or a fragment thereof; (iv) sequencing all or a portion of an identified nucleic acid portion of at least one peptide construct capable of specifically binding to an HLA protein or a fragment thereof; The pharmaceutical composition according to any one of claims 33 to 46, comprising:

48. The pharmaceutical composition of claim 47, wherein the library of peptide constructs comprises variant peptides designed from the analysis of multiple patient-specific tumor mutations that predict the effect of each mutation on the corresponding protein and exclude silent mutations and mutations in non-coding regions.

49. 49. The pharmaceutical composition of claim 48, wherein the variant peptide contains a mutation predicted to affect the structure of the corresponding protein.

50. The step of identifying neoantigens comprises: (i) generating a genetically encoded combinatorial library of polypeptides by phage display, ribosome display, mRNA display, bicistronic DNA display, P2A DNA display, CIS display, yeast display, or bacterial display, wherein the combinatorial library comprises polypeptides linked to corresponding nucleic acid molecules that encode the polypeptides; (ii) contacting the combinatorial library with an HLA protein or fragment thereof; (iii) isolating HLA proteins or fragments thereof that exhibit specific binding to the combinatorial library; (iv) sequencing all or a portion of the nucleic acid molecules of the combinatorial library bound to the HLA protein or fragment thereof to identify neoantigens; The pharmaceutical composition according to any one of claims 33 to 46, comprising:

51. The pharmaceutical composition of claim 50, wherein the combinatorial library of polypeptides comprises variant peptides designed from the analysis of multiple patient-specific tumor mutations that predict the effect of each mutation on the corresponding protein and exclude silent mutations and mutations in non-coding regions.

52. 52. The pharmaceutical composition of claim 51, wherein the variant peptide contains a mutation predicted to affect the structure of the corresponding protein.

53. The pharmaceutical composition according to any one of claims 33 to 52, wherein the step of administering the mRNA polynucleotide to the patient is intravenous, intraperitoneal, subcutaneous, intramuscular, or intratumoral.

54. The pharmaceutical composition of any one of claims 1 to 53, wherein the method further comprises administering an effective amount of a therapeutic population of tumor infiltrating lymphocytes (TILs).

55. 55. The pharmaceutical composition of claim 54, wherein the therapeutic population of TILs is administered simultaneously or sequentially with the peptide or mRNA polynucleotide.

56. 56. The pharmaceutical composition of claim 54 or 55, wherein the therapeutic population of TILs is activated and / or educated by at least one neoantigen presented by a peptide or encoded by an mRNA polynucleotide.

57. The pharmaceutical composition of any one of claims 54 to 56, wherein administering a peptide or mRNA polynucleotide together with a therapeutic population of TILs enhances the immunogenic response and / or anti-tumor activity in the patient.

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