COMPOSITIONS AND METHODS FOR IDENTIFYING FUNCTIONAL ANTI-TUMOR T CELL RESPONSES

Abstract

The invention features compositions and methods for identifying functional anti-tumor T cell responses.

Claims

1. A method of functionally evaluating a candidate antigen for the ability to induce a T cell response comprising: obtaining a test sample of blood or tumor-infiltrating lymphocytes from a subject having or at risk of developing a cancer or a viral infection; stimulating expansion of autologous T cells from the subject with the candidate antigen, said candidate antigen comprising a peptide, a protein or a minigene transfected into autologous monocytic cells; isolating deoxyribonucleic acid (DNA) from the T cells; amplifying the T cell receptor- (TCR-) complementarity-determining region 3 (CDR3) DNA; determining a level of antigen-specific T cell expansion; comparing the level of antigen-specific T cell expansion to a level of expansion of T cells in the absence of the candidate peptide; determining that the candidate antigen has the ability to induce a T cell response if the level of antigen-specific T cell expansion is higher than the level of expansion of T cells in the absence of the candidate peptide.

2. The method of claim 1, wherein the autologous T cells from the subject are stimulated to expand with the candidate antigen, said candidate antigen comprising a peptide or whole protein or with autologous peripheral blood mononuclear cells (PBMCs) which have been transfected with a tandem minigene construct encoding the candidate antigen(s).

3. The method of claim 2, wherein antigen-specific T cell expansion is determined by comparing TCR-V clonality prior to stimulation with the candidate antigen or PBMCs to TCR-V clonality after stimulation with the candidate antigen.

4. The method of claim 1, wherein the candidate antigen comprises a tumor antigen or a viral antigen.

5. The method of claim 4, wherein the candidate antigen, in the form of a peptide, protein or minigene transfected into autologous monocytic cells, comprises a tumor mutation-associated neoantigen (MANA), a viral antigen, or a non-mutated tumor-associated antigen.

6. The method of claim 5, wherein the viral antigen is expressed by an integrated cancer-associated virus or a non-oncogenic virus.

7. The method of claim 6, wherein the integrated cancer-associated virus comprises human papilloma virus (HPV) associated with cervical or head and neck cancer, Epstein Barr virus (EBV), Merkel Cell Polyomavirus, Hepatitis B virus (HBV) or Hepatitis C virus (HCV).

8. The method of claim 6, wherein the virus comprises human immunodeficiency virus (HIV).

9. The method of claim 1, wherein the sample comprises a blood sample or a tumor infiltrating lymphocyte population.

10. A method of determining whether a given immunotherapy will inhibit a tumor in a subject comprising: functionally validating a candidate antigen for the ability to induce a T cell response according to the method of claim 1; and determining that immunotherapy will inhibit the tumor if the candidate antigen has the ability to induce a T cell response, thereby determining whether the given immunotherapy should be used to treat the patient.

11. The method of claim 10, wherein it is determined whether immunotherapy will inhibit a tumor prior to or subsequent to administration of the immunotherapy to the subject.

12. The method of claim 11, wherein the immunotherapy comprises administration of an immune checkpoint inhibitor alone or in combination with one or more additional anti-tumor treatments.

13. The method of claim 12, wherein the immune checkpoint inhibitor comprises an anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) antibody, an anti-programmed cell death protein 1 (PD-1) antibody, an anti-programmed death-ligand 1 (PD-L1) antibody, an anti-lymphocyte-activation 3 (LAG3) antibody, an anti-T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) antibody, an anti-T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibition motif (ITIM) domains (TIGIT) antibody, an anti-V domain-containing Ig suppressor of T-cell activation antibody, an anti-cluster of differentiation 47 (CD47) antibody, an anti-signal regulatory alpha (SIRP ) antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-neuritin antibody, an anti-neuropilin antibody, or an anti-interleukin-35 (IL-35) antibody, or any combination thereof.

14. The method of claim 12, wherein the immune checkpoint inhibitor comprises a drug that inhibits indoleamine-pyrrole 2,3-dioxygenase (IDO), A2A adenosine receptor (A2AR), arginase, or glutaminase, or any combination thereof.

15. The method of claim 12, further comprising administering an agonist of a co-stimulatory receptor.

16. The method of claim 15, wherein agonist comprises an anti-glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR) antibody, an anti-CD27 antibody, an anti-4-1BB antibody, an anti-OX40 antibody, an anti-inducible T-cell co-stimulator (ICOS) antibody, or an anti-CD40 antibody, or any combination thereof.

17. A method of determining whether a vaccine will inhibit a tumor or a virus in a subject comprising: functionally evaluating a candidate antigen for the ability to induce a T cell response according to the method of claim 1; and determining that the vaccine will inhibit the tumor or virus if the candidate antigen has the ability to induce a T cell response, wherein the vaccine incorporates comprises the candidate antigen, thereby determining whether the vaccine will inhibit the tumor or virus.

18. The method of claim 17, further comprising administering the vaccine to the subject.

19. The method of claim 17, wherein the vaccine comprises the candidate peptide or a tandem minigene or full gene encoding the candidate antigen incorporated into a recombinant viral or bacterial vaccine.

20. The method of claim 17, wherein the candidate antigen comprises a tumor antigen or a viral antigen.

21. The method of claim 17, wherein the candidate antigen comprises a mutation-associated neoantigen (MANA) or a non-mutated tumor-associated antigen.

22. The method of claim 20, wherein the viral antigen is expressed by an integrated cancer-associated virus or a non-oncogenic virus.

23. The method of claim 1, wherein the subject is a human.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0103] FIG. 1 is a schematic showing an overview of MANAFEST assay development. Exome data are applied in the neoantigen prediction pipeline to generate candidate MANAs. Putative neoantigens are used to generate peptides and stimulate autologous T cells, followed by TCR next-generation sequencing. Intra-tumoral neoantigen-specific TCR expansion is subsequently evaluated. The peptides shown in FIG. 1 have been assigned SEQ ID NOs: 1, 2, and 3, sequentially.

[0104] FIG. 2 is a bar chart showing the results of neoantigen-specific TCR expansion in stimulated T cell cultures. Peptides generated from neoantigen candidates were synthesized and used to pulse autologous peripheral T cells. Reactive TCR clones were matched to clones found in tumor-infiltrating lymphocytes (TILs). Neoantigen-specific TCR reactivity (CDR3: CASSRDRGRGNSPLHF (SEQ ID NO: 4)) was observed for the mutant peptides associated with mutant HELB. Adjusted p value is given for pairwise comparisons between productive frequencies in peptide stimulated versus unstimulated T cells. Solid bars represent mutant and shaded bars denote wild type peptides. The peptides shown in FIG. 2 have been assigned SEQ ID NOs: 5-16, sequentially.

[0105] FIGS. 3A-3C are graphs showing that IFN ELISpot underestimates the breadth of the antigen-specific T cell response. T cells from healthy donor JH014 were stimulated with one of 13 known MHC class I-restricted epitopes and cultured for 10 days. IFN ELISpot was performed on an aliquot of cultured T cells (left panels) and TCR VP CDR3 sequencing was performed on the remaining T cells (right panels). ELISpot data are shown as the number of spot forming cells (SFC) per 10.sup.6 T cells with background subtracted for 3 tested epitopes. Accompanying clonotypic expansions are also shown in response to these 3 epitopes, EBV EBNA 4NP (FIG. 3A), EBV EBNA 3A (FIG. 3B), and EBV 1 (FIG. 3C). Background is the mean number of SFC detected without peptide stimulation in the ELISpot plus two standard deviations. TCR sequencing data are shown as the frequency of each clone among all T cells detected in the relevant culture.

[0106] FIGS. 4A-4D show that FEST assays detect antigen-specific T cell responses with high specificity. T cells from healthy donor JH014 were stained with an EBV EBNA 4NP (Table 1) pentamer (FIG. 4A). TCR V CDR3 sequencing was performed on epitope-specific T cells and the V gene segment usage was evaluated (FIG. 4B). Clonotypes identified in the pentamer-sorted population were compared with those found in the same peptide-stimulated 10 day culture before (FIG. 4C, top) and after (FIG. 4C, bottom) the FEST biostatistical filtering to identify antigen-specific clones. The frequency (%) of each clonotype in the pMHC.sup.+ population, in bulk uncultured T cells, and after the 10 day culture is also shown (FIG. 4D).

[0107] FIGS. 5A-5C are graphs showing the sensitivity of the FEST assay. Titrating numbers of T cells from healthy donors JH014 and JH016 were stimulated with a known EBV and flu peptide epitope, respectively, for 10 days or 20 days after a peptide re-stimulation on day 10. FEST-positive clones were identified in each condition. The number of FEST-positive clones (FIG. 5A), as well as the percent of the culture/productive reads that consisted of FEST-positive clones (FIG. 5B) are shown for each titrating cell number and culture length. The correlation between clonality and the percent of productive reads that were FEST-positive is shown for each donor (FIG. 5C).

[0108] FIGS. 6A-6D show that the MANAFEST assay identifies multiple recognized MANAs and provides TCR barcodes to enable tracking of the anti-tumor immune response in tissue and peripheral blood. Recognition of candidate MANAs was evaluated by the MANAFEST assay in a patient with NSCLC being treated with anti-PD-1. A heatmap generated by the FEST analysis platform shows all MANA/clone pairs to which significant antigen-specific expansion was detected, with expansions to MANA #7 outlined in red (FIG. 6A). T cell clonotypes specific for MANA #7 as determined by the FEST analysis platform are shown as the frequency after culture (FIG. 6B), as well as in the primary tumor (FIG. 6C), and in longitudinal pre- and post-treatment peripheral blood T cells (FIG. 6D). Data are shown as the percent among all TCR reads detected.

[0109] FIG. 7 is a scatterplot showing the replicate pMHC.sup.+ CD8.sup.+ T cell sort. Additional flow cytometry and sorting experiment on EBV EBNA 4NP-pentamer positive (pMHC.sup.+) T cells, followed by TCR V CDR3 sequencing of antigen-specific cells.

[0110] FIG. 8 are scatterplots showing the TCR V CDR3 sequences observed in replicate pMHC.sup.+ sort experiments. Replicate sorting and CDR3 sequencing experiments were performed on EBV EBNA 4NP-positive T cells from healthy donor JH014. Data are shown as the frequency (percent of total productive reads) of each clone detected in each sort. Each circle represents a unique V CDR3 sequence, with the large circle representing 3 overlapping datapoints.

[0111] FIG. 9 are graphs showing the FEST-positive expansions detected to HIV-1 and ebola epitopes after 10-day and 20-day cultures. T cells from a healthy donor were cultured for 10 days in the presence of the HLA A*11:01-restricted ebolavirus AY9.sub.AAGIAWIPY (left), the HLA A*02:01-restricted HIV-1 SL9.sub.SLYNTVATL (middle), and the HLA A*02:01-restricted HIV-1 TV9.sub.TLNAWVKVV (right) epitopes, followed by TCR sequencing of the expanded T cells. In tandem cultures, cells were restimulated with irradiated peptide-loaded, autologous T cell-depleted PBMC and were cultured for an additional 10 days followed by TCR sequencing of the expanded T cells. Within each graph, each symbol represents a unique significantly and specifically expanded T cell clone as determined by the FEST analysis platform. Data are shown as the percent among all T cells after expansion in the relevant culture

DETAILED DESCRIPTION

[0112] The invention is based, at least in part, on the surprising identification of a sensitive, specific, scalable, and simple method to identify functional anti-tumor T cell responses, i.e., mutation associated neoantigens (MANA) functional expansion of specific T cells (MANAFEST), virus antigen functional expansion of specific T cells (VIRAFEST), and tumor-associated antigen (TAAFEST). The invention integrates a relatively short in vitro T cell stimulation with candidate tumor-specific peptides or transfected minigenes with TCRseq to monitor peptide-specific responses with clonal expansion (as assayed by TCRseq) rather than by cytokine production or proliferation. The methods described herein demonstrate superior sensitivity over the conventional method of enzyme-linked immunospot (ELISPOT).

[0113] Tumor cells contain nonsynonymous somatic mutations that alter the amino acid sequences of the proteins encoded by the affected genes. Those alterations are foreign to the immune system and may therefore represent tumor-specific neoantigens capable of inducing anti-tumor immune responses. Somatic mutational and neoantigen density has recently been shown to correlate with long-term benefit from immune checkpoint blockade in non-small cell lung cancer and melanoma suggesting that a high density of neoepitopes stemming from somatic mutations may enhance clinical benefit from blockade of immune checkpoints that unleash endogenous responses to these mutation-associated neoantigens (MANAs).

[0114] However, prior to the invention described herein, in most cases, the specific neoantigens that are responsible for tumor-specific immune responses were not known and computational methods for predicting neoantigens were limited. Described herein is a method that sensitively and specifically evaluates candidate tumor neoantigens for their ability to induce T cell responses. This method is broadly useful for functional evaluation of neoantigens in research and clinical settings, including for biomarker prediction in checkpoint blockade therapy and for identification of functional MANAs for personalized immunotherapy approaches.

[0115] With the emergence of immunotherapy as a major clinical treatment for cancer, it has become clear that T cells are the primary cells that provide specific recognition of the cancer and mediate both direct killing and orchestrate innate anti-tumor responses. Given the number of mutations carried by tumors, it is clear that mutation associated neoantigens (MANA) are a major source of tumor antigenicity that allows T cells to distinguish them from normal cells. Additional antigenicity can come from viral antigens in tumors that are caused by virus infection and contain integrated viruses with oncogenes driving the cancer (ie HPV in cervical and head and neck cancer). Tumor associated antigensself antigens upregulated in tumorsmay also be a source of antigenicity.

[0116] As described herein, guidance of precision immunotherapy requires assessment of tumor-specific T cell responses, most particularly in the blood. These biomarkers define which patients will respond to a given immunotherapy and who might require additional therapies. Additionally, approaches that use personalized vaccines consisting of MANA that are specific to an individual's tumor will require knowledge of the antigens that T cells are responsive against. Prior to the invention described herein, algorithms applied to MANA or viral genes for predicting T cell antigen recognition gave a general list of possibilities, but were highly imperfect. The common functional assayELISPOTworks well for viral infections, but is not sensitive enough to detect the weaker T cell responses against tumor antigens. Tetramer staining is cumbersome and thus cannot easily be applied to analyze all the antigens that tumor specific T cells could potentially recognize, nor does it determine functionality.

[0117] To address these issues, described herein is an assay system, termed MANAFEST (MANA Functional Expansion of Specific T cells). Related assays for viral antigens in virus-associated tumors and for tumor-associated antigens are termed VIRAFEST and TAAFEST. The assay begins by using prediction algorithms to identify and provide a broad list of candidate antigens and then tests the individual candidates as peptides, or as a minigene that encodes the peptide, for functional recognition. Instead of assaying cytokine production, which is done with the ELISPOT, MANAFEST (and VIRAFEST and TAAFEST) use TCRseq to analyze for expansion of T cell clones (with unique TCRbeta CDR3 sequences) specifically with only one of the peptides.

[0118] Accordingly, in certain embodiments, method of functionally evaluating a candidate antigen for the ability to induce a T cell response comprises obtaining a test sample of blood or tumor-infiltrating lymphocytes from a subject having or at risk of developing a cancer or a viral infection; stimulating expansion of autologous T cells from the subject with the candidate antigen, said candidate antigen comprising a peptide, a protein or a minigene transfected into autologous monocytic cells; isolating deoxyribonucleic acid (DNA) from the T cells; amplifying the T cell receptor- (TCR-) complementarity-determining region 3 (CDR3) DNA; determining a level of antigen-specific T cell expansion; comparing the level of antigen-specific T cell expansion to a level of expansion of T cells in the absence of the candidate peptide; determining that the candidate antigen has the ability to induce a T cell response if the level of antigen-specific T cell expansion is higher than the level of expansion of T cells in the absence of the candidate peptide.

[0119] In certain embodiments, the autologous T cells from the subject are stimulated to expand with the candidate antigen, said candidate antigen comprising a peptide or whole protein or with autologous peripheral blood mononuclear cells (PBMCs) which have been transfected with a tandem minigene construct encoding the candidate antigen(s). Antigen-specific T cell expansion can be determined, for example, by comparing TCR-VB clonality prior to stimulation with the candidate antigen or PBMCs to TCR-VP clonality after stimulation with the candidate antigen.

