Cancer neoepitopes

Abstract

To provide a method of generating a pharmaceutical agent for cancer immune therapy.SOLUTION: The method comprises the steps of: using matched normal omics data of a tumor to generate in silico a plurality of peptides each of which contains a patient- and cancer-specific cancer neoepitope and has a length of between 7 and 11 amino acids; filtering in silico the peptides having a length of between 7 and 11 amino acids to obtain a subset of neoepitope sequences; preparing a synthetic peptide having a length of between 7 and 11 amino acids by using sequence information from the subset of neoepitope sequences; using the synthetic peptide to isolate a recombinant antibody; obtaining sequence information of the complementarity determining region of the recombinant antibody; generating a synthetic antibody using the sequence information of the complementarity determining region of the recombinant antibody; and coupling the synthetic antibody to a therapeutic or diagnostic agent to obtain a pharmaceutical agent.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The field of this invention is cancer neoepitopes, and in particular the identification and use of neoepitopes in the production of neoepitope-specific antibodies, and the use of neoepitopes and neoepitope-specific antibodies in prophylaxis and treatment. [Background technology]

[0002] The background discussion includes information that may be useful in understanding the invention. It is not an admission that any of the information provided herein is prior art or relevant to the claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] It is well known in the art that most, if not all, neoplastic diseases involve a relatively large number of mutations, such as point mutations, insertions, deletions, and translocations. Therefore, it is at least conceptually reasonable to assume that it is also possible to characterize neoplastic cells by the presence of one or more mutant proteins. More recently, as a result of numerous research efforts, a small group of T-cell-recognized human tumor antigens has become available for a limited number of tumor types (see, e.g., "T-cell Recognition and Tumor Immunoglobulins," vol. 1, no. 1, pp. 111-114, 2003). Unfortunately, these antigens have not yielded effective therapeutic agents for specific tumor types. All publications cited herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Furthermore, if a definition or use of a term in an incorporated reference contradicts or is contrary to the definition of that term provided herein, the definition of that term provided herein applies, and the definition of that term in the reference does not apply.

[0004] Although there are other possible reasons for the failure to produce immunotherapeutic agents for certain tumors, the seemingly random distribution of mutations in most tumors among various patients diagnosed with the same type of tumor has made the search for identifying one or more antigens that can be used as immunologically effective agents extremely complicated.In addition, the immune response to a specific antigen also depends on the individual's ability to bind to the antigen and present the antigen through HLA complex, so the statistical probability of identifying an antigen that is suitable for treating tumors in a large number of patients is very low.Therefore, the identification of patient-specific tumor antigens is likely to potentially lead to therapeutic agents, at least conceptually.

[0005] Unfortunately, many tumors have developed various immune evasion mechanisms, so that potentially useful patient- and tumor-specific antigens typically fail to elicit a therapeutically effective immune response or even the production of antibodies that bind to the patient- and tumor-specific antigens. Furthermore, even if a patient generates B cells that produce antibodies with specificity for the patient- and tumor-specific antigen, isolating such B cells is quite complex and time-consuming. Similarly, isolating large quantities of therapeutically useful antibodies from such a patient would be equally cumbersome and time-consuming, likely exceeding the patient's life expectancy. Additionally, even if sufficient quantities of antibodies against a single patient- and tumor-specific antigen could be obtained, the heterogeneity of many tumors may still render the treatment ineffective because not all cells in the tumor mass may express the same antigen. Furthermore, even if it were possible to produce or isolate a variety of therapeutic antibodies from a mammal for use in a single patient, such an approach would be entirely unsuitable as a platform for mass production for the vast number of patients requiring immunotherapy. In fact, traditional production of monoclonal antibodies against a single antigen often takes many months.

[0006] Thus, despite antibody production being generally well known in the art, there remains a need for systems and methods that allow for the rapid identification of patient-specific tumor antigens and the rapid production of antibodies targeting such antigens for diagnostic or therapeutic use. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Cancer Immunity, July 15, 2013, Vol. 13, p. 15 Summary of the Invention

[0008] The subject of the present invention relates to the identification and use of various cancer neoepitopes in patients, particularly when such neoepitopes fail to elicit a protective immune response in the patient.

[0009] In one embodiment of the present subject matter, a method for generating a drug for cancer immunotherapy is provided. A particularly preferred method includes using matched normal omics data for a tumor to in silico generate a plurality of n-mers (n-mers) containing at least one patient-specific and cancer-specific cancer neoepitope, and further filtering the n-mers in silico to obtain a subset of neoepitope sequences. In a further step, at least one synthetic n-mer peptide is prepared using sequence information from the subset of neoepitope sequences, and the synthetic n-mer peptide is then used to isolate a recombinant antibody. Sequence information of the complementarity-determining regions of the recombinant antibody is obtained, and a synthetic antibody is generated using the sequence information of the complementarity-determining regions of the recombinant antibody. In a further step, the synthetic antibody is then linked to a therapeutic or diagnostic agent, thereby obtaining a drug.

[0010] It is generally contemplated that the matched normal omics data is whole genome sequencing data, exome sequencing data, and / or transcriptome data, and that the matched normal omics data is matched to normal data prior to the patient's treatment. It is further contemplated that each of the plurality of n-mer peptides has a length of 7-11 amino acids and / or that the plurality of n-mer peptides comprises at least 1,000 n-mer peptides. Most typically, different ones of the plurality of n-mer peptides have different neoepitopes. While not limiting the subject matter of the present invention, it is further contemplated that the filtering step may include filtering by mutation type, expression intensity, subcellular location, and / or binding affinity to the patient's HLA type.

[0011] Additionally, it is contemplated that the step of using the synthetic n-mer peptide to isolate a recombinant antibody may comprise phage panning, which may further comprise a step of affinity maturation. It is also contemplated that the sequence information of the complementarity determining regions of the recombinant antibody may comprise CDR1-H, CDR2-H, and CDR3-H, and optionally CDR1-L, CDR2-L, and CDR3-L, and / or that the synthetic antibody is generated using grafting of CDRs or SDRs onto a human antibody scaffold.