[0120] In certain embodiments, the candidate antigen comprises a tumor antigen or a viral antigen. In certain embodiments, the candidate antigen, in the form of a peptide, protein or minigene transfected into autologous monocytic cells, comprises a tumor mutation-associated neoantigen (MANA), a viral antigen, or a non-mutated tumor-associated antigen. In some embodiments, the viral antigen is expressed by an integrated cancer-associated virus or a non-oncogenic virus. The integrated cancer-associated virus comprises human papilloma virus (HPV) associated with cervical or head and neck cancer, Epstein Barr virus (EBV), Merkel Cell Polyomavirus, Hepatitis B virus (HBV) or Hepatitis C virus (HCV). In certain embodiments, the virus comprises human immunodeficiency virus (HIV).

[0121] In certain embodiments, the sample comprises a blood sample or a tumor infiltrating lymphocyte population.

[0122] In other embodiments, a method of determining whether a given immunotherapy will inhibit a tumor in a subject comprises functionally validating a candidate antigen for the ability to induce a T cell response as described herein, thereby determining that immunotherapy will inhibit the tumor if the candidate antigen has the ability to induce a T cell response, and that the given immunotherapy will inhibit the tumor and should be used to treat the patient. It is also determined whether immunotherapy will inhibit a tumor prior to or subsequent to administration of the immunotherapy to the subject.

[0123] In certain embodiments, the immunotherapy comprises administration of an immune checkpoint inhibitor alone or in combination with one or more additional anti-tumor treatments. The immune checkpoint inhibitor comprises an anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) antibody, an anti-programmed cell death protein 1 (PD-1) antibody, an anti-programmed death-ligand 1 (PD-L1) antibody, an anti-lymphocyte-activation 3 (LAG3) antibody, an anti-T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) antibody, an anti-T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibition motif (ITIM) domains (TIGIT) antibody, an anti-V domain-containing Ig suppressor of T-cell activation antibody, an anti-cluster of differentiation 47 (CD47) antibody, an anti-signal regulatory alpha (SIRP ) antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-neuritin antibody, an anti-neuropilin antibody, or an anti-interleukin-35 (IL-35) antibody, or any combination thereof. In certain embodiments, the immune checkpoint inhibitor comprises a drug that inhibits indoleamine-pyrrole 2,3-dioxygenase (IDO), A2A adenosine receptor (A2AR), arginase, or glutaminase, or any combination thereof. In certain embodiments, the immunotherapy further comprises administering an agonist of a co-stimulatory receptor. The agonist comprises an anti-glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR) antibody, an anti-CD27 antibody, an anti-4-1BB antibody, an anti-OX40 antibody, an anti-inducible T-cell co-stimulator (ICOS) antibody, or an anti-CD40 antibody, or any combination thereof.

[0124] In another embodiment, a method of determining whether a vaccine will inhibit a tumor or a virus in a subject comprises functionally evaluating a candidate antigen for the ability to induce a T cell response; determining that the vaccine will inhibit the tumor or virus, if the candidate antigen has the ability to induce a T cell response, wherein the vaccine incorporates comprises the candidate antigen, thereby determining whether the vaccine will inhibit the tumor or virus. In embodiments, the vaccine is administered to the subject. In certain embodiments, the vaccine comprises the candidate peptide or a tandem minigene or full gene encoding the candidate antigen incorporated into a recombinant viral or bacterial vaccine. In some embodiments, the candidate antigen comprises a tumor antigen or a viral antigen. In certain embodiments, the the candidate antigen comprises a mutation-associated neoantigen (MANA) or a non-mutated tumor-associated antigen. In certain embodiments, the viral antigen is expressed by an integrated cancer-associated virus or a non-oncogenic virus.

T Cells

[0125] A T cell or T lymphocyte is a type of lymphocyte (a subtype of white blood cell) that plays a central role in cell-mediated immunity. T cells are distinguished from other lymphocytes, such as B cells and natural killer cells, by the presence of a T-cell receptor on the cell surface. T cells are so named because they mature in the thymus from thymocytes (although some also mature in the tonsils).

[0126] The several subsets of T cells each have a distinct function. There are many types of T cells including effector T cells, T helper cells, cytotoxic (killer) T cells, memory T cells, regulatory T cells, natural killer cells, gamma delta T cells, and mucosal associated invariant T cells. The majority of human T cells rearrange their alpha and beta chains on the cell receptor and are termed alpha beta T cells ( T cells) and are part of the adaptive immune system. Specialized gamma delta T cells, a small minority of T cells in the human body, more frequent in ruminants, have invariant T cell receptors with limited diversity that can effectively present antigens to other T cells and are considered to be part of the innate immune system.

[0127] A unique feature of T cells is their ability to discriminate between healthy and abnormal (e.g. infected or cancerous) cells in the body. Healthy cells typically express a large number of self derived peptide-loaded major histocombatibility complex (pMHC) on their cell surface and although the T cell antigen receptor can interact with at least a subset of these self pMHC, the T cell generally ignores these healthy cells. However, when these very same cells contain even minute quantities of pathogen derived pMHC, T cells are able to become activated and initiate immune responses. The ability of T cells to ignore healthy cells, but respond when these same cells contain pathogen (or cancer) derived pMHC is known as antigen discrimination.

T Cell Receptor

[0128] The T-cell receptor, or TCR, is a molecule found on the surface of T cells, or T lymphocytes, that is responsible for recognizing fragments of antigen as peptides bound to MHC molecules. The binding between TCR and antigen peptides is of relatively low affinity and is degenerate: that is, many TCRs recognize the same antigen peptide and many antigen peptides are recognized by the same TCR. The TCR is composed of two different protein chains (that is, it is a heterodimer). In humans, in 95% of T cells the TCR consists of an alpha () and beta () chain, whereas in 5% of T cells the TCR consists of gamma and delta (/) chains. This ratio changes during ontogeny and in diseased states as well as in different species.

[0129] When the TCR engages with antigenic peptide and MHC (peptide/MHC), the T lymphocyte is activated through signal transduction, that is, a series of biochemical events mediated by associated enzymes, co-receptors, specialized adaptor molecules, and activated or released transcription factors.

[0130] The TCR is a disulfide-linked membrane-anchored heterodimeric protein normally consisting of the highly variable alpha () and beta () chains expressed as part of a complex with the invariant CD3 chain molecules. T cells expressing this receptor are referred to as : (or ) T cells, though a minority of T cells express an alternate receptor, formed by variable gamma () and delta () chains, referred as T cells. Each chain is composed of two extracellular domains: Variable (V) region and a Constant (C) region, both of Immunoglobulin superfamily (IgSF) domain forming antiparallel -sheets. The constant region is proximal to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the Variable region binds to the peptide/MHC complex. The constant domain of the TCR domain consists of short connecting sequences in which a cysteine residue forms disulfide bonds, which forms a link between the two chains.

[0131] The variable domain of both the TCR -chain and -chain each have three hypervariable or complementarity determining regions (CDRs), whereas the variable region of the -chain has an additional area of hypervariability (HV4) that does not normally contact antigen and, therefore, is not considered a CDR. The residues are located in two regions of the TCR, at the interface of the - and -chains and in the -chain framework region that is thought to be in proximity to the CD3 signal-transduction complex. CDR3 is the main CDR responsible for recognizing processed antigen, although CDR1 of the alpha chain has also been shown to interact with the N-terminal part of the antigenic peptide, whereas CDR1 of the n-chain interacts with the C-terminal part of the peptide. CDR2 recognizes the MHC. CDR4 of the 1-chain does not participate in antigen recognition, but has been shown to interact with superantigens.

[0132] Processes for the generation of TCR diversity are based mainly on genetic recombination of the DNA encoded segments in individual somatic T cellseither somatic V(D)J recombination using recombinant activating gene 1 (RAG1) and RAG2 recombinases or gene conversion using cytidine deaminases (AID). Each recombined TCR possess unique antigen specificity, determined by the structure of the antigen-binding site formed by the and chains in case of T cells or and chains on case of T cells. The TCR alpha chain is generated by VJ recombination, whereas the beta chain is generated by VDJ recombination (both involving a somewhat random joining of gene segments to generate the complete TCR chain). Likewise, generation of the TCR gamma chain involves VJ recombination, whereas generation of the TCR delta chain occurs by VDJ recombination. The intersection of these specific regions (V and J for the alpha or gamma chain; V, D, and J for the beta or delta chain) corresponds to the CDR3 region that is important for peptide/MHC recognition.

TCRseq

[0133] The recent development of single-cell RNA sequencing (scRNAseq) allows the transcriptomes of thousands of cells to be processed simultaneously, bringing a way to identify subpopulations of cells and provide functional insights such as the identification of each cell's unique TCRs and paired alpha and beta heterodimers that were previously masked in the analysis of an ensemble of multiple cells. However, scRNAseq is not devoid of biases and noise. For example, scRNAseq can only quantify the expression of most highly expressed genes and likely suffers from PCR amplification biases.

[0134] Many PCR-based methods for the amplification of V(D)J segments either use primer sets that introduce amplification artifacts owing to the differential amplification of some DNA templates over others, requiring the usage of complex normalization methods or require complex protocols based on template-switching effect of reverse transcriptase for the unbiased preparation of TCR cDNA libraries.

[0135] The single-cell sequencing model for TCRs described recently (Redmond et al., 2016 Genome Medicine, 8:80, incorporated herein by reference) avoids these issues and recovers these complex repertoires alongside the rest of the transcriptome present in T-cells.

[0136] Redmon addresses the accurate characterization of T-cell repertoires from scRNAseq data. A computational method single-cell TCRseq (scTCRseq) was generated to identify and count RNA reads mapping to specific TCR V and C region genes. scTCRseq facilitates the identification of productive and paired alpha and beta chain V(D)J TCR rearrangements and enables the recovery of full TCR including the nucleotide insertions and deletions at junctions in single T-cells. As described by Redmon, single-cell TCRseq provides an avenue for phenotypic investigation of T-cells in conjunction with the accompanying whole-transcriptome data.

ELISPOT

[0137] The Enzyme-Linked ImmunoSpot (ELISPOT) assay is a widely used method for monitoring cellular immune responses in humans and other animals, and has found clinical applications in the diagnosis of tuberculosis and the monitoring of graft tolerance or rejection in transplant patients (Czerkinsky et al., J Immunol Methods, 65 (1-2): 109-121, incorporated herein by reference). Prior to the invention described herein, the ELISPOT technique was among the most useful means available for monitoring cell-mediated immunity, due to its sensitive and accurate detection of rare antigen-specific T cells (or B cells) and its ability to visualize single positive cells within a population of peripheral blood mononuclear cells (PBMCs). The ELISPOT assay is also used for the identification and enumeration of cytokine-producing cells at the single cell level, but is still used for detection of antigen-specific antibody-secreting cells (ASC).

[0138] At appropriate conditions, the ELISPOT assay allows visualization of the secretory product(s) of individual activated or responding cells. Each spot that develops in the assay represents a single reactive cell. Thus, the ELISPOT assay provides both qualitative (regarding the specific cytokine or other secreted immune molecule) and quantitative (the frequency of responding cells within the test population) information.

[0139] The ELISPOT assays employ a technique very similar to the sandwich enzyme-linked immunosorbent assay (ELISA) technique. In an ELISPOT assay, the membrane surfaces in a 96-well polyvinylidene fluoride (PVDF)-membrane microtiter plate are coated with capture antibody that binds a specific epitope of the cytokine being assayed. During the cell incubation and stimulation step, cells, e.g., peripheral blood mononuclear cells (PBMCs), are seeded into the wells of the plate along with the antigen, and form a monolayer on the membrane surface of the well. As the antigen-specific cells are activated, they release the cytokine, which is captured directly on the membrane surface by the immobilized antibody. The cytokine is thus captured in the area directly surrounding the secreting cell, before it has a chance to diffuse into the culture media, or to be degraded by proteases and bound by receptors on bystander cells. Subsequent detection steps visualize the immobilized cytokine as an ImmunoSpot; essentially the secretory footprint of the activated cell.

[0140] As described herein, ELISPOT works well for vial infections, but is not sensitive enough to detect the weaker T cell responses against tumor antigens.

Immune Checkpoint Inhibitors

[0141] Immune checkpoint inhibitors block certain proteins made by some types of immune system cells, such as T cells, and some cancer cells. These proteins help keep immune responses in check; however, they can also keep T cells from killing cancer cells. When these proteins are blocked, the brakes on the immune system are released and T cells are able to kill cancer cells more effectively. Examples of checkpoint proteins found on T cells or cancer cells include PD-1/PD-L1 and CTLA-4/B7-1/B7-2.

Pharmaceutical Therapeutics

[0142] The invention provides pharmaceutical compositions for use as a therapeutic. In one aspect, the composition is administered systemically, for example, formulated in a pharmaceutically-acceptable buffer such as physiological saline. Preferable routes of administration include, for example, instillation into the bladder, subcutaneous, intravenous, intraperitoneal, intramuscular, or intradermal injections that provide continuous, sustained levels of the composition in the patient. Treatment of human patients or other animals is carried out using a therapeutically effective amount of a therapeutic identified herein in a physiologically-acceptable carrier. Suitable carriers and their formulation are described, for example, in Remington's Pharmaceutical Sciences by E. W. Martin. The amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the clinical symptoms of the neoplasia. Generally, amounts will be in the range of those used for other agents used in the treatment of other diseases associated with neoplasia or infection, although in certain instances lower amounts will be needed because of the increased specificity of the compound. A compound is administered at a dosage that enhances an immune response of a subject, or that reduces the proliferation, survival, or invasiveness of a neoplastic cell as determined by a method known to one skilled in the art.

Formulation of Pharmaceutical Compositions

[0143] The administration of compositions for the treatment of cancer may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in ameliorating, reducing, or stabilizing cancer. The composition may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously, intramuscularly, intravesicularly or intraperitoneally) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

[0144] Human dosage amounts can initially be determined by extrapolating from the amount of compound used in mice or nonhuman primates, as a skilled artisan recognizes it is routine in the art to modify the dosage for humans compared to animal models. In certain embodiments it is envisioned that the dosage may vary from between about 0.1 g compound/kg body weight to about 5000 g compound/kg body weight; or from about 1 g/kg body weight to about 4000 g/kg body weight or from about 10 g/kg body weight to about 3000 g/kg body weight. In other embodiments this dose may be about 0.1, 0.3, 0.5, 1, 3, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 g/kg body weight. In other embodiments, it is envisaged that doses may be in the range of about 0.5 g compound/kg body weight to about 20 g compound/kg body weight. In other embodiments the doses may be about 0.5, 1, 3, 6, 10, or 20 mg/kg body weight. Of course, this dosage amount may be adjusted upward or downward, as is routinely done in such treatment protocols, depending on the results of the initial clinical trials and the needs of a particular patient.

[0145] Pharmaceutical compositions are formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.

Kits

[0146] The invention provides kits for the treatment or prevention of a cancer. In one embodiment, the kit includes a therapeutic or prophylactic composition containing an effective amount of an agent described herein. In some embodiments, the kit comprises a sterile container that contains a therapeutic or prophylactic composition; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0147] If desired an agent of the invention is provided together with instructions for administering the agent to a subject having or at risk of developing a cancer. The instructions will generally include information about the use of the composition for the treatment or prevention of a cancer. In other embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of a cancer or symptoms thereof; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container

[0148] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook, 1989); Oligonucleotide Synthesis (Gait, 1984); Animal Cell Culture (Freshney, 1987); Methods in Enzymology Handbook of Experimental Immunology (Weir, 1996); Gene Transfer Vectors for Mammalian Cells (Miller and Calos, 1987); Current Protocols in Molecular Biology (Ausubel, 1987); PCR: The Polymerase Chain Reaction, (Mullis, 1994); Current Protocols in Immunology (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

[0149] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

Example 1: Functional Analysis of Candidate Neoantigens

[0150] With the growth of cancer immunotherapy dependent on enhancing anti-tumor T cell responses, it has become important to rapidly, sensitively and specifically assess functional anti-tumor T cell responses, mostly in the blood. Described herein is a methodology to accomplish this.