[0012] Contemplated synthetic antibodies are typically produced by recombinant expression as IgG, F(ab')2, Fab', Fab, or scFv, and contemplated therapeutic or diagnostic agents include noncellular agents (e.g., chemotherapeutic agents, radioisotopes, PET-detectable isotopes, SPECT-detectable isotopes, affinity agents, etc.) and immunocompetent cells (e.g., T cells, NK cells, etc.). For example, if the cell is a T cell, the T cell may express a chimeric receptor having an scFv as an ectodomain, and the synthetic antibody is an scFv. Alternatively or additionally, if the cell is a NK cell, the NK cell may express a high-affinity Fcγ receptor (CD16), and the synthetic antibody may then be an IgG that binds to the NK cell via the high-affinity Fcγ receptor. Most notably, it should be recognized as a result that patient-specific and cancer-specific antibodies and compositions comprising such antibodies can be prepared in a significantly shorter time (e.g., less than 8 weeks, or less than 6 weeks, or even less than 4 weeks), even if the patient does not mount an effective and protective immune response to the neoepitope.

[0013] Therefore, the inventors also contemplate methods for generating synthetic antibodies against cancer neoepitopes in cases where the patient's cancer neoepitopes fail to elicit a protective immune response. The contemplated methods will typically involve using cancer neoepitopes (typically entirely synthetic) to select binding recombinant antibodies from a library of recombinant antibodies, where the cancer neoepitopes are patient- and cancer-specific. In a separate step, the hypervariable loops of the binding recombinant antibodies are then analyzed, thereby obtaining specificity information about the binding recombinant antibodies, which is then used to modify a gene encoding at least a portion of a human antibody. Finally, the gene is then recombinantly expressed to produce the synthetic antibody. Notably, cancer- and patient-specific antibodies are thus produced without the use of a mammalian immune system.

[0014] In particularly contemplated methods, the cancer neoepitopes are HLA-matched cancer neoepitopes, and / or the recombinant antibody library is a phage display library. If desired, the method may further comprise affinity maturation of the binding recombinant antibodies to derive recombinant antibodies with optimized binding. Regardless of the additional step, the hypervariable loops are preferably analyzed by sequencing the DNA encoding the hypervariable loops, and the modifying step will comprise CDR or SDR grafting (e.g., if the portion of the human antibody is an scFv). Recombinant expression of the modified genes will then result in a synthetic antibody in the form of an IgG, F(ab')2, Fab', Fab, or scFv. Most typically, the cancer neoepitopes are expressed in the patient's cancer, and / or the neoepitopes are unique to the patient and the cancer within the patient.

[0015] Viewed from another perspective, the inventors therefore also contemplate compositions comprising synthetic antibodies having binding affinity for patient-specific and cancer-specific HLA-matched cancer neoepitopes, said neoepitopes being unique to the patient and the patient's cancer.

[0016] In a particularly preferred embodiment, the HLA-matched cancer neoepitope is matched for MHC-I presentation, and the synthetic antibody is selected from IgG, F(ab')2, Fab', Fab, and scFv. If desired, the synthetic antibody may be linked to a therapeutic agent, which may be a non-cellular agent (e.g., a chemotherapeutic agent, a radioisotope, a PET-detectable isotope, a SPECT-detectable isotope, or an affinity agent). Alternatively, the therapeutic agent may be a cell, particularly an immunocompetent cell (e.g., a T cell or an NK cell). For example, if the cell is a T cell, the T cell may express a chimeric receptor having an scFv as an ectodomain, and the synthetic antibody is an scFv. On the other hand, if the cell is an NK cell, the NK cell may express a high-affinity Fcγ receptor (CD16), and the synthetic antibody is an IgG that binds to the NK cell via the high-affinity Fcγ receptor. Among other compositions, contemplated cancer neoepitopes can have a sequence selected from SEQ ID NO: 1 through SEQ ID NO: 1,408,729.

[0017] In yet another aspect of the present subject matter, the inventors also contemplate compositions comprising solid phases bearing patient-specific and cancer-specific HLA-matched cancer neoepitopes, the cancer neoepitopes being unique to the patient and the patient's cancer. For example, suitable solid phases include the interior walls of reagent vessels, magnetic beads, or individually addressable elements, with particularly preferred cancer neoepitopes having a length of 7-9 amino acids. Among other neoepitopes, contemplated neoepitopes include those having any one of the sequences of SEQ ID NO: 1 through SEQ ID NO: 1,408,729. If desired, the cancer neoepitopes are contemplated to be bound by synthetic antibodies (e.g., IgG, F(ab')2, Fab', Fab, and scFv), which may then be linked to viral particles.

[0018] Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments taken in conjunction with the accompanying drawings, in which like numerals represent like elements and in which: [Brief explanation of the drawings]

[0019] [Figure 1] 1 is an exemplary schematic flow diagram for one embodiment of the present subject matter. [Figure 2] 10 is an example plot showing filtering results for calculated neoepitopes. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present inventors have discovered that a conceptually simple yet powerful approach that combines in silico and in vitro techniques can be used to prepare a variety of diagnostic and therapeutic compositions and agents.

[0021] Using our systems and methods, it should be appreciated that it is possible to generate fully synthetic antibodies against one or more patient-specific neoepitopes, particularly against antigens for which no protective immune response was elicited and / or for which a suppressed immune response was demonstrated. Even more advantageously, it should be noted that such synthetic antibodies can be prepared by in silico analysis of a patient's omics data, thereby deriving a relatively large number of potentially useful neoepitopes, which can then be further filtered to enhance therapeutic efficacy. From another perspective, contemplated synthetic monoclonal antibodies are prepared without the time-consuming isolation of tumor-specific peptides from tumors and without any immunization procedures in mammals (or other animals equipped with immune systems). Furthermore, it should be noted that the methods contemplated herein will enable rapid production of synthetic antibodies, typically within a few days to a few weeks (e.g., 5 to 21 days).

[0022] Briefly, and as illustratively shown in the flow chart of FIG. 1 , one contemplated method 100 will comprise an in silico analysis section 110 and an in vitro synthesis section 120. Most typically, the in silico analysis begins with an omics analysis 112 to identify mutations in the tumor compared to normal tissue from the same patient, followed by a step 114 of calculating one or more neoepitopes to define tumor- and patient-specific neoepitopes. The neoepitopes thus obtained are further subjected to a filtering step 116, e.g., to eliminate weakly or not expressed neoepitopes. The identified expressed neoepitopes may then be subjected to an additional filtering algorithm 118, e.g., to limit the neoepitopes to those exposed extracellularly or bound to MHC-I. FIG. 2 exemplarily illustrates the results of the series of filtering steps. Here, whole-genome sequencing analysis of triple-negative breast cancer samples contrasted with matched normal samples (i.e., compared with non-diseased tissue from the same patient) revealed a relatively large number (~18,000) of neoepitopes in the tumor samples. Notably, an initial filtering step eliminated over 50% of all identified neoepitopes based on their intensity of expression. Here, neoepitope sequences were eliminated at expression levels less than 20% of those in the matched normal samples. The remaining sequences were subjected to in silico analysis to determine sequences that would bind to a single specific HLA type in the same sample (e.g., with an affinity less than 500 nM). Notably, again, a significant proportion of neoepitopes were eliminated, and ultimately, less than 1.3% of all neoepitopes were found to be suitable for use.