[0151] In lieu of the traditional ELISpot assay, which lacks sensitivity and only assesses cytokine production, described herein is a sensitive approach for assessing T cell response to candidate tumor antigens that utilizes next-generation sequencing of TCR-V CDR3 regions as a measure of antigen-specific T cell expansion. This technique, termed MANA functional expansion of specific T-cells (MANAFEST), has been applied to the functional analysis of T cell responses to MANAs, but can also be applied to responses against viral antigens, including those expressed in virus-induced cancers such as HPV-associated cervical and head-and-neck cancer (termed VIRAFEST), and non-mutated tumor-associated antigens, such as cancer-testes antigens or mesothelin (termed TAAFEST). In the case of MANA detection, whole exome sequencing data from tumor and matched normal samples are applied in a neoantigen prediction pipeline that evaluates antigen processing, MHC binding and gene expression to generate MANAs specific to the patient's HLA haplotype.

[0152] Peptides representing known and/or candidate MANAs based on in silico predictions are used to stimulate autologous T cells in a 10-day culture system (FIG. 1). DNA from cultured T cells is subsequently isolated and T cell receptor- (TCR-) CDR3 regions are amplified in a multiplex PCR method using 45 forward primers specific to TCR-V gene segments and 13 reverse primers specific to TCR J gene segments (Adaptive Biotechnologies). Synthesized tandem minigene constructs transfected into peripheral blood monocytes could be used in place of peptides for the T cell stimulation component of these assays.

[0153] In the MANAFEST approach, TCR-V clonality is compared pre-stimulation to post-stimulation in vitro with MANA peptides and the expansion of MANA-stimulated cultures is compared to the expansion observed in T cell cultures without peptide and/or uncultured T cells. A differential expansion TCR analysis is performed, where productive frequencies in the peptide stimulated T cells are compared to unstimulated T cells and/or uncultured T cells by Fisher's exact test. The p-values are corrected for multiple hypothesis testing using the Benjamini-Hochberg procedure (FIG. 2).

[0154] FIG. 2 shows neoantigen-specific TCR expansion in stimulated T cell cultures. Peptides generated from neoantigen candidates were synthesized and used to pulse autologous peripheral T cells. Reactive TCR clones were matched to clones found in tumor-infiltrating lymphocytes (TILs). Neoantigen-specific TCR reactivity (CDR3: CASSRDRGRGNSPLHF (SEQ ID NO: 4)) was observed for the mutant peptides associated with mutant helicase (DNA) B (HELB). Adjusted p value is given for pairwise comparisons between productive frequencies in peptide stimulated versus unstimulated T cells. Solid bars represent mutant and shaded bars denote wild type peptides.

[0155] The combination of the MANA stimulated T cell cultures and TCR- CDR3 sequencing followed by differential expansion analysis together compose the MANA functional expansion of specific T-cells (MANAFEST) assay. Identical analyses are used for VIRAFEST and TAAFEST, except the neoantigen prediction algorithms are applied to the relevant virus, viral antigens expressed by the virus-associated tumor, or to the shared genes up-regulated in the cancer.

[0156] The strategy is based on previous findings that antigen-specific T cells undergo rapid expansion upon stimulation by a cognate peptide-major histocompatibility complex (MHC) complex. Most importantly, as described in detail herein, this expansion may occur in the absence of detectable cytokine production and antigen recognition would therefore not be detected by conventional approaches, e.g., ELISPOT. In addition to being more sensitive than the conventional enzyme linked immunospot assay, the MANAFEST approach described herein allows us to match cMANA expanded TCR-V CDR3s with those found in the patients' tumors themselves, identified by TCR-V CDR3 deep sequencing from the same DNA used for mutational analysis. Thus, this approach can evaluate MANA-specific responses by T cells known to be present within the tumor microenvironment.

[0157] Also provided is the optimization of culture conditions to use a smaller number of cells, as well as optimization of biostatistical and bioinformatics analysis of TCRseq.

Example 2: Detection and Monitoring of Anti-MANA T Cell Repertoire

[0158] A set of sensitive and specific functional expansion of specific T cells (FEST) assays were developed herein to experimentally and bioinformatically evaluate antigen-specific clonal expansion using next generation TCR V CDR3 sequencing of T cells cultured with peptides representing candidate viral antigens (ViraFEST), TAAs (TAAFEST), or MANAs (MANAFEST). The FEST assays utilize this TCR quantification to identify antigen-specific T cell clonotypes based on clonal expansion after short-term stimulation and not only operate independently of cytokine production, but have enhanced sensitivity, specificity, and throughput capacity compared to other methods. The FEST platform works with all HLA haplotypes, and allows for tracking of antigen-specific T cells in FFPE and/or frozen tissue based on the ability of CDR3 regions to be used as a barcode for clones whose specificity is defined in the FEST assay.

[0159] This example shows that the MANAFEST assay, supported by a web-based biostatistical analytic platform, identifies MANA-specific TCR V clones that can be matched with clones detected in tumor tissue and in the blood of cancer patients treated with checkpoint blockade. MANAFEST can therefore validate the tumor specificity of TCR V clonotypes, interrogate the dynamics of the antigen-specific T cell response over time, and monitor the efficacy of checkpoint blockade using a simple liquid biopsy. Most importantly, the FEST assays can detect low frequency antigen-specific T cells in cases where other methods cannot.

[0160] Methods

[0161] Healthy Donors and Patients:

[0162] All healthy donors and patients described in this study provided informed consent as approved by the IRB of Johns Hopkins University. The patient described in this study was treated at the Sidney Kimmel Comprehensive Cancer Center.

[0163] Non-Small Cell Lung Cancer (NSCLC) Patient:

[0164] Patient JH124 was diagnosed with Stage IIB squamous non-small cell lung cancer in November 2015 and enrolled on JHU IRB protocol NA_00092076. He received 2 doses of anti-PD-1 immunotherapy and underwent surgical resection in December 2015. Pathology demonstrated a complete pathologic response in the 9 cm primary tumor and N1 nodes positive for tumor, final pathology stage was IIA. The patient received adjuvant platinum-based chemotherapy from February 2016 to May 2016. He had no evidence of recurrence of his cancer at last follow up in September 2017.

[0165] Whole Exome Sequencing and Putative MANA Identification:

[0166] Whole exome sequencing and identification of candidate neoantigens was performed as previously described using the VariantDx and ImmunoSelect-R pipelines (Personal Genome Diagnostics, Baltimore, Md.) (Anagnostou, V. et al. Cancer Discov 7, 264-276, doi:10.1158/2159-8290.CD-16-0828 (2017)).

[0167] Whole exome sequencing was performed on pre-treatment tumor and matched normal samples. The tumor sample underwent pathological review for confirmation of lung cancer diagnosis and assessment of tumor purity. Slides from the FFPE block were macrodissected to remove contaminating normal tissue and peripheral blood was used as matched normal. DNA was extracted from tumor and matched peripheral blood using the Qiagen DNA FFPE and Qiagen DNA blood mini kit respectively (Qiagen, CA). Fragmented genomic DNA from tumor and normal samples was used for Illumina TruSeq library construction (Illumina, San Diego, Calif.) and exonic regions were captured in solution using the Agilent SureSelect v.4 kit (Agilent, Santa Clara, Calif.) according to the manufacturers' instructions as previously described (Sausen, M. et al. Nature genetics 45, 12-17, doi:10.1038/ng.2493 (2013); Jones, S. et al. Science translational medicine 7, 283ra253, doi:10.1126/scitranslmed.aaa7161 (2015); Bertotti, A. et al. Nature 526, 263-267, doi:10.1038/nature14969 (2015); Anagnostou, V. et al. Cancer Discov 7, 264-276, doi:10.1158/2159-8290.CD-16-0828 (2017)). Paired-end sequencing, resulting in 100 bases from each end of the fragments for the exome libraries was performed using Illumina HiSeq 2000/2500 instrumentation (Illumina, San Diego, Calif.). Depth of coverage was 209 and 80 for the tumor and matched normal respectively.

[0168] Primary Processing of Next-Generation Sequencing Data and Identification of Putative Somatic Mutations:

[0169] Somatic mutations were identified using the VariantDx custom software for identifying mutations in matched tumor and normal samples as previously described (Jones, S. et al. (2015)). In brief, prior to mutation calling, primary processing of sequence data for both the tumor and normal sample was performed using Illumina CASAVA software (version 1.8), including masking of adapter sequences. Sequence reads were aligned against the human reference genome (version hg19) using ELAND with additional realignment of select regions using the Needleman-Wunsch method (Needleman, S. B. & Wunsch, C. D. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol 48, 443-453 (1970)). Candidate somatic mutations, consisting of point mutations, insertions and deletions were then identified using VariantDx across the whole exome. VariantDx examines sequence alignments of tumor samples against a matched normal while applying filters to exclude alignment and sequencing artifacts. In brief, an alignment filter was applied to exclude quality failed reads, unpaired reads, and poorly mapped reads in the tumor. A base quality filter was applied to limit inclusion of bases with reported Phred quality score >30 for the tumor and >20 for the normal. A mutation in the pre or post treatment tumor samples was identified as a candidate somatic mutation only when (1) distinct paired reads contained the mutation in the tumor; (2) the fraction of distinct paired reads containing a particular mutation in the tumor was at least 10% of the total distinct read pairs and (3) the mismatched base was not present in >1% of the reads in the matched normal sample as well as not present in a custom database of common germline variants derived from dbSNP and (4) the position was covered in both the tumor and normal. Mutations arising from misplaced genome alignments, including paralogous sequences, were identified and excluded by searching the reference genome.

[0170] Candidate somatic mutations were further filtered based on gene annotation to identify those occurring in protein coding regions. Functional consequences were predicted using snpEff and a custom database of CCDS, RefSeq and Ensembl annotations using the latest transcript versions available on hg19 from UCSC (https://genome.ucsc.edu/). Predictions were ordered to prefer transcripts with canonical start and stop codons and CCDS or RefSeq transcripts over Ensembl when available. Finally, mutations were filtered to exclude intronic and silent changes, while retaining mutations resulting in missense mutations, nonsense mutations, frameshifts, or splice site alterations. A manual visual inspection step was used to further remove artefactual changes.

[0171] Neoantigen Predictions:

[0172] To assess the immunogenicity of somatic mutations, exome data combined with the patient's MHC class I haplotype, were applied in a neoantigen prediction platform that evaluates binding of somatic peptides to class I MHC, antigen processing, self-similarity and gene expression. Detected somatic mutations, consisting of nonsynonymous single base substitutions, insertions and deletions, were evaluated for putative neoantigens using the ImmunoSelect-R pipeline (Personal Genome Diagnostics, Baltimore, Md.). To accurately infer the germline HLA 4-digit allele genotype, whole-exome-sequencing data from paired tumor/normal samples were first aligned to a reference allele set, which was then formulated as an integer linear programming optimization procedure to generate a final genotype (Szolek, A. et al. Bioinformatics 30, 3310-3316, doi:10.1093/bioinformatics/btu548 (2014)). The HLA genotype served as input to netMHCpan to predict the MHC class I binding potential of each somatic and wild-type peptide (IC.sub.50 nM), with each peptide classified as a strong binder (SB), weak binder (WB) or non-binder (NB) (Nielsen, M. & Andreatta, M. Genome Med 8, 33, doi:10.1186/s13073-016-0288-x (2016); Lundegaard, C. et al. Nucleic Acids Res 36, W509-512, doi:10.1093/nar/gkn202 (2008); Lundegaard, C., Lund, O. & Nielsen, M. Bioinformatics 24, 1397-1398, doi:10.1093/bioinformatics/btn128 (2008)). Peptides were further evaluated for antigen processing by netCTLpan (Stranzl, T., et al. Immunogenetics 62, 357-368, doi:10.1007/s00251-010-0441-4 (2010)). and were classified as cytotoxic T lymphocyte epitopes (E) or non-epitopes (NA). Paired somatic and wild-type peptides were assessed for self-similarity based on MHC class I binding affinity (Kim, Y., Sidney, J., Pinilla, C., Sette, A. & Peters, B. Derivation of an amino acid similarity matrix for peptide: MHC binding and its application as a Bayesian prior. BMC Bioinformatics 10, 394, doi:10.1186/1471-2105-10-394 (2009)). Neoantigen candidates meeting an IC.sub.50 affinity <500 nM were subsequently ranked based on MHC binding and T-cell epitope classifications. Tumor-associated expression levels derived from TCGA were used to generate a final ranking of candidate immunogenic peptides. Putative MANAs were synthesized using the PEPscreen platform (Sigma-Aldrich; St. Louis, Mo.). Lyophilized peptides were dissolved in minimal DMSO, resuspended in 100 &g/ml aliquots in AIM V media, and stored at 80 C.

[0173] T Cell Culture:

[0174] T cells were cultured and evaluated for significant antigen-specific expansions as previously described, with minor modifications (Anagnostou, V. et al. Cancer Discov 7, 264-276, doi:10.1158/2159-8290.CD-16-0828 (2017); Le, D. T. et al. Science 357, 409-413, doi:10.1126/science.aan6733 (2017)).

[0175] On day 0, frozen PBMC from healthy donors or patients were thawed and counted. T cells were isolated using the EasySep Human T Cell Enrichment Kit (Stemcell Technologies; Vancouver, Canada). T cells were washed, counted, and resuspended at 2.010.sup.6/ml in AIM V media supplemented with 50 g/ml gentamicin (ThermoFisher Scientific; Waltham, Mass.). The T cell-negative fraction was washed, counted, and irradiated at 3,000 -rads. The irradiated T cell-depleted fraction was washed and resuspended at 2.010.sup.6/ml in AIM V media supplemented with 50 g/ml gentamicin. Irradiated T cell-depleted cells were added to a 96-well, 48-well, 24-well, or 12-well plate at 125 l, 250 l, 500 l, or 1,000 l per well, respectively. An equal volume of T cells was then added to each well, along with 1 g/ml of one of 13 HLA-matched CMV, EBV, or flu peptide epitopes (Sigma-Aldrich, St. Louis, Mo.) or without peptide. Cells were cultured for 10 days at 37 C. in a 5% CO.sub.2 atmosphere, replacing half the culture media with fresh culture media containing 100 IU/ml IL-2, 50 ng/ml IL-7, and 50 ng/ml IL-15 (for final concentrations of 50 IU/ml IL-2, 25 ng/ml IL-7, and 25 ng/ml IL-15) on day 3 and replacing half the culture media with fresh media containing 200 IU/ml IL-2, 50 ng/ml IL-7, and 50 ng/ml IL-15 (for final concentrations of 100 IU/ml IL-2, 25 ng/ml IL-7, and 25 ng/ml IL-15) on day 7. If cells were to be used in IFN ELISpot or IFN/granzyme B fluorospot assays, cells were rested on day 9 by removing half the media and replacing with fresh media without cytokines. For cells to be used in TCR sequencing/FEST analysis, cells were not rested and were harvested on day 10. CD8.sup.+ cells were further isolated from T cells cultured with putative MANAs using the EasySep Human CD8.sup.+ T Cell Enrichment Kit (Stemcell Technologies) and plate magnet for added throughput.

[0176] For the generation of 20-day, restimulated cultures, autologous PBMC were incubated with 1 g/ml relevant peptide for 2 h at 37 C. in a 5% CO.sub.2 atmosphere, irradiated at 3,000 -rads, and were added to cultures at a 1:1 T cell:PBMC ratio on day 10 of the culture. Cells were fed on culture days 13 and 17 by replacing half the culture media with fresh media containing 200 IU/ml IL-2, 50 ng/ml IL-7, and 50 ng/ml IL-15 (for final concentrations of 100 IU/ml IL-2, 25 ng/ml IL-7, and 25 ng/ml IL-15). On day 20, T cells were harvested and washed for DNA extraction.

[0177] IFN ELISpot Assays:

[0178] 10-day cultured cells or uncultured PBMC obtained from the same stock of cells used in culture were evaluated for IFN production by a standard overnight enzyme-linked immunosorbent spot (ELISpot) assay. Briefly, 96-well nitrocellulose plates (EMD Millipore, Billerica, Mass.) were coated with anti-IFN monoclonal antibody (10 jig/ml; Mabtech, Stockholm, Sweden) and incubated overnight at 4 C. Plates were washed and blocked with IMDM supplemented with 10% heat-inactivated FBS for 2 h at 37 C. T cells stimulated for 10 days with CMV, EBV, and flu peptides were added to wells in duplicate at 50,000 cells per well and were stimulated overnight with PBMC pre-loaded with 1 jig/ml relevant peptide, a cytomegalovirus (CMV), Epstein-Barr virus (EBV), and influenza virus peptide pool (CEF), or no peptide in AIM V media. Cultured T cells with PBMC alone served as the background/negative control condition. Fresh-thawed PBMC were added to wells in singlet at 100,000 cells/well and were stimulated overnight with 1 g/ml of the same peptides used in the T cell culture assays. PBMC alone in duplicate wells served as the background/negative control condition.