[0023] Referring again to FIG. 1 , and following in silico identification of suitable neoepitope sequences, corresponding synthetic peptides are then prepared in vitro (e.g., using solid phase synthesis) and typically synthesized into large, highly diverse libraries (e.g., at least 10 distinct members) of scFv constructs as shown in step 122. 910 scFv) are used in phage display assays. Strongly binding phage will naturally bind via a matching scFv displayed by the phage, which scFv is encoded by the corresponding nucleic acid found in the bound phage. Sequence analysis of the phage DNA will then reveal the sequences of the complementarity-determining regions in the scFv (typically at least CDR1-H, CDR2-H, and CDR3-H, and CDR1-L, CDR2-L, and CDR3-L), as shown in step 124. This information can then be used as guidance for modifying nucleic acids encoding human or humanized scFvs or other antibodies. Once expressed in a suitable expression system, the synthetic antibody ("synbody") is then formulated into a desired therapeutic entity, for example, by binding the antibody to NK cells or by grafting the corresponding scFv onto a chimeric T cell receptor.

[0024] More specifically, and with respect to obtaining omics information from a patient to identify one or more neoepitopes, it is generally contemplated that omics data be obtained from one or more patient biopsies according to standard tissue processing and sequencing protocols. While not limiting the subject matter of the present invention, it is typically preferred that the data be patient-matched tumor data (e.g., tumor versus normal data from the same patient), and that the data format be SAM, BAM, GAR, or VCF format. However, unmatched or matched versus other references (e.g., previous normals or tumors from the same patient, or homo statistic data) are also considered suitable for use herein. Thus, the omics data may be "fresh" omics data or omics data obtained from a previous procedure (or even from a different patient). For example, neoepitopes can be identified in a first step from a patient's tumor by analysis of the whole genome and / or exome of a tumor biopsy (or lymphatic biopsy or metastatic biopsy) and matched normal tissue (i.e., non-diseased tissue from the same patient, such as peripheral blood), via location-guided synchronous comparison of the omics information thus obtained.

[0025] Among other options, it is contemplated that genomic analysis can be performed by a number of analytical methods, with particularly preferred analytical methods including WGS (whole genome sequencing) and exome sequencing of tumor and matched normal samples using next-generation sequencing such as massively parallel sequencing, ion torrent™ sequencing, pyrosequencing, and the like. Similarly, it should be appreciated that computational analysis of sequence data can be performed in a number of ways. However, in most preferred methods, analysis is performed in silico by location-guided synchronous alignment of tumor and normal samples, as disclosed, for example, in U.S. Patent Application Publication Nos. 2012 / 0059670A1 and 2012 / 0066001A1, using BAM files and a BAM server. Of course, alternative file formats for sequence analysis (e.g., SAM, GAR, FASTA, etc.) are expressly contemplated herein.

[0026] It should be noted that any reference to computers should be read to include any appropriate combination of computing devices, such as servers, interfaces, systems, databases, agents, peers, engines, controllers, or other types of computing devices operating individually or collectively. It should be understood that a computing device includes a processor configured to execute software instructions stored on a tangible, non-transitory, computer-readable storage medium (e.g., a hard drive, solid-state drive, RAM, flash, ROM, etc.). The software instructions preferably configure the computing device to provide roles, responsibilities, or other functionality as discussed below with respect to the disclosed apparatus. Furthermore, the disclosed technology can be embodied as a computer program product, including a non-transitory, computer-readable medium storing software instructions that cause a processor to perform the disclosed steps associated with implementing a computer-based algorithm, process, method, or other instructions. In particularly preferred embodiments, the various servers, systems, databases, or interfaces exchange data using standardized protocols or algorithms, perhaps based on HTTP, HTTPS, AES, public-private key exchange, web services APIs, known financial transaction protocols, or other methods of exchanging electronic information. Data exchange between devices can occur over a packet-switched network, the Internet, a LAN, a WAN, a VPN, or other type of packet-switched network, a circuit-switched network, a cell-switched network, or other type of network.

[0027] Of course, it should be recognized that downstream analysis of the sequence differences so identified can be performed to identify those that lead to new peptide sequences based on cancer- and patient-specific mutations. Thus, neoepitopes may be identified by considering the type of mutation (e.g., deletion, insertion, transversion, transition, translocation) and effect (e.g., nonsense, missense, frameshift, etc.), and as such can act as a content filter to filter out silent mutations and other irrelevant (e.g., non-expressed) mutations.

[0028] It should be further understood that neoepitope sequences as contemplated herein can be defined as relatively short (e.g., 5-30 mers, more typically 7-11 mers, or 12-25 mers) sequence stretches containing alteration(s) in the amino acid sequence. Most typically, the alteration(s) are located in or near the center (e.g., less than 4, 5, or 6 amino acids from the center). Thus, and viewed from a different perspective, neoepitope sequences contemplated herein will particularly include those in which a single amino acid has been exchanged with respect to the matching normal sequence, and the site of the altered amino acid is in the center or near the center of the neoepitope sequence (e.g., in the case of a 9 mer, the altered amino acid is at position 2, 3, 4, or 5, more typically at position 3, 4, or 5, and most typically at position 4 or 5). Thus, it will be appreciated that a single amino acid change can be represented in multiple neoepitope sequences containing the altered amino acid, depending on the location of the altered amino acid. Advantageously, such sequence diversity allows for multiple selection of neoepitopes, thereby increasing the number of potentially useful targets, which can then be selected based on one or more desirable traits (e.g., highest affinity for the patient's HLA type, highest structural stability, etc.). Most typically, neoepitopes will be predicted to have a length of 2-50 amino acids, more typically 5-30 amino acids, and most typically 9-15 amino acids, with the altered amino acid preferably centrally located or otherwise positioned to improve binding to MHC. For example, if the epitope is to be presented by the MHC-I complex, the typical epitope length will be about 8-11 amino acids, while the typical epitope length for presentation via the MHC-II complex will be about 13-17 amino acids. As will be readily appreciated, the site of the altered amino acid in the neoepitope may be other than central, so the actual peptide sequence and, with that sequence, the actual topology of the neoepitope may vary considerably.Furthermore, when neoepitopes are presented to immunocompetent (or other) cells as synthetic peptides, it will be appreciated that the synthetic peptides may be significantly longer than the portion of the peptide ultimately bound by the MHC-I or MHC-II system, thus allowing for proteolytic processing within the cell. For example, contemplated synthetic peptides may thus have 8-15 amino acids upstream and downstream of the altered amino acid.

[0029] Regarding filtering identified neoepitopes, it is generally contemplated that neoepitopes are particularly suitable for use herein if omics (or other) analysis reveals that the neoepitopes are actually expressed. Identifying neoepitopes' expression and expression levels can be performed in any manner known in the art, with preferred methods including quantitative RNA (hnRNA or mRNA) analysis and / or quantitative proteomics analysis. Most typically, the threshold for incorporating neoepitopes will be an expression level that is at least 20%, and more typically at least 50%, of the expression level of the corresponding matching normal sequence, thereby ensuring that the (neo)epitope is at least potentially "visible" to the immune system. Therefore, it is generally preferred that the omics analysis further include analysis of gene expression (transcriptome analysis), thus helping to identify the expression levels of genes with mutations. Numerous methods for transcriptome analysis are known in the art, and all known methods are considered suitable for use herein. For example, preferred materials include mRNA and primary transcripts (hnRNA), where the RNA sequence information is reverse transcribed poly(A) + - RNA, which may also be obtained from tumor samples and matched normal (healthy) samples from the same patient. Also noteworthy is the polyA +The point is that, although -RNA is typically preferred as a representation of the transcriptome, other forms of RNA (hnRNA, non-polyadenylated RNA, siRNA, miRNA, etc.) are also considered suitable for use herein. Preferred methods include quantitative RNA (hnRNA or mRNA) analysis and / or quantitative proteomic analysis. Most typically, RNA quantification and sequencing is performed using qPCR and / or rtPCR-based methods, although other methods (e.g., solid-phase hybridization-based methods) are also considered suitable. From another perspective, transcriptome analysis may be suitable (alone or in combination with genomic analysis) for identifying and quantifying genes with cancer- and patient-specific mutations.

[0030] Similarly, proteomic analysis can be performed in a number of ways to confirm neoepitope expression, and any known method or proteomic analysis is contemplated herein. However, particularly preferred proteomic methods include antibody-based methods and mass spectrometry. Furthermore, it should be noted that proteomic analysis may not only provide qualitative or quantitative information about proteins in nature, but may also include protein activity data if the protein has catalytic or other functional activity. One example of a technique for performing proteomic assays is U.S. Patent No. 7,473,532, filed March 10, 2004, to Darfler et al., entitled "Liquid Tissue Preparation from Histopathologically Processed Biological Samples, Tissues, and Cells."

[0031] Additionally, neoepitopes may be further subjected to detailed analysis and filtering using predetermined structural and / or subcellular location parameters. For example, it is contemplated that a neoepitope sequence may be selected for further use if the sequence is identified as being in a membrane-associated location (e.g., located on the outside of the cell's plasma membrane) and / or if in silico structural prediction confirms that the neoepitope is likely to be solvent-exposed or represents a structurally stable epitope.

[0032] It should be recognized, therefore, that patient- and cancer-specific neoepitopes can be identified from omics information only in an in silico environment, ultimately predicting potential epitopes specific to the patient and tumor type. Such identified and selected neoepitopes can then be further filtered in silico against the identified patient HLA type. Such HLA matching is believed to ensure strong binding of the neoepitopes to the MHC-I complex of nucleated cells and the MHC-II complex of specific antigen-presenting cells. Targeting both antigen-presenting systems is particularly believed to generate therapeutically effective and durable immune responses involving both the cellular and humoral branches of the immune system. Of course, it should also be recognized that the HLA-matched neoepitopes identified in this way can be biochemically validated in vitro.

[0033] HLA determination for both MHC-I and MHC-II can be performed using a variety of wet chemistry methods well known in the art, and all of these methods are considered suitable for use herein. However, in a particularly preferred method, HLA types can also be predicted in silico from omics data using a reference sequence containing most or all of the known and / or common HLA types, as described in more detail below. Briefly, a patient's HLA type is identified (using wet chemistry or in silico determination), and a structural solution for the HLA type is calculated or obtained from a database, which is then used as a docking model in silico to determine the binding affinity of the neoepitope to the HLA structural solution. Suitable systems for determining binding affinity include the NetMHC platform (see, e.g., Nucleic Acids Res., July 1, 2008, Vol. 36 (Web Server Version), W509-W512), HLA Matchmaker (http: / / www.epitopes.net / downloads.html), and the IEDB Analysis Resource (http: / / tools.immuneepitope.org / mhcii / ). Neoepitopes with high affinity for the previously determined HLA type (e.g., less than 100 nM, less than 75 nM, or less than 50 nM for MHC-I and less than 500 nM, less than 300 nM, or less than 100 nM for MHC-II) are then selected. Upon predicting the highest affinity, neoepitope modification may be performed by N- and / or C-terminal modifications to further enhance binding of the synthetic neoepitope to the patient's HLA type. Thus, neoepitopes may be naturally identified or may be further modified to better suit particular HLA types.

[0034] In yet another aspect of filtering, neoepitopes may be compared against a database containing known human sequences, thus avoiding the use of human-identical sequences. Furthermore, filtering may also include removing neoepitope sequences resulting from patient SNPs. For example, the Single Nucleotide Polymorphism Database (dbSNP) is a free, public archive of genetic variation within and across various species developed and hosted by the National Center for Biotechnology Information (NCBI) in collaboration with the National Human Genome Research Institute (NHGRI). Although the name of the database implies a collection of only one type of polymorphism (i.e., single nucleotide polymorphisms (SNPs)), it actually contains a relatively wide range of molecular variations, namely: (1) SNPs, (2) short deletion-insertion polymorphisms (indels / DIPs), (3) microsatellite markers or short tandem repeats (STRs), (4) multinucleotide polymorphisms (MNPs), (5) heterozygous sequences, and (6) named variants. dbSNP clearly lists neutral polymorphisms, polymorphisms corresponding to known phenotypes, and regions without mutations. Using such a database, patient- and tumor-specific neoepitopes may be further filtered to remove these known sequences, resulting in therapeutic sequences designed with multiple neoepitope sequences.

[0035] Consequently, while of course such identified cancer neoepitopes are unique to the patient and their particular cancer (e.g., having a frequency of less than 0.1%, and more typically less than 0.01%, of all neoepitopes in a population of cancer patients diagnosed with the same cancer), such identified cancer neoepitopes are likely to be presented in tumors and therefore likely to be specifically targeted by synthetic antibodies, even if the cancer has an immunosuppressive microenvironment.