[0179] Staining and Sorting of Pentamer Positive Populations:

[0180] T cells obtained from healthy donors were evaluated for specificity of known viral antigens. Fluorochrome-conjugated pentamers were synthesized (ProImmune, Oxford, UK) and used to stain PBMC from healthy donor JH014 per the manufacturer's instructions. Cells were co-stained with CD3, CD4, CD8, and CD45RO to identify antigen-specific memory CD.sup.8+ T cells for sorting. The pentamer-positive population of interest was sorted using a BD FACSAria II and DNA was immediately extracted for TCR sequencing.

[0181] T Cell Receptor (TCR) Sequencing and Assessment of Significant Antigen-Specific Expansions:

[0182] DNA was extracted from peptide-stimulated T cells, tumor tissue, and longitudinal pre- and post-treatment PBMC and pentamer-sorted T cells using a Qiagen DNA blood mini kit, DNA FFPE kit, or DNA blood kit, respectively (Qiagen). TCR V CDR3 sequencing was performed using the survey (tissue, cultured cells, and pentamer-sorted cells) or deep (PBMC) resolution Immunoseq platforms (Adaptive Biotechnologies, Seattle, Wash.) (Carlson, C. S. et al. Nat Commun 4, 2680, doi:10.1038/ncomms3680 (2013); Robins, H. S. et al. Blood 114, 4099-4107, doi:10.1182/blood-2009-04-217604 (2009)). Bioinformatic and biostatistical analysis of productive clones was performed to identify and evaluate antigen-specific expansions. Antigen-specific T cell clones were identified using the following criteria: 1) significant expansion of the relevant clone (Fisher exact test with Benjamini-Hochberg FDR, p<0.01) compared to T cells cultured without peptide, 2) significant expansion (Fisher exact test with Benjamini-Hochberg FDR, p<0.01) of this clone compared to every other peptide-stimulated culture, 3) and odds ratio >10 for the relevant clone, and 4) at least 10 reads in the relevant T cell culture. The peripheral and intratumoral representation of these antigen-specific clones was further analyzed.

[0183] Statistical and Bioinformatic Analysis:

[0184] A custom script was developed in R/Bioconductor (R: A language and environment for statistical computing (R Foundation for Statistical Computing, Vienna, Austria, 2014); Gentleman, R. C. et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5, R80, doi:10.1186/gb-2004-5-10-r80 (2004)) to load TCR sequencing data directly exported from Adaptive Biotechnologies ImmunoSEQ platform in V2 in the tab-delimited format, perform the analysis, and visualize and save results. For analysis, only productive clones were used and read counts were summarized for nucleotide sequences that translated into the same amino acid sequence. For each clone, Fisher's exact test was applied to compare the number of reads in a culture of interest (with peptide) and a reference culture (without peptide). The p-value adjusted by Benjamini-Hochberg procedure (FDR) (Benjamini, Y. & Hochberg, Y. J Roy Stat Soc B Met 57, 289-300 (1995)) was used to determine statistically significant specific expansions (FEST assay positive clones) that met the following criteria: 1) significantly expanded in the culture of interest compared to the reference culture (T cells cultured with cytokines but without peptide) at an FDR less than the specified threshold (<0.01), 2) significantly expanded in the culture of interest compared to every other culture performed in tandem (FDR <0.01), 3) have a positive odds ratio compared to the reference culture (e.g. >10), and 4) not significantly expanded in any other peptide-stimulated culture compared to the reference culture. All clones were subject to a 10-read lower threshold for consideration in the statistical analysis. FEST assay positive clones were saved in the output table and plotted at output heat maps using build-in R functions. The script was wrapped into a web application using Shiny Server (shiny: Web Application Framework for R. R package version 1.0.0 (2017)).

[0185] Results

[0186] In Vitro TCRV CDR3 Clonotype Amplification as a Functional Readout of T Cell RecognitionSensitivity Comparison with ELISpot:

[0187] Quantitative TCR sequencing is characterized by high sensitivity and elucidates the clonotypic hierarchy and composition of T cell populations (Carlson, C. S. et al. Nat Commun 4, 2680, doi:10.1038/ncomms3680 (2013); Robins, H. S. et al. Blood 114, 4099-4107, doi:10.1182/blood-2009-04-217604 (2009)). The molecular characterization of antigen-specific clonotypes can further provide a barcode tag to track and quantify antigen-specific T cells and allow for spatio-temporal characterization of the anti-tumor immune response, which is not achievable with ELISpot or flow cytometry-based approaches. To validate TCR sequencing as a metric of T cell recognition, viral antigens were first used and compared IFNg ELISpot with TCR V CDR3 sequencing in healthy donors. Cytomegalovirus (CMV)-, influenza (flu)-, and Epstein Barr virus (EBV)-derived HLA-I epitopes are well-defined and induce CD8.sup.+ T cell responses detectable by IFN ELISpot and tetramer/pentamer staining. ELISpot, used as a reference assay, was compared with the FEST assay to technically validate this new test. It was initially tested if peptide-induced T cell expansion could be observed in the absence of ELISpot positively (no detectable antigen-specific IFN production). T cells from healthy donor JH014 were cultured for 10 days with multiple HLA-matched viral peptide epitopes (Table 1) or no peptide as a control. At the term of the culture, one aliquot of the cells was used to perform IFN ELISpot and the remaining cells were evaluated by TCR V CDR3 sequencing. High-magnitude IFN production was observed that was associated with expansion of several T cell clones after a 10 day culture with the HLA A11-restricted EBV EBNA 4NP AVFDRKSDAK (FIG. 3A) and HLA B8-restricted EBV EBNA 3A FLRGRAYGL epitopes (FIG. 3B). Notably, while there was no IFN ELISpot signal for the HLA A11-restricted EBV EBNA-4 epitope, IVTDFSVIK, 9 T cell clones showed clear expansion (FIG. 3C). These ELISpot results were recapitulated in uncultured T cells. One of the features of this assay format that provides robust statistical specificity is that, for a set the T cell clones expanded upon stimulation with a given peptide, cultures with all the other peptides in the assay represent negative controls. As seen in FIGS. 3A-3C, the absence of amplification of the clonotypes in the other cultures indeed demonstrates the specificity of peptide-specific clonotypic expansion.

[0188] FEST Associated Biostatistics Platform:

[0189] A high throughput statistical analysis platform was developed, to combine with the experimental approach of the FEST assays in order to efficiently evaluate the specificity of the clonotypic expansion and therefore antigen-specific T cell recognition. To optimize data analysis and to streamline a stringent statistical analysis unlikely to result in false positives, TCR sequencing data were uploaded into a web-based biostatistics application that integrates the clonotypic amplification in each peptide-stimulated culture to determine the positivity and specificity of antigen-specific T cell recognition (stat-apps.onc.jhmi.edu/FEST). A clonotype was considered antigen-specific if it 1) was significantly expanded in the relevant culture compared to T cells cultured without peptide at FDR <0.01, 2) was significantly expanded in the relevant culture compared to T cells cultured with every other peptide at FDR <0.01, 3) had greater than 10 reads, and 4) had an odds ratio >10 compared to the no peptide control. These criteria are stringent and were chosen to minimize false positives, given the sensitivity of the assay platform. In the analyses below, clones satisfying these criteria were considered to be FEST assay positive and were saved as an output of analysis (Table 3). However, each parameter can be adapted according to the investigator preferences. The web analysis platform also generates heatmaps showing odds ratio compared to the no peptide control for each peptide to which antigen recognition was detected and for all specifically and significantly expanded clones detected across all cultures. The FEST assays, comprised of an experimental T cell culture and computerized analytical tool, allows for efficient monitoring and analysis of antigen-specific T cell responses in a high throughput, turnkey fashion.

[0190] Specificity of the FEST Assay:

[0191] The high sensitivity of the FEST assay might be associated with decreased specificity and an increased false positive rate. To address this, the composition of the EBV EBNA 4NP-specific repertoire in healthy donor JH014 was first evaluated by performing duplicate sorting and TCR CDR3 V sequencing experiments (sort #1 and sort #2) on pentamer-positive (pMHC.sup.+) CD8.sup.+ T cells. The EBV EBNA 4NP-specific population was detected at 0.2% of total T cells in both experiments (FIGS. 4A-4D, 7). TCR sequencing of pMHC.sup.+ T cells demonstrated dominance of V 28-01 within this antigen-specific population (FIG. 4B, Table 2), which is consistent with prior findings that different T cell clonotypes specific for the same antigen often preferentially utilize the same V gene segment (Price, D. A. et al. J Exp Med 206, 923-936, doi:10.1084/jem.20081127 (2009); Valkenburg, S. A. et al. Proc Natl Acad Sci USA 113, 4440-4445, doi:10.1073/pnas.1603106113 (2016); Du, V. Y. et al. J Immunol 196, 3276-3286, doi:10.4049/jimmunol.1502411 (2016); Kloverpris, H. N. et al. J Immunol 194, 5329-5345, doi:10.4049/jimmunol.1400854 (2015); Hill, B. J. et al. J Immunol 193, 5626-5636, doi:10.4049/jimmunol.1401017 (2014)). In comparison to 92.5% of T cells utilizing V 28-01 in pMHC.sup.+ T cells, only 7.0% of pMHC.sup. CD8.sup.+ T cells used this gene segment.

[0192] Two hundred thirteen and 104 unique CDR3 sequences were identified from sort #1 and sort #2, respectively, however only 9 of these sequences were shared between the two replicate pMHC.sup.+ populations (FIG. 8, Table 2), with consistency in frequency between 4 of the most dominant clones, making up 90.0% and 88.9% of the total pentamer-positive T cells from each sort experiment. All 4 of these clones used the V 28-01 gene. The remaining pMHC.sup.+ T cells expressed over 40 different V genes, with no clone making up more than 0.9% of the total pentamer-positive population (0.002% of total T cells).

[0193] To evaluate the specificity of the FEST assay, the 9 common pMHC.sup.+ CDR3 V sequences identified in sorts #1 and #2 were first compared with those identified in bulk T cells after a 10 day culture and stimulation with the EBV EBNA 4NP epitope. Four unique clones, representing 48.1% of the T cell culture matched pMHC.sup.+ CDR3 V sequences. When the FEST associated biostatistical filtering was applied as described above to the clonotypic amplifications in this same 10 day culture, the specificity of these 4 clones were confirmed, which now made up 87.4% of the T cells that were identified as being antigen-positive by the FEST analysis (FIG. 4C). Of note, all 4 dominant clones used the V 28-01 gene and matched the most frequent clones detected in the pMHC.sup.+ populations (FIGS. 4D, 8 and Table 2). Thirty eight low frequency clonotypes that were positive by FEST analysis but absent in the pMHC.sup.+ T cell fraction were neither in the uncultured nor 10 day cultured pMHC.sup. CD8.sup.+ T cell population, suggesting the presence of in vitro artifacts. To minimize these artifacts, CD8.sup.+ T cells were isolated at the end of the 10 day culture before DNA isolation and TCR sequencing.

[0194] Because each peptide-stimulated culture serves as a negative control for all other cultures, the confidence in the specificity of T cell recognition can be improved by increasing the number of distinct peptide cultures. With 46 cultures, the estimated specificity of a unique clonotype would be nearly 98% (45/46), and a one-sided 95% confidence interval would run from 90%-100%. Therefore, with at least 46 cultures there is 95% confidence that specificity is above 90%. With 93 cultures, a unique clonotype has an estimated specificity of approximately 99% (95% C.I.=(95%-100%)).

[0195] Sensitivity of the FEST Assay:

[0196] The FEST assays rely on the identification of antigen-specific V CDR3 clonotypes and on their frequency following a 10 day in vitro expansion. Sensitivity of the FEST assays (i.e the detection of low frequency clonotypes) is expected to be highly dependent on the starting number of CD8.sup.+ T cells in the 10 day culture. It was sought to determine 1) the optimal number of starting T cells required to accurately capture the breadth of the antigen-specific repertoire and 2) if clonotypes that were undetectable after a 10 day culture could be detected in 20 day cultures using a peptide restimulation step. Titrating numbers of T cells (from 1.2510.sup.5 to 1.010.sup.6) obtained from two healthy donors were cultured for 10 and 20 days. T cells from donor JH014 were stimulated with the HLA A11-restricted EBV EBNA 4NP AVFDRKSDAK epitope shown in FIG. 3A and T cells from donor JH016 were stimulated with the HLA A2-restricted influenza M peptide GILGFVFTL. Peptide epitopes were chosen based on previously-documented reactivity in these two donors. The absolute number of unique clones that were expanded decreased as the starting cell number was decreased (FIG. 5A). Therefore, a higher number of starting cells results in the identification of a higher number of unique antigen-specific clonotypes that can then be tracked in peripheral blood lymphocytes or tissue. In both donors, the percent of productive reads (frequency of total T cells in the culture) that were significantly expanded compared to the no peptide control was consistently higher after a 20 day culture regardless of starting T cell number (FIG. 5B). This provides evidence that significantly expanded clones make up a greater percentage of the total T cell population after 20 days compared to 10 days. Indeed, there was a direct correlation between the percent of total productive reads that were significantly expanded and the clonality metric of the cultures (FIG. 5C), showing that clones expanded and detected via the FEST analytic platform directly contribute to the clonality of the culture. The percent of T cells that were significantly expanded in cultures without peptide stimulation had no correlation with clonality, highlighting the ability of the 10 day peptide-stimulated culture to specifically enrich for antigen-specific T cells. Despite the observance of more unique clones and a higher percent of clones that are FEST positive in the 20 day assay, clones were still expanded in all 10 day peptide cultures even at the lowest starting cell number of 1.2510. Therefore, a 10 day culture with as few as 1.2510.sup.5 starting T cells per condition is sufficient to accurately screen a library of peptides for recognition of peripheral T cells with frequencies as low as 0.008% (10 cells in 125,000), with the sensitivity increasing to 0.001% when starting with 1.010.sup.6 T cells (10 cells in 1.010.sup.6).

[0197] The possibility that the 20 day culture was identifying clones expanded from nave precursors and was not quantifying the endogenous recall response, was next assessed. T cells from healthy donor JH014 were cultured with two well-documented HIV-1 and one Ebola HLA A*02:01-restricted peptides. After 10 days, there were no clones that significantly expanded in response to any of the peptides compared to the no peptide control. Strikingly, after a restimulation and 20 days of culture, there was significant expansion of 61, 108, and 111 clones in response to the HIV-1 gag SL9, HIV-1 gag TV9, and ebola AY9 epitopes, respectively (FIG. 9). These data demonstrate that a restimulation and 20 day culture can result in the detection of primary T cell responses and is therefore not suitable when evaluating the endogenous memory repertoire, but may inform on the repertoire that is available for vaccination.

[0198] Validation of MANAFEST to Detect Anti-Tumor Immune Responses in Patients Receiving Anti-PD-1:

[0199] While T cell responses to viral antigens are often immunodominant (Motozono, C. et al. J Immunol 192, 3428-3434, doi:10.4049/jimmunol.1302667 (2014); Wu, C. et al. Proc Natl Acad Sci USA 108, 9178-9183, doi:10.1073/pnas.1105624108 (2011); Steven, N. M. et al. J Exp Med 185, 1605-1617 (1997); Betts, M. R. et al. Blood 107, 4781-4789, doi:10.1182/blood-2005-12-4818 (2006)), MANA-specific T cells could potentially be diverse and subdominant as well as functionally compromised (low cytokine production). It was therefore considered that the breadth and magnitude of the endogenous immune response in cancer patients may be substantially underestimated using ELISpot or multimer-based assays and that improved characterization of this response could be attained by using the FEST assay approach. Moreover, immune monitoring of the clinical response to checkpoint blockade requires T cell clonotype tracking in tissue and longitudinal peripheral blood samples to confirm the amplification of MANA-specific TCR V clonotypes upon treatment, a parameter that is not achievable by ELISpot. As a proof of principle, MANAFEST was performed on cells obtained from JH124, a patient with stage IIB squamous NSCLC who achieved a complete pathological response following two doses of nivolumab (anti-PD-1 therapy). Whole exome sequencing was performed in pre-treatment tumor and matched normal tissue and tumor-specific alterations were analyzed using a neoantigen prediction pipeline to identify candidate MANAs specific to the patient's HLA haplotype. T cells obtained 4 weeks post initiation of nivolumab were cultured for 10 days with one of 47 putative MANAs (Table 3) and resulting expanded T cells were isolated for TCR V CDR3 sequencing and MANAFEST analysis. TCR sequencing data was also used from tumor infiltrating lymphocytes to identify intra-tumoral clones.