[0036] In practical terms, neoepitopes can be scored / ranked based on allele frequency multiplied by the number of transcripts per million to obtain a likelihood score. This score can then be further enhanced using HLA information and calculated or actual binding affinity for the patient's HLA type. For example, an exemplary ranking format is: >254 NM_001000.3 RPL39 Missense p.M29K A->T Normal: WIRMKTGNK, AF: 0.179104477612 TPM: 1023.96 TPM_MEDIAN: 7.35 LL: 183.395820896 netMHC: 242.96 Allele: HLA-A0301 WIRKKTGNK It may be.

[0037] Here, the file is a FASTA format file, and entries start with the character ">", which simply conveys sample information. The next line is the neoepitope. The sample information line includes the number used to index the sample (e.g., 254), the Refseq gene ID (e.g., NM_001000.3), the HUGO synonym (e.g., RPL39), the variant classification (e.g., Missense), the protein change (e.g., p.M29K), the base pair change (e.g., A->T), the normal epitope (e.g., Normal: WIRMKTGNK ), the allele frequency (e.g., AF: 0.179104477612), the transcripts per million for this gene (e.g., TPM: 1023.96), the TPM_MEDIAN, which is the mean expression level of all genes (e.g., TPM_MEDIAN: 7.35), the LL score, which is just AF x TPM (e.g., LL: 183.395820896), and the netMHC predicted binding value (e.g., netMHC: 242.96), and the specific HLA allele to which the neoepitope binds (e.g., Allele: HLA-A0301). The next line is the neoepitope (e.g., WIRKKTGNK).

[0038] The feasibility of such an approach was demonstrated by the inventors using omics information from the publicly available TCGA database covering a number of cancers, each with data available for a large number of patients. Table 1 below lists the names and cancer types, followed by the neoepitopes found for each cancer type.

[0039] [Table 1]

[0040] Thus, it should be recognized that it is feasible to create an entire rationally designed collection of neoepitopes for a particular patient with a particular cancer, a collection that can then be further tested in vitro to discover or generate high-affinity antibodies. Indeed, contemplated collections may include 1, 2, 3, 4, 5, 6-10, 10-50, 50-150, 1,000, or more neoepitopes specific to the patient and cancer. Viewed from different perspectives, a rationally designed collection of neoepitopes can include 1-10%, or 10-25%, or 25-60%, or 60-100% of all neoepitopes expressed and binding to the patient's HLA type. Thus, contemplated collections would comprise at least 15%, at least 25%, at least 50%, at least 70%, or at least 90% of the cancer immunome (neoepitopes expressed and binding to the patient's HLA type). Consequently, it should also be recognized that multiple targets for immunotherapy are now available, even for patients with immune systems damaged by tumor immunosuppression or chemotherapy.

[0041] To obtain synthetic antibodies against identified neoepitopes, it is contemplated that those identified in silico may be prepared in vitro to obtain synthetic peptides. Numerous methods for preparing synthetic peptides are known in the art, and all known methods are considered suitable for use herein. For example, peptides comprising cancer neoepitope sequences can be prepared on solid phase (e.g., using Merrified synthesis), by liquid phase synthesis, or from smaller peptide fragments. In a less preferred embodiment, peptides may be produced by expression of recombinant nucleic acids in a suitable host (especially when multiple neoepitopes are on a single peptide chain, optionally with spacers or cleavage sites between the neoepitopes).

[0042] Therefore, the structure of a synthetic peptide corresponding to or comprising a neoepitope sequence is X-L1-(A n -L2) m -Q, where X is an optional linking group or moiety suitable for covalently or non-covalently attaching the synthetic peptide to a solid phase, and L1 is an optional linker that covalently attaches the synthetic peptide to the solid phase or linking group. nis a synthetic peptide having a neoepitope sequence, where A is a natural (proteinogenic) amino acid and n is an integer between 7 and 30, most typically between 7 and 11 or 15 and 25. L2 is an optional linker that may be present, particularly when multiple synthetic peptide sequences (identical or different) are present in the construct, and m is typically an integer between 1 and 30, most typically between 2 and 15. Finally, Q is a terminal group that may be used to link the end of the synthetic peptide to a solid phase (e.g., to sterically constrain the peptide) or to a reporter group (e.g., a fluorescent marker) or other functional moiety (e.g., an affinity marker). Consequently, it should be noted that when synthetic peptides are used for direct MHC-I binding, the total length will be between 8 and 10 amino acids. Similarly, when synthetic peptides are used for direct MHC-II binding, the total length will be between 14 and 20 amino acids. On the other hand, if the synthetic peptide is processed intracellularly (typically by proteasomal processing) prior to MHC presentation, the total length will typically be 10-40 amino acids, with the altered amino acid at or near the central position in the synthetic peptide.

[0043] For example, X could be a non-covalent affinity moiety (e.g., biotin) that binds to a corresponding binding agent (e.g., avidin) on the solid phase, or a chemical group (with or without a spacer) that reacts with amino or carboxyl groups at the N- or C-terminus of the peptide, or a selectively reactive group (e.g., an iodoacetyl group or a maleimide group) that reacts with sulfhydryl groups in the peptide or linker L1. L1 may be used to increase the distance of the synthetic peptide from the solid phase and thus will typically comprise a flexible linear portion (e.g., comprising a glycol group, an alkoxy group, glycine, etc.) having a length equivalent to about 2-20 carbon-carbon bonds (e.g., 0.3 nm-3 nm). Of course, it should also be recognized that the synthetic peptide may use the solid phase on which it was produced and, as such, may not require a separate linking group or linker.

[0044] The nature of the solid phase can, of course, vary considerably depending on the particular synthetic peptide and coupling method; all known solid phases for attaching peptides are considered suitable for use herein. For example, suitable solid phases include agarose beads, polymer beads (colored or otherwise individually addressable), the walls of microtiter plate wells, paper, nitrocellulose, glass, and the like. Those skilled in the art will readily appreciate the appropriate choice of solid phase and attachment chemistry. In a further preferred embodiment, it is further noted that the solid phase will be generally suitable for protocols associated with phage display methods, for example, to allow peptides displayed on phage (or other scaffold carriers) to be reversibly bound to the solid phase via the synthetic peptide. It should also be recognized that in contemplated applications, the solid phase may be a carrier protein used in vaccination (e.g., albumin, KLH, tetanus toxoid, diphtheria toxin, etc.), particularly when the synthetic protein is used as a vaccine in mammals or as an immunogenic compound for antibody production in mammals other than humans. Similarly, the synthetic proteins may be used as vaccines or immunogenic compounds without a carrier.