[0200] Twenty-one out of 47 putative MANAs induced significant and specific T cell expansions and can be visualized as a heatmap output from the MANAFEST Web Application (FIG. 6A). Of the 28 clones that were expanded in response to these 21 MANAs, 2 were detected in the primary tumor and were both specific for the putative HLA A*25:01-restricted EVIVPLSGW MANA derived from a somatic sequence alteration in the ARVCF gene (FIGS. 6B, 6C and Table 3). Longitudinal analysis of these clones demonstrated a peripheral expansion upon nivolumab administration that decreased by 10 weeks post-first dose and was reminiscent of acute responses to viral infections (FIG. 6D). Aside from the frequency after culture, in tissue, and in serial peripheral blood samples, additional parameters are outputted from the FEST analysis that can be correlated with treatment response (see output for patient JH124 in Tables 4, 5 and 6). These parameters include the magnitude of in vitro expansion compared to uncultured T cells and to the no peptide control condition, the number of clones that are significantly expanded in response to a given candidate MANA, and the sum frequency of FEST-positive clones in response to each MANA after culture.

DISCUSSION

[0201] The rapid development of personalized cancer immunotherapies as well as the imperative to develop biomarkers predictive of immunotherapy responses calls for routine high throughput assays that monitor the anti-tumor immune response (Couzin-Frankel, J. Science 342, 1432-1433, doi:10.1126/science.342.6165.1432 (2013)). These assays will likely be a critical biomarker for the efficiency of the immunotherapeutic treatment and can also determine the eligibility of patients for immunotherapy based on detection of a preexisting anti-tumor immune response (Topalian, S. L., et al. Nat Rev Cancer 16, 275-287, doi:10.1038/nrc.2016.36 (2016)). With the recent development of NGS technologies it has become feasible to characterize mutations in the tumor exome and the TCR recognizing the neoantigens derived from these mutations (Kirsch, I., et al. Mol Oncol 9, 2063-2070, doi:10.1016/j.molonc.2015.09.003 (2015); Vogelstein, B. et al. Cancer genome landscapes. Science 339, 1546-1558, doi:10.1126/science.1235122 (2013)). Because amplification of selective TCRV clonotypes in tumor tissue has been proposed as a surrogate biomarker of MANA recognition (Pasetto, A. et al. Cancer Immunol Res 4, 734-743, doi:10.1158/2326-6066.CIR-16-0001 (2016)), the MANAFEST assay introduced herein, is based on tumor exome-guided identification of putative MANAs and the measure of the MANA-specific TCR clonotypic amplification following patient T cell stimulation. It was shown that epitope-triggered clonal expansion can be observed in the absence of detectable IFN production, and that ELISpot likely underestimates the peripheral T cell response. Furthermore, TCR sequencing underscores the diversity of the T cell response to a single HLA-restricted epitope. Altogether, these results validate TCR sequencing of a 10 day peptide-stimulated culture as the experimental core of the functional expansion of specific T-cells (FEST) assays to monitor antigen-specific T cell responses.

[0202] In comparison with existing methods, the FEST assays are highly sensitive and specific, and enable the tracking of antigen-specific TCR clonotype dynamics in T cell DNA derived from tissues and peripheral blood. These methods can be used to detect virus- and MANA-specific responses with greater sensitivity and throughput than current methods, and can be expanded to a variety of antigens including tumor associated antigens (TAAFEST), viral antigens (VIRAFEST), bacterial antigens (BactiFEST), and autoantigens (AutoFEST). This assay works independently of the limitations often met in traditional tests such as the low frequency and functional state of the T cells (ELISpot), HLA availability for multimer approaches (combinatorial encoding multimer), and the inadequacy of routine high throughput clinical monitoring (ELISpot). The molecular characterization of each TCR clonotype amplified in response to the specific MANA provides a convenient T cell clone-associated barcode, or molecular tag, to enable tracking of anti-tumor T cells in a multitude of fresh, fixed, and frozen cell and tissue types. This approach can inform on the spatiotemporal distribution of the anti-mutanome in serial peripheral blood samples or in differential geographic regions of the tumor. The MANAFEST method has already been used to detect and monitor peripheral and intratumoral MANA-specific T cell responses in NSCLC patients with acquired resistance to checkpoint blockade (Anagnostou, V. et al. Cancer Discov 7, 264-276, doi:10.1158/2159-8290.CD-16-0828 (2017)) and a colorectal cancer patient with a sustained partial response to pembrolizumab (Le, D. T. et al. Science 357, 409-413, doi:10.1126/science.aan6733 (2017)).

[0203] Consequently, FEST-based monitoring provides critical information in terms of the intensity (magnitude of expansion), diversity (distinct unique CDR3 sequences), dynamics (unique sequence reads at different time points), and geographic distribution (tissue-resident and periphery) of the anti-tumor immune response at a magnitude that will never be reached with any of the traditional assays. In comparison with traditional existing methods, such as ELISpot and multimer approaches, it is shown herein, that the setup of the test is easily feasible, using direct incubation of peptides with patient T cells, does not require specialized equipment such as a multiparameter flow cytometer or an ELISpot reader, permits a higher throughput, and also facilitates a multi-center standardization for data sharing, databasing, and computational identification of biomarkers. The throughput of this assay can be dramatically increased by identifying clonotypic amplifications to pools prior to testing individual peptides present in positive pools.

[0204] Additionally, because the test does not require the derivation of autologous antigen presenting cells as required for the TMG approach, relatively fewer numbers of PBMC and therefore smaller samples are necessary to detect MANA-specific T cells with high sensitivity. Although whole exome sequencing and TCR sequencing are currently costly methods, NGS has become relatively affordable and routine in patients receiving immunotherapy and clinical use of whole exome sequencing may be envisaged in the future. In the context of widespread use of immunotherapy, the characteristics aforementioned may facilitate the compatibility with clinical practice (liquid biopsy) and improved patient comfort (non-invasive sampling). Importantly, the computational pipeline to predict HLA-restricted MANAs and the web-based biostatistics used to identify immunogenic MANAs by FEST-based assays allow flexibility in the decision making regarding the selection of MANAs to accommodate high or low mutational density and in the determination of a positive MANA-specific response by modifying the desired alpha and odds ratio threshold.

[0205] While the assays described here evaluated MHC class I-restricted responses, this assay can be easily adapted to assess CD4.sup.+/MHC class II-restricted responses as well. Additionally, because antigen-specific T.sub.reg are of particular interest in cancer patients, this T cell subpopulation can be assayed using the FEST approach.

[0206] Importantly, the scientific application and translational perspectives of MANAFEST are indispensable, owing to the capacity for molecular characterization of the TCR sequences associated with MANA recognition that can be coordinated across patients or histologies and between institutions to identify common genomic features associated with immunogenicity of tumors and common structural motifs of the TCR (Faham, M. et al. Arthritis Rheumatol, doi:10.1002/art.40028 (2016)). A central repository of these data would help define molecular motifs that could inform on the capacity of cancer patients to mount immune responses to their cancer and on their eligibility for immune checkpoint modulation. The MANAFEST assay is therefore expected to become a unique tool that could serve as a pan-cancer predictive biomarker of response to immunotherapy.

TABLE-US-00001 TABLE1 ViralpeptideepitopestestedinhealthydonorJH014 PeptideName Source Protein AASequence HLARestriction FluM influenza matrix GILGFVFTL A2 FluA influenza polymoraseacidic FMYSDFHFI A2 EBVLMP2A humanherpesvirus4 latentmembraneprotein2A CLGGLLTMV A2 EBVBMLF1 humanherpesvirus4 BMLF1 GLCTLVAML A2 HcMVpp65 humancytomegalovirus phosphoprotein65 NLVPMVATV A*02:01 FluM influenza matrix SIIPSGPLK A11 EBVEBNA4NP humanherpesvirus4 Epstein-Barrnuclearantigen1 AVFDRKSDAK A11 EBV1 humanherpesvirus4 Epstein-Barrnuclearantigen1 IVTDFSVIK A11 EBV2 humanherpesvirus4 BRLF1 ATIGTAMYK A11 FluNP influenza nucleoprotein ELRSRYWAI B8 EBVBZLF-1 humanherpesvirus4 BZLF1 RAKFKQLL B8 EBVEBNA3A humanherpesvirus4 Epstein-Barrnuclearantigen3 FLRGRAYGL B8 EBVEBNA3 humanherpesvirus4 Epstein-Barrnuclearantigen3 QAKWRLQTL B8

TABLE-US-00002 TABLE2 Clonotypesdetectedinpentamer-sortedTcells CDR3FrequencyinEBV CDR3FrequencyInEBV CDR3AminoAddSequence* V Gene EBNA4NPpMHC+ sort#1 EBNA4NPpMHC+ sort#2 CASSLTSATGELFF TCRBV28-01*01 43.982301 41.706161 CASSLTSAAGELFF TCRBV28-01*01 37.654867 38.704581 CASSPTSATGELFF TCRBV28-01*01 7.787611 7.740916 CASSLKGTRDQETQYF TCRBV28-01*01 0.57521 0.789889 CASSRHHNKETQYF TCRBV28-01*01 0.044248 0.157978 CSAKLAGYQTGELFF TCRBV20 0.176991 0.157978 CSARTLGPGDEQYF TCRBV20 0.044248 0.157978 CSVEIGGEQYF TCRBV29-01*01 0.044248 0.157978 CSVLGGTSGAQETQYF TCRBV29-01*01 0.088496 0.157978 CASSIPNPIWHHQPQHF TCRBV03 0 0.315956 CASSKATSSQSRANVLTF TCRBV07-04*01 0 0.315956 CASSLGGMDYGYTF TCRBV05-06*01 0 0.315956 CASSLGVGYTF TCRBV07-09 0 0.315956 CASSLRQGTTEAFF TCRBV28-01*01 0 0.315956 CSASAGLVTEAFF TCRBV20 0 0.315956 CARGQGDGYTF TCRBV30-01*01 0 0.157978 CASNREPNSPLHF TCRBV13-01*01 0 0.157978 CASQLGLGYEQYF TCRBV27-01*01 0 0.157978 CASRAPDLAPYEQYF TCRBV28-01*01 0 0.157978 CASRGYDREAFF TCRBV06-06 0 0.157978 CASRLKQSRANVLTF TCRBV09-01 0 0.157978 CASRPTGVLEQYF TCRBV03 0 0.157978 CASSAQGVIGDEQFF TCRBV09-01 0 0.157978 CASSCDHLHARNTIYF TCRBV03 0 0.157978 CASSDQGSDYEQYF TCRBV02-01*01 0 0.157978 CASSFGLAGLKTGELFF TCRBV06 0 0.157978 CASSFGQMNTEAFF TCRBV11-02*02 0 0.157978 CASSFGSHTEAFF TCRBV06-06 0 0.157978 CASSFLTQETQYF TCRBV13-01*01 0 0.157978 CASSFPGEASYEQYF TCRBV28-01*01 0 0.157978 CASSGADSNSPLHF TCRBV03 0 0.157978 CASSGGTADEQFF TCRBV13-01*01 0 0.157978 CASSLLASGNTIYF TCRBV07-01*01 0 0.157978 CASSLNSQGQSRANVLTF TCRBV07-01*01 0 0.157978 CASSLQGTSGRDNEQFF TCRBV27-01*01 0 0.157978 CASSLTPGQMNTEAFF TCRBV05-06*01 0 0.157978 CASSNQGLWHHQPQHF TCRBV03 0 0.157978 CASSNTGTVYGYTF TCRBV06-05*01 0 0.157978 CASSPDWGTGELFF TCRBV04-01*01 0 0.157978 CASSPGGLAKNIQYF TCRBV28-01*01 0 0.157978 CASSPGTANQPQHF TCRBV09-01 0 0.157978 CASSQDGGTVEQYF TCRBV04-02*01 0 0.157978 CASSQEDEYNEQFF TCRBV04-01*01 0 0.157978 CASSQEFNQPQHF TCRBV03 0 0.157978 CASSQHTQQSRANVLTF TCRBV23-01*01 0 0.157978 CASSQQETYEQYF TCRBV06-05*01 0 0.157978 CASSRASSYEQYF TCRBV27-01*01 0 0.157978 CASSRFVTMTSRANVLTF TCRBV07-09 0 0.157978 CASSSRGNEQFF TCRBV09-01 0 0.157978 CASSTLGQLNTEAFF TCRBV09-01 0 0.157978 CASSTTTGPSDNEQFF TCRBV18-01*01 0 0.157978 CASSWVANIQYF TCRBV28-01*01 0 0.157978 CASTLRRNEQYF TCRBV06-06 0 0.157978 CASVGPPNYGYTF TCRBV11-02*02 0 0.157978 CATGNKDEQYF TCRBV30-01*02 0 0.157978 CATRGGQGKGLADYNEQFF TCRBV24 0 0.157978 CESQGKRKTQYF TCRBV09-01 0 0.157978 CSAKPAGYQTGELFF TCRBV20 0 0.157978 CSAREGQGRARQETQYF TCRBV20 0 0.157978 CSASSDKNIQYF TCRBV20 0 0.157978 CSATSGYSYEQYF TCRBV0 0 0.157978 CSGLSDWAQYF TCRBV29-01*01 0 0.157978 CSVARSGKNYEQYF TCRBV29-01*01 0 0.157978 CSVEEAASGSPYEQYF TCRBV29-01*01 0 0.157978 CSVGTGGTNEKLFF TCRBV29-01*01 0 0.157978 CSVLGVLNTEAFF TCRBV29-01*01 0 0.157978 CSVVLGAGYEQYF TCRBV29*01*01 0 0.157978 RAIRVQGDWTEAFF TCRBV10-03*01 0 0.157978 CAIRGTSGRTGELFF TCRBV10-03*01 0.044248 0 CAISGDSSGANVLTF TCRBV10-03*01 0.044248 0 CAISPQVGEQYF TCRBV10-03*01 0.044248 0 CAPNGGGNTIYF TCRBV30-01*01 0.044248 0 CASATGVRAYEQYF TCRBV09-01 0.044248 0 CASGPDRTYEQYF TCRBV02-01*01 0.044248 0 CASGPFPRDSSYNEQFF TCRBV06 0.044248 0 CASGQGNQDTQYF TCRBV06-06 0.044248 0 CASHGAPGRAGKATQYF TCRBV03 0.044248 0 CASLGGIEAFF TCRBV16-01 0.044248 0 CASMRREVYEQFF TCRBV07-09 0.044248 0 CASNRSTQSRANVLTF TCRBV05-02*01 0.044248 0 CASQTSGSYEQYF TCRBV28-01*01 0.044248 0 CASRATMGTGQETQYF TCRBV06 0.044248 0 CASRDGDTGELFF TCRBV06-01*01 0.088496 0 CASRGRQAYEQYF TCRBV02-01*01 0.044248 0 CASRRIRELLTDTQYF TCRBV28-01*01 0.044248 0 CASRRPGLDNYGYTF TCRBV28-01*01 0.044248 0 CASRRSGLDNYGYTF TCRBV28-01*01 0.044248 0 CASRTAGEPHEQYF TCRBV13-01*01 0.088496 0 CASSAGLNTEAFF TCRBV06-04 0.088496 0 CASSALGNQPQHF TCRBV03 0.088496 0 CASSAPGHVGHGYTF TCRBV25-01*01 0.088496 0 CASSEATGRSEKLFF TCRBV02-01*01 0.044248 0 CASSEGFGEKLFF TCRBV25-01*01 0.088496 0 CASSFAGGGNTEAFF TCRBV05-01*01 0.044248 0 CASSFATSGFTDTQYF TCRBV12 0.044248 0 CASSFDGSLNTEAFF TCRBV06 0.044248 0 CASSFGDSAYNEQFF TCRBV06-06 0.044248 0 CASSFGGAHTGELFF TCRBV06 0.044248 0 CASSFKNRIGTEAFF TCRBV13-01*01 0.044248 0 CASSFLLHMISTDTQYF TCRBV05-06*01 0.265487 0 CASSFRTSGIDTQYF TCRBV28-00*01 0.044248 0 CASSGGGSGSDTQYF TCRB3V06-04 0.044248 0 CASSIGLLEQFF TCRBV07-02*01 0.044248 0 CASSIVDPIIHHNSPLHF TCRBV03 0.044248 0 CASSIVGSWGSNQPQHF TCRBV19-01 0.044248 0 CASSKGHPFHTYNSPLHF TCRBV03 0.044248 0 CASSLDNSYEQYF TCRBV07-06*01 0.044248 0 CASSLDRAGQPQHF TCRBV06 0.088496 0 CASSLEAGSYNEQFF TCRBV07-06*01 0.044248 0 CASSLELASSYEQYF TCRBV07-06*01 0.044248 0 CASSLGAWWEQYF TCRBV07-06*01 0.044248 0 CASSLGLGYYGYTF TCRBV07-02*01 0.044248 0 CASSLGRGLEQYF TCRBV07-07*01 0.044248 0 CASSLGRWERGETQYF TCRBV09-01 0.044248 0 CASSLGVDGLAYEQYF TCRBV07-02*01 0.044248 0 CASSLGVSPTDTQYF TCRBV07-09 0.044248 0 CASSLLGNSYNEQFF TCRBV27-01*01 0.044248 0 CASSLPPTGQETQYF TCRBV07-02*01 0.044248 0 CASSLSRGARTYEQYF TCRBV27-01*01 0.044248 0 CASSLVVESYEQYF TCRBV07-09 0.044248 0 CASSPDFGRLSYEQYF TCRBV27-01*01 0.044248 0 CASSPFGRGQDTQYF TCRBV11-02*02 0.088496 0 CASSPGETYEQYF TCRBV06 0.176991 0 CASSPGGQPFGYEQYF TCRBV09-01 0.044248 0 CASSPGISEQYF TCRBV04-02*01 0.044248 0 CASSPPGGVKEKLFF TCRBV04-01*01 0.044248 0 CASSPPNTGELFF TCRBV07-07*01 0.088496 0 CASSPRGTEAFF TCRBV06 0.044248 0 CASSPRTRGGGLNEQFF TCRBV07-03*01 0.044248 0 CASSPSGGLYF TCRBV06 0.044248 0 CASSPSGGNNYEQYF TCRBV28-01*01 0.044248 0 CASSPSPGQLTYEQYF TCRBV28-01*01 0.088496 0 CASSPWTGSEQYF TCRBV05-08*01 0.088496 0 CASSQAAGDQPQHF TCRBV03 0.044248 0 CASSQDLRGTRKNIQYF TCRBV11-02*02 0.044248 0 CASSQGRDAYEQYF TVRBV04-03*01 0.044248 0 CASSQKRDYEQYF TCRBV19-01 0.044248 0 CASSQLTSGDYNEQFF TCRBV04-03*01 0.088496 0 CASSQRDPNSPLHF TCRBV14-01*01 0.044248 0 CASSRAIATIRTEAFF TCRBV21-01*01 0.088496 0 CASSRGRGDGRTIYF TCRBV14-01*01 0.044248 0 CASSRKTVLNTEAFF TCRBV04-03*01 0.044248 0 CASSRVGQSYEQYF TCRBV12 0.044248 0 CASSSFRVHLYEQYF TCTBV28-01*01 0.044248 0 CASSSLRGGLTNTGELFF TCRBV07-07*01 0.044248 0 CASSSNHPQSRANVLTF TCRBV07-08*01 0.088496 0 CASSSQTSGSWTGELFF TCRBV07-06*01 0.044748 0 CASSSSISGPRSIYF TCRBV05-06*01 0.044248 0 CASSSSQSRANVLTF TCRBV07-04*01 0.044248 0 CASSSTGGTDTQYF TCRBV07-07*01 0.044248 0 CASSSTGVGETQYF TCRBV06-05*01 0.044248 0 CASSSTLAGYEQYV TCRBV14-01*01 0.044248 0 CASSSTSGLAKNIQYF TCRBV07-09 0.044248 0 CASSSVRGIQPPLHF TCRBV11-02*02 0.309735 0 CASSSVRGTQPPLHF TCRBV11-02*02 0.044248 0 CASSTLDRGVAGYTF TCRBV27-01*01 0.884956 0 CASSVAGAGELFF TCRBV09-01 0.044248 0 CASSVGGDQPQHF TCRBV09-01 0.044248 0 CASSVGVGVSGNTIYF TCRBV09-01 0.044248 0 CASSWAGGINEQFF TCRBV12 0.044248 0 CASSWDGNEAFF TCRBV13-01*01 0.044248 0 CASSYDRTGADTEAFF TCRBV06 0.044248 0 CASSYMGSGANVLTF TCRBV06-05*01 0.044248 0 CASSYSGRISRGYTS TCRBV06-05*01 0.044248 0 CASSYSRPGSGRAKDTQYF TCRBV06 0.044248 0 CASSYTRMPPTFNEKLFF TCRBV06-09*01 0.088496 0 CASTGGNRGVNEQFF TCRBV25-01*01 0.39823 0 CASTGGNRGVNERFF TCRBV25-01*01 0.044248 0 CASTLSQLGPLYYEQYF TCRBV04-01*01 0.044248 0 CASTPDRAVWNTEAFF TCRBV09-01 0.044248 0 CASTPGMGGYYEQYF TCRBV09-01 0.044248 0 CASTSGTANNEQFF TCRBV27-01*01 0.044248 0 CASTYGSGDYEQYF TCRBV19-01 0.044248 0 CATSAPGGYNEQFF TCRBV24 0.044248 0 CATSDSLAKNIQYF TCRBV24 0.044248 0 CATSDSQVLAGLNQETQYF TCRBV24 0.044248 0 CATSGYRELAFF TCRBV15-01*01 0.044248 0 CATSRDPLQEQFF TCRBV15-01*01 0.088496 0 CATSRDSNNEQFF TCRBV15-01*01 0.044248 0 CATSRDYEQYF TCRBV15-01*01 0.044248 0 CATSRGLAGGFEQFF TCRBV15-01*01 0.044248 0 CATSSLLASPNEQFF TCRBV15-01*01 0.442478 0 CAWGDMIRSQYF TCRBV30-01*01 0.044248 0 CAWSEDRDEQYF TCRBV30-01*01 0.044248 0 CAWSERDGGEQFF TCRBV30-01*01 0.044248 0 CAWSGDAGGYTLHF TCRBV30-01*01 0.044248 0 CAWSPGTGSQYF TCRBV30-01*01 0.088496 0 CAWTRTDTQYF TCRBV30-01*01 0.044248 0 CSAAPSGYSPLHF TCRBV20 0.044248 0 CSAGGQGSYSYGYTF TCRBV29-01*01 0.044248 0 CSAHLIGGRYNEQFF TCRBV20 0.044248 0 CSARDNRAEISPLHF TCRBV20 0.044248 0 CSARDQDEKLFF TCRBV20 0.088496 0 CSARDQGQHAPYEQYF TCRBV20 0.044248 0 CSARGLAGGGQFF TCRBV20 0.044248 0 CSARNRVNTGELFF TCRBV20 0.044248 0 CSARQLPYGYTF TCRBV20 0.044248 0 CSARVGVNQPQHF TCRBV20 0.044248 0 CSARVMTSGSMRETQYF TCRBV20 0.265487 0 CSARATMTSRSMREAQYF TCRBV20 0.044248 0 CSASEAAGANVLTF TCRBV20 0.044248 0 CSASPGTSTQTQYF TCRBV20 0.044248 0 CSASVTGTAYEQYF TCRBV20 0.044248 0 CSVAVTGTGEQYF TCRBV29-01*01 0.044248 0 CSVERGTEAFF TCRBV29-01*01 0.044248 0 CSVGPGLASPLHF TCRBV29-01*01 0.088496 0 CSVSGRSYNEQFF TCRBV29-01*01 0.044248 0 CSVTGTDYSYEQYF TCRBV29-01*01 0.044248 0 CSVVQGAGYTF TCRBV29-01*01 0.088496 0 RASSGADSNSPLHF TCRBV03 0.044248 0 *Bolded clonotypes were positive by FEST analysis

TABLE-US-00003 TABLE3 CandidateMANAstestedbyMANAFESTinpatientJH124 Candidate MANA Predicted Candidate CandidateMANA % MHC MANA Candidate AminoAcid PredictedHLA Gene mutant ClassI Affinity% MANAID Sequence Restriction Symbol reads Affinity Rank MANA2 HVIENIYF HLA-A*25:01 OPA1 46% 171.5 0.1 MANA3 DVAAHLQPL HLA-A.25:01 HMGXB4 35% 185 0.1 MANA4 ETPNLDLM HLA-A*25:01 GALNS 73% 1085.7 0.5 MANA5 SVFNTWNPM HIA-A*25:01 PLEK 49% 738 0.3 MANA5 SVFNTWNPM HLA-C*12:03 PLEK 49% 21.6 0.3 MANA6 EVQQFLRY HLA-A*25:01 LARGE 38% 665.3 0.3 MANA7 EVIVPLSGW HLA-A*25:01 ARVCF 39% 339.7 0.17 MANA8 ETMQCSELY HLA-A*25:01 KLHL29 79% 85.2 0.05 MANA9 ETMQCSELYHM HLA-A*25:01 KLHL29 79% 208.9 0.12 MANA10 ETMQCSEL HLA-A*25:01 KLHL29 79% 773.2 0.4 MANA18 ITRTVSANTV HLA-A*30:01 TNC 40% 43.4 0.8 MANA19 ATKNNKVIMA HLA-A*30:01 PSMC5 36% 28.4 0.5 MANA20 VAHFQLQMLK HLA-A*30:01 SLC4A2 25% 49.5 1 MANA23 EEDTFSYLI HLA-B*38:01 ANKLE2 44% 1421.3 0.5 MANA24 AHFQLQML HLA-B*38:01 SLC4A2 25% 628.5 0.25 MANA24 AHFQLQML HLA-B*39:01 SLC4A2 25% 83.9 0.4 MANA25 LHAMIQAAGKL HLA-B*38:01 CHD6 34% 777.4 0.3 MANA26 LHEAQPWFEFL HLA-B*38:01 SPG11 31% 517.6 0.2 MANA27 LHEAQPWFEF HLA-B*38:01 SPG11 31% 822.9 03 MANA28 EHLSCPDNFL HLA-B*38:01 ATG5 33% 583.5 0.25 MANA29 NHARIDAAKV HLA-B*38:01 CDKN2A 67% 1323.2 0.5 MANA30 QHQPNPFEV HLA-B*38:01 SGK223 54% 978.5 0.4 MANA33 TQLEKEAL HLA-B*39:01 NIN 70% 106 0.5 MANA34 TRARNEYLLSL HLA-B*39:01 ARHGAP4 23% 56.4 0.25 MANA35 NPMWVVLL HLA-B*39:01 PLEK 49% 60.8 0.3 MANA36 KHILVWAL HLA-B*39:01 C22orf39 14% 81.4 0.4 MANA36 KHILVWAL HLA-B*38:01 C22orf3 14% 406.1 0.17 MANA37 SQSDYIPM HLA-B*39:01 GAB2 39% 44.4 0.2 MANA38 VHDYFSVI HLA-B*39:01 SACS 14% 114.7 0.5 MANA43 IYFPAAQTM HLA-C*12:03 OPA1 46% 18.6 0.25 MANA44 ISYLIWSNPRY HLA-C*12:03 ANKLE2 44% 32.8 0.5 MANA45 YSWSAQRQAL HLA-C*12:03 GAK 18% 39.2 0.8 MANA46 FAVWTLAETI HLA-C*12:03 IPO4 39% 17.4 0.2 MANA47 FASLALARRYL HLA-C*12:03 TMEM64 45% 17.1 0.2 MANA48 DVIQQDELDSY HLA-A*25:01 PVRL3 39% 151 0.1 MANA49 KNRSSGTVSA HLA-A*30:01 HBP1 24% 27.5 0.5 MANA50 KLKRFNLSA HLA-A*30:01 APEH 48% 8.6 0.1 MANA51 KSFAVWTLA HLA-A*30:01 IPO4 39% 23.6 0.4 MANA52 KWRLSLCTV HLA-A*30:01 C3orf17 44% 31.6 0.8 MANA53 RSRPVAATAK HLA-A*30:01 IQCB1 45% 3 0.01 MANA54 RSPVAATA HLA-A*30:01 IQCB1 45% 4.8 0.03 MANA55 TAKQAHLTTLK HLA-A*30:01 IQCB1 45% 33.6 0.8 MANA56 SHCPSAMGI HLA-B*38:01 NFKBIL1 47% 925.8 0.4 MANA57 FHASEGWL HLA-B*39:01 CENPBD1 75% 54.5 0.25 MANA58 THEVIVPL HLA-B*39:01 ARVCF 39% 42.5 0.2 MANA59 SRHCLQPL HLA-B*39:01 FRS3 43% 58.5 0.25 MANA60 FASLALARRY HLA-C*12:03 TMEM64 45% 41.7 0.8 MANA61 SVFNTWNPMWV HLA-C*12:03 PLEK 49% 32.8 0.5 MANA62 LTHEVIVPL HLA-C*12:03 ARVCF 39% 36.7 0.5 MANA63 YTVMARKSPV HLA-C*12:03 RAD54L2 74% 34.5 0.5 Candidate MANA WTPredicted WT Median Candidate Binding WTAminoAcid MHCClassI Affinity WTBinding Tumor MANAID Classification Sequence Affinity %Rank Classification Expression* MANA2 SB HVIENIYL 686.2 0.3 SB 2671.6 MANA3 SB DVAAHLQLL 551.1 0.25 SB 857.6 MANA4 SB ETPNLDRM 1796.9 0.8 WB 779.5 MANA5 SB SVFNTWKPM 2722 1 WB 516.3 MANA5 SB SVFNTWKPM 56.3 0.8 WB 516.3 MANA6 SB EAQQFLRY 3442.6 1.5 WB 365.8 MANA7 SB EVIVPHSGW 1530.2 0.8 WB 253.7 MANA8 SB EAMQCSELY 900.2 0.4 SB 207.2 MANA9 SB EAMQCSELYHM 2362.4 0.8 WB 207.2 MANA10 SB EAMQCSEL 5586.5 2 WB 207.2 MANA18 SB ITRTVSGNTV 69.9 1.5 WB 6942.8 MANA19 SB ATKNIKVIMA 23.2 0.4 SB 2784.6 MANA20 SB VAHFQRQMLK 28.7 0.5 SB 1906.6 MANA23 SB EEDTFSDLI 2976.8 1 WB 1946.6 MANA24 SB AHFQRQML 1933 0.8 WB 1906.6 MANA24 SB AHFQRQML 129.8 0.8 WB 1906.6 MANA25 SB LQAMIQAAGKL 8269.3 4 NB 1380.9 MANA26 SB LHEAHPWFEFL 448.7 0.17 SB 1306 MANA27 SB LHEAHPWFEP 626.8 0.25 SB 1306 MANA28 SB EHLSYPDNFL 465.8 0.17 SB 998 MANA29 SB NHARIDAAEG 17959.6 15 NB 576.8 MANA30 SB LHQPNPFEV 745.4 0.3 SB 542.1 MANA33 SB TQQEKEAL 162.7 0.8 WB 1078.6 MANA34 SB TKARNEYLLSL 142 0.8 WB 538.9 MANA35 SB KPMWVVLL 334.1 1.5 WB 516.3 MANA36 SB KHILVWAP 10929.7 15 NB 441.3 MANA36 SB KHILVWAP 19040.8 15 NB 441.3 MANA37 SB SQSVYIPM 127.9 0.8 WB 375.9 MANA38 SB VHDDFSVI 177.5 0.8 WB 358.2 MANA43 SB IYLPAAQTM 174.5 2 WB 2671.6 MANA44 SB FSDLIWSNPRY 833 6 NB 1946.6 MANA45 SB YSWSAQRRAL 30.5 0.4 SB 1492.1 MANA46 SB FAVGTLAETI 24.7 0.3 SB 1293.3 MANA47 SB FASLALVRRYL 19.2 0.25 SB 653 MANA48 SB DVLQQDELDSY 3171.1 1.5 WB 93.3 MANA49 SB KNHSSGTVSA 180.6 3 WB 1549.7 MANA50 SB KLKSFNLSA 9.3 0.12 SB 1448.6 MANA51 SB KSFAVGTLA 57 1 WB 1293.3 MANA52 SB KWRLSHCTV 22.8 0.4 SB 1213.3 MANA53 SB RSRPVAAKAK 4.1 0.03 SB 722.9 MANA54 SB RSRPVAAKA 5.3 0.05 SB 722.9 MANA55 SB KAKQAHLTTLK 4.8 0.03 SB 722.9 MANA56 SB SRCPSAMGI 15334.5 9 NB 400.2 MANA57 SB FHASQGWL 31.9 0.12 SB 292.7 MANA58 SB THEVIVPH 9995.6 15 NB 253.7 MANA59 SB GRHCLQPL 365.5 1.5 WB 120.4 MANA60 SB FASLALVRRY 32.8 0.5 SB 653 MANA61 SB SVFNTWKPMWV 71 1 WB 516.3 MANA62 SB LTHEVIVPH 747.9 6 NB 253.7 MANA63 SB VYRYGQKKPC 38060.3 50 NB 172.4