[0045] It should be appreciated that in an even more preferred method, when synthetic peptides (comprising or corresponding to cancer neoepitopes) are immobilized on a solid phase, affinity agents, and in particular antibodies, directed against the neoepitopes can be isolated and / or purified. Most preferably, such isolation will include a pre-made high diversity antibody library. As used herein, and unless the context indicates otherwise, the term "antibody" or "antibodies" includes all antibody isotypes and subtypes (e.g., IgG, IgM, IgE, etc.) and all fragments thereof, such as monovalent IgG, F(ab')2, Fab', Fab, scFv, scFv-Fc, VhH, etc. Furthermore, contemplated antibodies may be humanized, of human or non-human (e.g., rodent) origin, or chimeric antibodies. In a typical method, a high diversity library will contain at least 10 diverse antibodies. 9 Each different member of a species, or at least 10 10 Phage display libraries having different members of each species, or even more, typically based on M13 phage, displayed via pIII, pVIII, pVI, or pIX, or libraries based on T7 phage and g10 capsid proteins, may be used. As should be readily appreciated, the use of large diversity libraries will provide several binding candidate antibodies in a relatively short time, which can be further selected for best binding. In practice, it should be recognized that if the binding affinity to the immobilized synthetic peptide is lower than desired, the affinity can be improved by affinity maturation using protocols well known in the art. For example, the low affinity (K D >10 -7Binders or members of the M) may be subjected to affinity maturation to improve binding affinity and / or binding kinetics using methods well known in the art (see, e.g., Briefings In Functional Genomics And Proteomics, July 2002, Vol. 1, No. 2, pp. 189-203). In addition, it should be noted that while antibody libraries are generally preferred, other scaffolds may also be suitable, including β-barrel, ribosome display, cell surface display, etc. (see, e.g., Protein Sci., January 2006, Vol. 15, No. 1, pp. 14-27). Thus, it should be appreciated that in a preferred embodiment, synthetic peptides are used as bait in antibody libraries, thus enabling the generation of high affinity (K D <10 -7 M, and typically K D <10 -8 Antibodies that bind to the antibody are identified.

[0046] It should be further appreciated that because antibodies are directly linked to cells carrying nucleic acids encoding these antibodies, such nucleic acids can then be analyzed to identify sequence elements encoding the hypervariable loops, CDR1, CDR2, and CDR3, and / or SDRs (specificity-determining residues) for the light and heavy chains, respectively. Most typically, this determination is performed using standard sequencing methods. Once determined, it is next contemplated that the hypervariable loops, or CDR1-H, CDR2-H, and / or CDR3-H, and / or CDR1-L, CDR2-L, and / or CDR3-L, and / or SDRs, can be grafted onto a human or humanized antibody scaffold or antibody. As will be readily appreciated, grafting can be achieved by genetic engineering of the nucleic acid encoding the human or humanized antibody scaffold or antibody. For example, within each CDR, there are highly variable sites, i.e., specificity-determining residues (SDRs), that are directly involved in the interaction with the antigen, while there are highly conserved residues that maintain the conformation of the CDR loop. SDRs can be identified from the 3D structure of an antigen-antibody complex and / or mutational analysis of the CDRs. SDR-grafted humanized antibodies are constructed by grafting SDRs and residues that maintain the conformation of the CDRs onto a human template. Consequently, it should be recognized that human or humanized antibodies with specificity for cancer neoepitopes can be prepared in a completely artificial manner, where the antibodies are expressed in cells that have not previously been exposed to the antigen. Furthermore, the contemplated methods enable the production of patient- and cancer-specific antibodies for the treatment of patients who have failed to produce or effectively utilize antibodies against neoepitopes.

[0047] Without limiting the subject matter of the present invention, the synthetic antibodies so prepared can be used directly as IgG (or other isotypes), as fragments (e.g., bispecific Fab or other bispecific fragments), and / or as chimeric proteins (e.g., scFv as the ectodomain of a chimeric T-cell receptor), alone or in combination with therapeutic or diagnostic agents, and / or as hybrid proteins with a transmembrane domain ensuring cell membrane anchoring of the antibody to the cell. Thus, the inventors contemplate a method for generating agents for cancer immunotherapy, in which the synthetic antibodies so identified are linked to a therapeutic or diagnostic agent (which may have a cellular or non-cellular component), thus obtaining the agent.

[0048] For example, contemplated non-cellular agents include various chemotherapeutic agents for directly delivering chemotherapeutic agents to cancer cells. For example, suitable chemotherapeutic agents include kinase inhibitors (e.g., erlotinib, imatinib, bortezomib, etc.), topoisomerase inhibitors (e.g., topotecan, etoposide, teniposide, etc.), nucleotide analogs (e.g., fluorouracil, gemcitabine, azacitidine, etc.), platinum-based agents (e.g., cisplatin, carboplatin, etc.), alkylating agents (e.g., cyclophosphamide, chlorambucil, temozolomide, etc.), taxanes (e.g., docetaxel, paclitaxel, etc.), and microtubulin inhibitors (e.g., vincristine, vinblastine, etc.). On the other hand, directed and site-specific radiotherapy may be used by linking a radioactive agent to an antibody to selectively destroy cancer cells. Suitable radioactive agents include all radioactive agents suitable for brachytherapy, especially 125 I, 103 Pd, or 192 Another example is Ir. If neutron capture therapy using low-energy thermal neutrons is desired, 10 B may also be used. Similarly, imaging agents may be linked to antibodies or fragments thereof, and particularly preferred imaging agents include PET labels (e.g. 11 C. 13 N,15 O and 18 F) and SPECT labels (e.g. 123 I, 99m Tc, 133 Xe, 201 Tl and 18 F). As used herein, and unless the context indicates otherwise, the term "coupled to" is intended to include both direct coupling (the two elements coupled to each other are in contact with each other) and indirect coupling (there is at least one additional element between the two elements). Thus, the terms "coupled to" and "coupled with" are used interchangeably. In a further contemplated embodiment, the antibody may be modified with an antigen known to be an immunogenic antigen. Such modification is particularly advantageous if the patient has previously been immunized with the same antigen. In such a situation, it is contemplated that neoepitope-bearing cancer cells may be "painted" with the modified antibody presenting the immunogenic antigen, which is particularly advantageous if the immune response to the original neoepitope was not immunogenic or was suppressed.

[0049] It should be noted that when a neoepitope is used to target immune cells to tumors, the antibody can also be linked to a portion of a T cell receptor or to a cytotoxic T cell or NK cell. For example, when the antibody is used in a chimeric T cell receptor of a cytotoxic T cell, the antigen-binding portion of the chimeric T cell receptor can have an scFv as an ectodomain, and the scFv has binding affinity for one of the neoepitopes (e.g., those of SEQ ID NOs: 1 to 1,408,729). On the other hand, when the antibody is used in conjunction with NK cells, preferred NK cells are NK-92 derivative cells modified to reduce or abolish expression of at least one killer cell immunoglobulin-like receptor (KIR), resulting in constitutive activation of such cells (due to the absence or reduction of inhibition). Such NK cells are available as aNK cells ("activated NK cells") from NantKwest (see nantkwest.com) and can be further engineered to express membrane-bound synthetic antibodies with binding affinity to neoepitopes (e.g., those of SEQ ID NO: 1 to SEQ ID NO: 1,408,729).

[0050] As another example, NK cells may be NK-92 derivative cells modified to express a high-affinity Fcγ receptor (CD16), and it is specifically contemplated that the antibodies contemplated herein may bind to such modified NK cells. Such cells are available from NantQuest as haNK cells ("high-affinity natural killer cells"). Similarly, NK cells may be genetically engineered to express chimeric T cell receptors. In particularly preferred embodiments, the chimeric T cell receptor will have an scFv portion or other ectodomain with binding specificity for a neoepitope (e.g., SEQ ID NO: 1 to SEQ ID NO: 1,408,729). Of course, it should also be noted that where a therapeutic agent has a cellular component, the cells may be autologous or xenogeneic cells derived from the patient.

[0051] Consequently, it should be appreciated that effective immune responses to cancer neoepitopes can be elicited using processes that do not require immunization in patients or other organisms, dramatically reducing response times and the use of therapeutic antibodies. Indeed, by using contemplated compositions and methods, it is possible to stimulate or even trigger an immune response to neoepitopes in a patient when the patient's immune system is insufficient to generate a protective response (e.g., due to chemotherapy, or due to immunosuppression by the tumor or Treg cells or myeloid-derived suppressor cells).

[0052] It will be appreciated, therefore, that depending on the particular composition, the agent may be administered to a patient in vivo or to cells or tissues in vitro. For example, if a synthetic antibody is used for diagnosis, the antibody may be added to a tissue sample ex vivo (e.g., to an FFPE sample using a fluorescently labeled antibody on a microscope slide) or in vivo (e.g., when the antibody is labeled with a PET label). On the other hand, if the antibody is bound to an immunocompetent cell, the antibody may be administered to a patient in vivo.

[0053] In some embodiments, numbers expressing properties such as quantities and concentrations of ingredients, reaction conditions, and the like, used to describe and claim certain embodiments of the present invention are to be understood as being modified in some cases by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the written description and accompanying claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as practicable. Unless the context indicates otherwise, all ranges set forth herein should be construed as inclusive of the endpoints of the range, and open-ended ranges should be construed to include commercially practical values. Similarly, all lists of values ​​should be construed to include intermediate values ​​unless the context indicates otherwise.