TABLE-US-00004 TABLE 4 positive_clones sum_freq JH124_04 1 2.737226 JH124_07 2 8.590604 JH124_08 1 6.757391 JH124_09 1 6.359032 JH124_18 1 1.067236 JH124_25 1 1.070579 JH124_28 1 0.804455 JH124_30 3 3.697834 JH124_33 1 0.705581 JH124_34 1 3.039874 JH124_35 1 2.122642 JH124_46 1 2.004008 JH124_51 1 0.668449 JH124_52 3 6.909895 JH124_53 1 3.774238 JH124_54 1 0.74206 JH124_55 1 4.546692 JH124_57 1 0.755192 JH124_58 1 3.218391 JH124_59 2 6.101104 JH124_63 2 2.952381

TABLE-US-00005 TABLE5 JH124baseline JH124_NoPep FC:JH124 Condition percent FC:JH124baseline percent NoPep CASSGQGGVSEKLFF JH124_04 0 29 0 34 CASSLTGGYTGELFF JH124_07 0.093458 53 0.079365 62 CASSLRSSSETQYF JH124_07 0 39 0 46 CASSLQAGSSYNEQFF JH124_63 0 23 0 28 CASSPSGGSLYNEQFF JH124_63 0 8 0 10 CASSGGISNTEAFF JH124_35 0 23 0 27 CATSTLPSGGEGYEQYF JH124_54 0.186916 4 0 9 CASSGTTYGYTF JH124_09 0 68 0 80 CSASRGLSGYTF JH124_53 0 40 0 48 LGPGTFTSYEQYF JH124_28 0 9 0 10 CASSTGTGFGEQYF JH124_57 0 8 0 10 CASSEGGSDEQYF JH124_25 0 11 0 13 CASSLDPRSSYNSPLHF JH124_33 0 8 0 9 CASTAGTVHSNQPQHF JH124_55 0 49 0 57 CASRDTRELNTEAFF JH124_59 0 34 0 40 CASSLGDLRGFTEAFF JH124_59 0 31 0.15873 18 CATSDLVARDEQFF JH124_18 0 11 0 13 CASSYRGTGGGGYTF JH124_08 0 72 0 85 CASSLQGGMGNQPQHF JH124_30 0 22 0 26 CASSLGLGDEQFF JH124_30 0 11 0 13 CASFRQVNYGYTF JH124_30 0 7 0 8 CASSFRQGSYEQYF JH124_46 0 21 0 25 CASSPDIQYF JH124_51 0 7 0 8 CASSETGWGAFF JH124_34 0.186916 16 0.238095 13 CASRRHSNQPQHF JH124_52 0 46 0 55 CASSLVGLAGNEQFF JH124_52 0 19 0 22 CASSPQDQHVYEQYF JH124_52 0 9 0 10 CASTLRVDTEAFF JH124_58 0 34 0 41