[0054] It should be apparent to those skilled in the art that many further modifications beyond those already described are possible without departing from the inventive concepts herein. The subject matter of the present invention, therefore, should not be limited except as by the scope of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted as broadly as possible consistent with the context. In particular, the terms "comprise" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that a referenced element, component, or step can be present in, utilized with, or combined with other elements, components, or steps not explicitly referenced. When a specification or claim refers to at least one something selected from a group consisting of A, B, C, ..., and N, the sentence should be interpreted as requiring just one element from the group, not A plus N, or B plus N, etc. (Appendix 1) 1. A method for producing a drug for cancer immunotherapy, comprising: using matched normal omics data for the tumor to generate in silico a plurality of n-mers containing at least one patient-specific and cancer-specific cancer neoepitope; filtering said n-mers in silico, thus obtaining a subset of neoepitope sequences; preparing at least one synthetic n-mer peptide using sequence information from said subset of neoepitope sequences; using the synthetic n-mer peptide to isolate a recombinant antibody; Obtaining sequence information of the complementarity determining regions of the recombinant antibody; generating a synthetic antibody using sequence information of the complementarity determining regions of said recombinant antibody; and b. linking said synthetic antibody to a therapeutic or diagnostic agent, thereby obtaining the agent. (Appendix 2) 2. The method of claim 1, wherein the matched normal omics data is at least one of whole genome sequencing data, exome sequencing data, and transcriptome data. (Appendix 3) 2. The method of claim 1, wherein the matched normal omics data is matched to normal data from before treatment of the patient. (Appendix 4) 2. The method of claim 1, wherein each of the n-mer peptides has a length of 7 to 11 amino acids. (Appendix 5) 2. The method of claim 1, wherein the plurality of n-mer peptides is at least 1,000 n-mer peptides. (Appendix 6) 2. The method of claim 1, wherein different ones of the plurality of n-mer peptides have different neoepitopes. (Appendix 7) 2. The method of claim 1, wherein the filtering step includes at least one of filtering by mutation type, filtering by expression intensity, filtering by intracellular location, and filtering by binding affinity to the patient's HLA type. (Appendix 8) 2. The method of claim 1, wherein the filtering step includes at least two of filtering by mutation type, filtering by expression intensity, filtering by intracellular location, and filtering by binding affinity to the patient's HLA type. (Appendix 9) 2. The method of claim 1, wherein the filtering step includes at least three of filtering by mutation type, filtering by expression intensity, filtering by intracellular location, and filtering by binding affinity to the patient's HLA type. (Appendix 10) 2. The method of claim 1, wherein the step of using the synthetic n-mer peptide to isolate a recombinant antibody comprises phage panning. (Appendix 11) 11. The method of claim 10, wherein the phage panning step further comprises affinity maturation. (Appendix 12) The method described in Appendix 1, wherein the sequence information of the complementarity determining regions of the recombinant antibody comprises CDR1-H, CDR2-H, and CDR3-H. (Appendix 13) 2. The method of claim 1, wherein the synthetic antibody is generated using grafting of CDRs or SDRs onto a human antibody scaffold. (Appendix 14) The synthetic antibodies can be produced by recombinant expression, including IgG, F(ab') 2 , Fab', Fab, or scFv. (Appendix 15) 2. The method of claim 1, wherein the therapeutic or diagnostic agent is a non-cellular agent. (Appendix 16) 16. The method of claim 15, wherein the non-cellular agent is a chemotherapeutic agent, a radioisotope, a PET-detectable isotope, a SPECT-detectable isotope, or an affinity agent. (Appendix 17) 2. The method of claim 1, wherein the therapeutic agent is a cell. (Appendix 18) 18. The method of claim 17, wherein the cell is a T cell or an NK cell. (Appendix 19) 19. The method of claim 18, wherein the cell is a T cell expressing a chimeric receptor having an scFv as an ectodomain, and the synthetic antibody is an scFv. (Appendix 20) 19. The method of claim 18, wherein the cells are NK cells that express a high affinity Fcγ receptor (CD16), and the synthetic antibody is an IgG and is bound to the NK cells via the high affinity Fcγ receptor. (Appendix 21) 2. The method of claim 1, wherein the agent is obtained in a therapeutically effective amount within less than six weeks of use of matched normal omics data. (Appendix 22) 1. A method for generating synthetic antibodies against a cancer neoepitope in a patient where the cancer neoepitope has not elicited a protective immune response, comprising: using cancer neoepitopes to select binding recombinant antibodies from a library of recombinant antibodies, wherein the cancer neoepitopes are patient-specific and cancer-specific; obtaining specificity information about said binding recombinant antibody by analyzing the hypervariable loops of said binding recombinant antibody; using said specificity information to modify a gene encoding at least a portion of a human antibody; and recombinantly expressing the modified gene to produce said synthetic antibody. (Appendix 23) 23. The method of claim 22, wherein the cancer neoepitope is an HLA-matched cancer neoepitope. (Appendix 24) 23. The method of claim 22, wherein the library of recombinant antibodies is a phage display library. (Appendix 25) 23. The method of claim 22, further comprising affinity maturation of the binding recombinant antibody to derive a recombinant antibody with optimized binding. (Appendix 26) 23. The method of claim 22, wherein the step of analyzing the hypervariable loops comprises sequencing DNA encoding the hypervariable loops. (Appendix 27) 23. The method of claim 22, wherein the modifying step comprises grafting CDRs or SDRs. (Appendix 28) 23. The method of claim 22, wherein at least a portion of the human antibody is an scFv. (Appendix 29) The step of recombinantly expressing the gene includes the step of recombinantly expressing IgG, F(ab') 2 23. The method of claim 22, wherein the synthetic antibody is produced in the form of Fab', Fab, or scFv. (Appendix 30) 23. The method of claim 22, wherein the cancer neoepitope is expressed in the patient's cancer. (Appendix 31) 23. The method of claim 22, wherein the neoepitope is unique to the patient and the patient's cancer. (Appendix 32) A composition comprising a synthetic antibody having binding affinity for a cancer-specific and patient-specific HLA-matched cancer neoepitope, wherein the HLA-matched cancer neoepitope is patient-specific and specific to the patient's cancer. (Appendix 33) 33. The composition of claim 32, wherein the HLA-matched cancer neoepitope is matched for MHC-I presentation. (Appendix 34) The synthetic antibody is an IgG, F(ab') 2 33. The composition of claim 32, wherein the antibody is selected from the group consisting of Fab', Fab, and scFv. (Appendix 35) 33. The composition of claim 32, wherein the synthetic antibody is linked to a therapeutic agent. (Appendix 36) 36. The composition of claim 35, wherein the therapeutic agent is a non-cellular agent. (Appendix 37) 37. The composition of claim 36, wherein the non-cellular agent is a chemotherapeutic agent, a radioisotope, a PET-detectable isotope, a SPECT-detectable isotope, or an affinity agent. (Appendix 38) 36. The composition of claim 35, wherein the therapeutic agent is a cell. (Appendix 39) 39. The composition of claim 38, wherein the cells are T cells or NK cells that optionally express a genetically modified CD16 receptor. (Appendix 40) 40. The composition of claim 39, wherein the cell is a T cell expressing a chimeric receptor having an scFv as an ectodomain, and the synthetic antibody is an scFv. (Appendix 41) 40. The composition of claim 39, wherein the cells are NK cells that express a high affinity Fcγ receptor (CD16), and the synthetic antibody is an IgG and binds to the NK cells via the high affinity Fcγ receptor. (Appendix 42) 33. The composition of claim 32, wherein the HLA-matched cancer neoepitope has a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 1,408,729. (Appendix 43) 1. A composition comprising a solid phase having bound thereto a cancer-specific and patient-specific HLA-matched cancer neoepitope, wherein the HLA-matched cancer neoepitope is specific to the patient and to the patient's cancer. (Appendix 44) 44. The composition of claim 43, wherein the solid phase comprises an inner wall of a reagent vessel, a magnetic bead, or an individually addressable element. (Appendix 45) 44. The composition of claim 43, wherein the HLA-matched cancer neoepitope has a length of 7 to 9 amino acids. (Appendix 46) 44. The composition of claim 43, further comprising a synthetic antibody binding to the HLA-matched cancer neoepitope. (Appendix 47) The conjugated synthetic antibody may be IgG, F(ab') 2 47. The composition of claim 46, wherein the antibody is selected from the group consisting of Fab', Fab, and scFv. (Appendix 48) 47. The composition of claim 46, wherein the synthetic antibody is linked to a viral particle. (Appendix 49) 47. The composition of claim 46, wherein the HLA-matched cancer neoepitope has a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 1,408,729.

Claims

1. 1. A method for producing a recombinant antibody that binds to a cancer neoepitope in a patient, comprising: obtaining nucleic acid sequence information from the patient's tumor tissue and matched normal tissue; identifying in silico one or more candidate cancer neoepitopes present in the tumor tissue and absent in the normal tissue, wherein the identification comprises predicting the binding affinity of the candidate neoepitopes to the patient's MHC alleles; screening a recombinant antibody library using the identified candidate cancer neoepitopes to select antibodies that bind to said candidate neoepitopes; obtaining specificity information about selected antibodies from said recombinant antibody library by analyzing the hypervariable loops of the selected antibodies; using said specificity information to modify a gene encoding at least a portion of a human antibody; recombinantly expressing the modified gene to produce the recombinant antibody; The method comprising:

2. 10. The method of claim 1, further comprising the step of affinity maturation of antibodies selected from said recombinant antibody library to derive optimized recombinant antibodies.

3. 2. The method of claim 1, wherein the step of analyzing the hypervariable loop comprises sequencing DNA encoding the hypervariable loop.

4. The method of claim 1 , wherein the modifying step comprises grafting a CDR or an SDR.

5. The step of recombinantly expressing the gene includes the step of recombinantly expressing IgG, F(ab'), ２ 10. The method of claim 1, wherein the synthetic antibody is produced in the form of a Fab', Fab, or scFv.

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