TABLE-US-00006 TABLE6 JH124_PBMC_ JH124 JH124_PBMC_ 68w_ Baseline 64w_ JH124_NoPep JH124_10_ condition abundance abundance abundance abundance abundance CASSGQGGVSEKLFF JH124_04 0 0 0 0 0 CASSLTGGYTGELFF JH124_07 1 1 0 1 0 CASSLRSSSETQYF JH124_07 0 0 0 0 0 CASSLQAGSSYNEQFF JH124_63 0 0 0 0 0 CASSPSGGSLYNEQFF JH124_63 0 0 0 0 0 CASSGGISNTEAFF JH124_35 0 0 0 0 0 CATSTLPSGGEGYEQYF JH124_54 0 2 0 0 0 CASSGTTYGYTF JH124_09 4 0 11 0 0 CSASRGLSGYTF JH124_53 0 0 0 0 0 LGPGTFTSYEQYF JH124_28 0 0 2 0 0 CASSTGTGFGEQYF JH124_57 0 0 0 0 0 CASSEGGSDEQYF JH124_25 0 0 0 0 0 CASSLDPRSSYNSPLHF JH124_33 0 0 0 0 0 CASTAGTVHSNQPQHF JH124_55 0 0 0 0 0 CASRDTRELNTEAFF JH124_59 0 0 0 0 0 CASSLGDLRGFTEAFF JH124_59 0 0 0 2 0 CATSDLVARDEQFF JH124_18 0 0 0 0 0 CASSYRGTGGGGYTF JH124_08 1 0 0 0 0 CASSLQGGMGNQPQHF JH124_30 2 0 1 0 0 CASSLGLGDEQFF JH124_30 0 0 1 0 0 CASFRQVNYGYTF JH124_30 0 0 0 0 0 CASSFRQGSYEQYF JH124_46 0 0 0 0 0 CASSPDIQYF JH124_51 0 0 0 0 0 CASSETGWGAFF JH124_34 8 2 27 3 0 CASRRHSNQPQHF JH124_52 0 0 0 0 0 CASSLVGLAGNEQFF JH124_52 0 0 0 0 0 CASSPQDQHVYEQYF JH124_52 0 0 0 0 0 CASTLRVDTEAFF JH124_58 0 0 0 0 0 JH124_49 JH124_50 JH124_04_ JH124_07_ JH124_19_ JH124_05_ JH124_27_ condition abundance abundance abundance abundance abundance abundance abundance CASSGQGGVSEKLFF JH124_04 0 0 60 0 0 0 0 CASSLTGGYTGELFF JH124_07 1 2 0 110 0 4 0 CASSLRSSSETQYF JH124_07 0 0 0 82 0 0 0 CASSLQAGSSYNEQFF JH124_63 0 0 0 0 0 0 0 CASSPSGGSLYNEQFF JH124_63 0 0 0 0 0 0 0 CASSGGISNTEAFF JH124_35 0 0 0 0 1 0 0 CATSTLPSGGEGYEQYF JH124_54 0 0 0 0 0 0 0 CASSGTTYGYTF JH124_09 0 0 0 0 0 0 0 CSASRGLSGYTF JH124_53 0 0 0 0 0 0 0 LGPGTFTSYEQYF JH124_28 0 0 0 0 0 0 0 CASSTGTGFGEQYF JH124_57 0 0 0 0 0 0 0 CASSEGGSDEQYF JH124_25 0 0 0 0 0 0 0 CASSLDPRSSYNSPLHF JH124_33 0 0 0 0 0 0 0 CASTAGTVHSNQPQHF JH124_55 0 0 0 0 0 0 0 CASRDTRELNTEAFF JH124_59 0 0 0 0 0 0 0 CASSLGDLRGFTEAFF JH124_59 0 0 0 0 0 0 0 CATSDLVARDEQFF JH124_18 0 0 0 0 0 0 0 CASSYRGTGGGGYTF JH124_08 0 0 0 0 0 0 0 CASSLQGGMGNQPQHF JH124_30 0 1 0 3 0 0 0 CASSLGLGDEQFF JH124_30 0 0 0 0 0 0 0 CASFRQVNYGYTF JH124_30 0 0 0 0 0 0 0 CASSFRQGSYEQYF JH124_46 0 0 0 0 0 0 0 CASSPDIQYF JH124_51 0 0 0 0 0 0 0 CASSETGWGAFF JH124_34 1 4 0 0 1 1 2 CASRRHSNQPQHF JH124_52 0 0 0 0 0 0 0 CASSLVGLAGNEQFF JH124_52 0 0 0 0 0 0 0 CASSPQDQHVYEQYF JH124_52 0 0 0 0 0 0 0 CASTLRVDTEAFF JH124_58 0 0 0 0 0 0 0 JH124_63_ JH124_35_ JH124_54_ JH124_61_ JH124_09_ JH124_45_ condition abundance abundance abundance abundance abundance abundance CASSGQGGVSEKLFF JH124_04 0 0 0 0 0 0 CASSLTGGYTGELFF JH124_07 4 1 2 1 2 4 CASSLRSSSETQYF JH124_07 0 0 0 0 0 0 CASSLQAGSSYNEQFF JH124_63 46 0 0 0 0 0 CASSPSGGSLYNEQFF JH124_63 16 0 0 0 0 0 CASSGGISNTEAFF JH124_35 0 54 0 0 0 0 CATSTLPSGGEGYEQYF JH124_54 0 0 25 0 0 0 CASSGTTYGYTF JH124_09 0 0 0 0 113 0 CSASRGLSGYTF JH124_53 0 0 0 0 0 0 LGPGTFTSYEQYF JH124_28 0 0 0 0 0 0 CASSTGTGFGEQYF JH124_57 0 0 0 0 0 0 CASSEGGSDEQYF JH124_25 0 0 0 0 0 0 CASSLDPRSSYNSPLHF JH124_33 0 0 0 0 0 0 CASTAGTVHSNQPQHF JH124_55 0 0 3 0 0 0 CASRDTRELNTEAFF JH124_59 0 0 0 0 0 0 CASSLGDLRGFTEAFF JH124_59 0 0 0 0 0 0 CATSDLVARDEQFF JH124_18 0 0 0 0 0 8 CASSYRGTGGGGYTF JH124_08 0 0 0 0 0 0 CASSLQGGMGNQPQHF JH124_30 0 0 1 0 0 0 CASSLGLGDEQFF JH124_30 0 1 0 0 0 0 CASFRQVNYGYTF JH124_30 0 0 0 0 0 0 CASSFRQGSYEQYF JH124_46 0 0 0 0 0 0 CASSPDIQYF JH124_51 0 0 0 0 0 0 CASSETGWGAFF JH124_34 0 0 0 0 1 1 CASRRHSNQPQHF JH124_52 0 0 0 0 0 0 CASSLVGLAGNEQFF JH124_52 0 0 0 0 0 0 CASSPQDQHVYEQYF JH124_52 0 0 0 0 0 0 CASTLRVDTEAFF JH124_58 0 0 0 0 0 0 JH124_53_ JH124_06_ JH124_28_ JH124_36_ JH124_47_ JH124_62_ condition abundance abundance abundance abundance abundance abundance CASSGQGGVSEKLFF JH124_04 0 0 0 0 0 0 CASSLTGGYTGELFF JH124_07 0 0 1 0 3 0 CASSLRSSSETQYF JH124_07 0 0 0 0 0 0 CASSLQAGSSYNEQFF JH124_63 0 0 0 0 0 0 CASSPSGGSLYNEQFF JH124_63 0 0 0 0 0 0 CASSGGISNTEAFF JH124_35 0 0 0 0 0 0 CATSTLPSGGEGYEQYF JH124_54 0 0 0 0 0 0 CASSGTTYGYTF JH124_09 0 0 2 0 0 0 CSASRGLSGYTF JH124_53 109 0 0 0 0 0 LGPGTFTSYEQYF JH124_28 0 0 13 0 0 0 CASSTGTGFGEQYF JH124_57 0 0 0 0 0 0 CASSEGGSDEQYF JH124_25 0 0 0 0 0 0 CASSLDPRSSYNSPLHF JH124_33 0 0 0 0 0 0 CASTAGTVHSNQPQHF JH124_55 0 0 0 0 0 0 CASRDTRELNTEAFF JH124_59 0 0 0 0 0 0 CASSLGDLRGFTEAFF JH124_59 0 0 0 0 0 0 CATSDLVARDEQFF JH124_18 0 0 0 0 0 0 CASSYRGTGGGGYTF JH124_08 0 0 0 0 0 0 CASSLQGGMGNQPQHF JH124_30 0 0 0 0 0 0 CASSLGLGDEQFF JH124_30 0 0 0 0 0 0 CASFRQVNYGYTF JH124_30 0 0 0 0 0 0 CASSFRQGSYEQYF JH124_46 0 0 0 0 0 0 CASSPDIQYF JH124_51 0 0 0 0 0 0 CASSETGWGAFF JH124_34 0 1 0 1 0 4 CASRRHSNQPQHF JH124_52 0 0 0 0 0 0 CASSLVGLAGNEQFF JH124_52 0 0 0 0 0 0 CASSPQDQHVYEQYF JH124_52 0 0 0 0 0 0 CASTLRVDTEAFF JH124_58 0 0 0 0 0 0 JH124_02_ JH124_26_ JH124_37_ JH124_57_ JH124_25_ JH124_33_ JH124_38_ condition abundance abundance abundance abundance abundance abundance abundance CASSGQGGVSEKLFF JH124_04 0 0 0 0 0 0 0 CASSLTGGYTGELFF JH124_07 2 0 1 1 0 2 1 CASSLRSSSETQYF JH124_07 0 0 0 0 0 0 0 CASSLQAGSSYNEQFF JH124_63 0 0 0 0 0 0 0 CASSPSGGSLYNEQFF JH124_63 0 0 0 0 0 0 0 CASSGGISNTEAFF JH124_35 0 0 0 0 0 0 0 CATSTLPSGGEGYEQYF JH124_54 0 1 0 1 0 0 0 CASSGTTYGYTF JH124_09 0 0 0 0 0 0 0 CSASRGLSGYTF JH124_53 0 0 5 0 0 0 0 LGPGTFTSYEQYF JH124_28 0 0 0 0 0 0 0 CASSTGTGFGEQYF JH124_57 0 0 0 12 0 0 0 CASSEGGSDEQYF JH124_25 0 0 0 0 27 0 0 CASSLDPRSSYNSPLHF JH124_33 0 0 0 0 0 11 0 CASTAGTVHSNQPQHF JH124_55 0 0 0 0 0 0 0 CASRDTRELNTEAFF JH124_59 0 0 0 0 0 0 0 CASSLGDLRGFTEAFF JH124_59 0 0 0 0 0 0 0 CATSDLVARDEQFF JH124_18 0 0 0 0 0 0 0 CASSYRGTGGGGYTF JH124_08 0 0 0 0 6 0 2 CASSLQGGMGNQPQHF JH124_30 0 0 0 1 1 0 0 CASSLGLGDEQFF JH124_30 0 0 0 0 0 0 0 CASFRQVNYGYTF JH124_30 0 0 0 0 0 0 0 CASSFRQGSYEQYF JH124_46 0 0 0 0 0 0 0 CASSPDIQYF JH124_51 0 0 0 0 0 0 0 CASSETGWGAFF JH124_34 0 1 0 1 0 0 0 CASRRHSNQPQHF JH124_52 0 0 0 0 0 0 0 CASSLVGLAGNEQFF JH124_52 0 0 0 0 0 0 0 CASSPQDQHVYEQYF JH124_52 0 0 0 0 0 0 0 CASTLRVDTEAFF JH124_58 0 0 0 0 0 0 0 JH124_60_ JH124_03_ JH124_24_ JH124_48_ JH124_43_ JH124_55_ condition abundance abundance abundance abundance abundance abundance CASSGQGGVSEKLFF JH124_04 0 5 0 0 0 0 CASSLTGGYTGELFF JH124_07 3 0 3 1 1 1 CASSLRSSSETQYF JH124_07 0 0 0 0 0 0 CASSLQAGSSYNEQFF JH124_63 0 0 0 0 0 0 CASSPSGGSLYNEQFF JH124_63 0 0 0 0 0 0 CASSGGISNTEAFF JH124_35 0 0 0 0 0 0 CATSTLPSGGEGYEQYF JH124_54 0 0 0 0 0 0 CASSGTTYGYTF JH124_09 0 0 0 0 0 0 CSASRGLSGYTF JH124_53 0 0 0 0 0 0 LGPGTFTSYEQYF JH124_28 0 0 0 0 0 0 CASSTGTGFGEQYF JH124_57 0 0 0 0 0 0 CASSEGGSDEQYF JH124_25 0 0 2 0 0 0 CASSLDPRSSYNSPLHF JH124_33 0 0 0 0 0 0 CASTAGTVHSNQPQHF JH124_55 0 0 0 0 1 167 CASRDTRELNTEAFF JH124_59 0 0 0 0 0 0 CASSLGDLRGFTEAFF JH124_59 0 0 0 0 0 0 CATSDLVARDEQFF JH124_18 0 0 0 0 0 0 CASSYRGTGGGGYTF JH124_08 0 0 0 0 0 0 CASSLQGGMGNQPQHF JH124_30 0 2 0 0 0 0 CASSLGLGDEQFF JH124_30 0 0 0 0 0 0 CASFRQVNYGYTF JH124_30 0 0 0 0 0 0 CASSFRQGSYEQYF JH124_46 0 0 0 0 0 0 CASSPDIQYF JH124_51 0 0 0 0 0 0 CASSETGWGAFF JH124_34 9 0 0 4 1 0 CASRRHSNQPQHF JH124_52 0 0 0 0 0 0 CASSLVGLAGNEQFF JH124_52 0 0 0 0 0 0 CASSPQDQHVYEQYF JH124_52 0 0 0 0 0 0 CASTLRVDTEAFF JH124_58 0 0 0 0 0 0 JH124_59_ JH124_18_ JH124_44_ JH124_56_ JH124_08_ JH124_29_ JH124_30_ condition abundance abundance abundance abundance abundance abundance abundance CASSGQGGVSEKLFF JH124_04 0 0 0 0 0 0 0 CASSLTGGYTGELFF JH124_07 4 0 0 3 0 2 0 CASSLRSSSETQYF JH124_07 0 0 0 0 0 0 0 CASSLQAGSSYNEQFF JH124_63 0 0 0 0 0 0 0 CASSPSGGSLYNEQFF JH124_63 0 0 0 0 0 0 0 CASSGGISNTEAFF JH124_35 0 0 0 0 0 0 0 CATSTLPSGGEGYEQYF JH124_54 0 0 0 0 0 0 0 CASSGTTYGYTF JH124_09 0 0 0 0 0 0 0 CSASRGLSGYTF JH124_53 0 0 0 0 0 0 0 LGPGTFTSYEQYF JH124_28 0 0 0 0 0 0 0 CASSTGTGFGEQYF JH124_57 0 0 0 0 0 0 0 CASSEGGSDEQYF JH124_25 0 0 0 0 0 0 0 CASSLDPRSSYNSPLHF JH124_33 0 0 0 0 0 0 0 CASTAGTVHSNQPQHF JH124_55 0 0 0 0 0 0 0 CASRDTRELNTEAFF JH124_59 109 0 0 0 0 0 0 CASSLGDLRGFTEAFF JH124_59 101 0 0 0 0 0 0 CATSDLVARDEQFF JH124_18 0 10 0 0 0 0 0 CASSYRGTGGGGYTF JH124_08 0 0 0 1 144 0 0 CASSLQGGMGNQPQHF JH124_30 0 0 0 0 0 0 39 CASSLGLGDEQFF JH124_30 0 0 0 0 0 0 19 CASFRQVNYGYTF JH124_30 0 0 0 0 0 0 12 CASSFRQGSYEQYF JH124_46 0 0 0 0 0 0 0 CASSPDIQYF JH124_51 0 0 0 0 0 0 0 CASSETGWGAFF JH124_34 1 1 0 0 1 0 1 CASRRHSNQPQHF JH124_52 0 0 0 0 0 0 0 CASSLVGLAGNEQFF JH124_52 0 0 0 0 0 0 0 CASSPQDQHVYEQYF JH124_52 0 0 0 0 0 0 0 CASTLRVDTEAFF JH124_58 0 0 0 0 0 0 0 JH124_46_ JH124_20_ JH124_23_ JH124_51_ JH124_34_ JH124_52_ condition abundance abundance abundance abundance abundance abundance CASSGQGGVSEKLFF JH124_04 0 0 0 0 0 0 CASSLTGGYTGELFF JH124_07 0 0 3 0 1 1 CASSLRSSSETQYF JH124_07 0 0 0 0 0 0 CASSLQAGSSYNEQFF JH124_63 0 0 0 0 0 0 CASSPSGGSLYNEQFF JH124_63 0 0 0 0 0 0 CASSGGISNTEAFF JH124_35 0 0 0 0 0 0 CATSTLPSGGEGYEQYF JH124_54 0 0 0 0 0 0 CASSGTTYGYTF JH124_09 0 0 0 0 0 0 CSASRGLSGYTF JH124_53 0 0 0 0 0 0 LGPGTFTSYEQYF JH124_28 0 0 0 0 0 0 CASSTGTGFGEQYF JH124_57 0 0 0 0 0 0 CASSEGGSDEQYF JH124_25 0 0 0 0 0 0 CASSLDPRSSYNSPLHF JH124_33 0 0 0 0 0 0 CASTAGTVHSNQPQHF JH124_55 0 0 0 0 0 0 CASRDTRELNTEAFF JH124_59 0 0 0 0 0 0 CASSLGDLRGFTEAFF JH124_59 0 0 0 0 0 0 CATSDLVARDEQFF JH124_18 0 0 0 0 0 0 CASSYRGTGGGGYTF JH124_08 0 0 0 0 0 2 CASSLQGGMGNQPQHF JH124_30 0 0 0 5 0 2 CASSLGLGDEQFF JH124_30 0 0 0 0 0 0 CASFRQVNYGYTF JH124_30 0 0 0 0 0 0 CASSFRQGSYEQYF JH124_46 40 0 0 0 0 0 CASSPDIQYF JH124_51 0 0 0 15 0 0 CASSETGWGAFF JH124_34 0 0 0 2 77 3 CASRRHSNQPQHF JH124_52 0 0 0 0 0 157 CASSLVGLAGNEQFF JH124_52 0 0 0 7 0 64 CASSPQDQHVYEQYF JH124_52 0 0 0 0 0 29 CASTLRVDTEAFF JH124_58 0 0 0 0 0 0 JH124_58_ JH124_PBMC_2w_ JH124_PBMC_10w_ JH124_PBMC_4w__ condition abundance abundance abundance abundance CASSGQGGVSEKLFF JH124_04 0 0 0 0 CASSLTGGYTGELFF JH124_07 0 10 4 10 CASSLRSSSETQYF JH124_07 0 0 0 0 CASSLQAGSSYNEQFF JH124_63 0 0 0 0 CASSPSGGSLYNEQFF JH124_63 0 0 0 0 CASSGGISNTEAFF JH124_35 0 0 0 0 CATSTLPSGGEGYEQYF JH124_54 0 0 0 0 CASSGTTYGYTF JH124_09 0 8 27 10 CSASRGLSGYTF JH124_53 0 0 0 0 LGPGTFTSYEQYF JH124_28 0 0 0 3 CASSTGTGFGEQYF JH124_57 0 0 0 0 CASSEGGSDEQYF JH124_25 0 0 0 0 CASSLDPRSSYNSPLHF JH124_33 0 0 0 0 CASTAGTVHSNQPQHF JH124_55 0 0 0 0 CASRDTRELNTEAFF JH124_59 0 0 0 0 CASSLGDLRGFTEAFF JH124_59 0 0 0 0 CATSDLVARDEQFF JH124_18 0 0 0 0 CASSYRGTGGGGYTF JH124_08 0 0 0 0 CASSLQGGMGNQPQHF JH124_30 0 0 11 7 CASSLGLGDEQFF JH124_30 0 0 2 0 CASFRQVNYGYTF JH124_30 0 0 0 0 CASSFRQGSYEQYF JH124_46 0 0 0 0 CASSPDIQYF JH124_51 0 0 0 0 CASSETGWGAFF JH124_34 1 0 0 0 CASRRHSNQPQHF JH124_52 0 0 0 0 CASSLVGLAGNEQFF JH124_52 0 0 0 0 CASSPQDQHVYEQYF JH124_52 0 0 0 0 CASTLRVDTEAFF JH124_58 42 0 0 0 JH124_PBMC_ JH124_Tumor_ JH124_PBMC_68w_ JH124_baseline JH124_PBMC_64w_ condition PreRx_abundance abundance percent percent percent CASSGQGGVSEKLFF JH124_04 0 0 0 0 0 CASSLTGGYTGELFF JH124_07 2 5 0.002048131 0.093457944 0 CASSLRSSSETQYF JH124_07 0 0 0 0 0 CASSLQAGSSYNEQFF JH124_63 0 0 0 0 0 CASSPSGGSLYNEQFF JH124_63 0 0 0 0 0 CASSGGISNTEAFF JH124_35 0 0 0 0 0 CATSTLPSGGEGYEQYF JH124_54 0 0 0 0.186915888 0 CASSGTTYGYTF JH124_09 0 0 0.008192524 0 0.016853072 CSASRGLSGYTF JH124_53 0 0 0 0 0 LGPGTFTSYEQYF JH124_28 3 0 0 0 0.003064195 CASSTGTGFGEQYF JH124_57 0 0 0 0 0 CASSEGGSDEQYF JH124_25 0 0 0 0 0 CASSLDPRSSYNSPLHF JH124_33 0 0 0 0 0 CASTAGTVHSNQPQHF JH124_55 0 0 0 0 0 CASRDTRELNTEAFF JH124_59 0 0 0 0 0 CASSLGDLRGFTEAFF JH124_59 0 0 0 0 0 CATSDLVARDEQFF JH124_18 0 0 0 0 0 CASSYRGTGGGGYTF JH124_08 0 0 0.002048131 0 0 CASSLQGGMGNQPQHF JH124_30 2 0 0.004096262 0 0.001532097 CASSLGLGDEQFF JH124_30 2 0 0 0 0.001532097 CASFRQVNYGYTF JH124_30 0 0 0 0 0 CASSFRQGSYEQYF JH124_46 0 0 0 0 0 CASSPDIQYF JH124_51 0 0 0 0 0 CASSETGWGAFF JH124_34 0 0 0.016385049 0.186915888 0.041366631 CASRRHSNQPQHF JH124_52 0 0 0 0 0 CASSLVGLAGNEQFF JH124_52 0 0 0 0 0 CASSPQDQHVYEQYF JH124_52 0 0 0 0 0 CASTLRVDTEAFF JH124_58 0 0 0 0 0 JH124_NoPep_ JH124_10_ JH124_49_ JH124_50_ JH124_04_ condition percent percent percent percent percent CASSGQGGVSEKLFF JH124_04 0 0 0 0 2.737226 CASSLTGGYTGELFF JH124_07 0.079365079 0 0.038153 0.075047 0 CASSLRSSSETQYF JH124_07 0 0 0 0 0 CASSLQAGSSYNEQFF JH124_63 0 0 0 0 0 CASSPSGGSLYNEQFF JH124_63 0 0 0 0 0 CASSGGISNTEAFF JH124_35 0 0 0 0 0 CATSTLPSGGEGYEQYF JH124_54 0 0 0 0 0 CASSGTTYGYTF JH124_09 0 0 0 0 0 CSASRGLSGYTF JH124_53 0 0 0 0 0 LGPGTFTSYEQYF JH124_28 0 0 0 0 0 CASSTGTGFGEQYF JH124_57 0 0 0 0 0 CASSEGGSDEQYF JH124_25 0 0 0 0 0 CASSLDPRSSYNSPLHF JH124_33 0 0 0 0 0 CASTAGTVHSNQPQHF JH124_55 0 0 0 0 0 CASRDTRELNTEAFF JH124_59 0 0 0 0 0 CASSLGDLRGFTEAFF JH124_59 0.158730159 0 0 0 0 CATSDLVARDEQFF JH124_18 0 0 0 0 0 CASSYRGTGGGGYTF JH124_08 0 0 0 0 0 CASSLQGGMGNQPQHF JH124_30 0 0 0 0.037523 0 CASSLGLGDEQFF JH124_30 0 0 0 0 0 CASFRQVNYGYTF JH124_30 0 0 0 0 0 CASSFRQGSYEQYF JH124_46 0 0 0 0 0 CASSPDIQYF JH124_51 0 0 0 0 0 CASSETGWGAFF JH124_34 0.238095238 0 0.038153 0.150094 0 CASRRHSNQPQHF JH124_52 0 0 0 0 0 CASSLVGLAGNEQFF JH124_52 0 0 0 0 0 CASSPQDQHVYEQYF JH124_52 0 0 0 0 0 CASTLRVDTEAFF JH124_58 0 0 0 0 0 JH124_19_ JH124_05_ JH124_27_ JH124_63_ JH124_35_ JH124_54_ condition percent percent percent percent percent percent CASSGQGGVSEKLFF JH124_04 0 0 0 0 0 0 CASSLTGGYTGELFF JH124_07 0 0.128991 0 0.190476 0.039308 0.059365 CASSLRSSSETQYF JH124_07 0 0 0 0 0 0 CASSLQAGSSYNEQFF JH124_63 0 0 0 2.190476 0 0 CASSPSGGSLYNEQFF JH124_63 0 0 0 0.761905 0 0 CASSGGISNTEAFF JH124_35 0.031786 0 0 0 2.122642 0 CATSTLPSGGEGYEQYF JH124_54 0 0 0 0 0 0.74206 CASSGTTYGYTF JH124_09 0 0 0 0 0 0 CSASRGLSGYTF JH124_53 0 0 0 0 0 0 LGPGTFTSYEQYF JH124_28 0 0 0 0 0 0 CASSTGTGFGEQYF JH124_57 0 0 0 0 0 0 CASSEGGSDEQYF JH124_25 0 0 0 0 0 0 CASSLDPRSSYNSPLHF JH124_33 0 0 0 0 0 0 CASTAGTVHSNQPQHF JH124_55 0 0 0 0 0 0.089047 CASRDTRELNTEAFF JH124_59 0 0 0 0 0 0 CASSLGDLRGFTEAFF JH124_59 0 0 0 0 0 0 CATSDLVARDEQFF JH124_18 0 0 0 0 0 0 CASSYRGTGGGGYTF JH124_08 0 0 0 0 0 0 CASSLQGGMGNQPQHF JH124_30 0 0 0 0 0 0.029682 CASSLGLGDEQFF JH124_30 0 0 0 0 0.039308 0 CASFRQVNYGYTF JH124_30 0 0 0 0 0 0 CASSFRQGSYEQYF JH124_46 0 0 0 0 0 0 CASSPDIQYF JH124_51 0.031786 0.032248 0.078958 0 0 0 CASSETGWGAFF JH124_34 0 0 0 0 0 0 CASRRHSNQPQHF JH124_52 0 0 0 0 0 0 CASSLVGLAGNEQFF JH124_52 0 0 0 0 0 0 CASSPQDQHVYEQYF JH124_52 0 0 0 0 0 0 CASTLRVDTEAFF JH124_58 0 0 0 0 0 0 JH124_61_ JH124_09_ JH124_45_ JH124_53_ JH124_06_ JH124_28_ condition percent percent percent percent percent percent CASSGQGGVSEKLFF JH124_04 0 0 0 0 0 0 CASSLTGGYTGELFF JH124_07 0.069589 0.112549 0.083857 0.207756 0 0.061881 CASSLRSSSETQYF JH124_07 0 0 0 0 0 0 CASSLQAGSSYNEQFF JH124_63 0 0 0 0 0 0 CASSPSGGSLYNEQFF JH124_63 0 0 0 0 0 0 CASSGGISNTEAFF JH124_35 0 0 0 0 0 0 CATSTLPSGGEGYEQYF JH124_54 0 0 0 0 0 0 CASSGTTYGYTF JH124_09 0 6.359032 0 0 0 0.123762 CSASRGLSGYTF JH124_53 0 0 0 3.774238 0 0 LGPGTFTSYEQYF JH124_28 0 0 0 0 0 0.804455 CASSTGTGFGEQYF JH124_57 0 0 0 0 0 0 CASSEGGSDEQYF JH124_25 0 0 0 0 0 0 CASSLDPRSSYNSPLHF JH124_33 0 0 0 0 0 0 CASTAGTVHSNQPQHF JH124_55 0 0 0 0 0 0 CASSGQGGVSEKLFF JH124_59 0 0 0 0 0 0 CASSLTGGYTGELFF JH124_59 0 0 0 0 0 0 CASSLRSSSETQYF JH124_18 0 0 0.167715 0 0 0 CASSLQAGSSYNEQFF JH124_08 0 0 0 0 0 0 CASSPSGGSLYNEQFF JH124_30 0 0 0 0 0 0 CASSGGISNTEAFF JH124_30 0 0 0 0 0 0 CATSTLPSGGEGYEQYF JH124_30 0 0 0 0 0 0 CASSGTTYGYTF JH124_46 0 0 0 0 0 0 CSASRGLSGYTF JH124_51 0 0 0 0 0 0 LGPGTFTSYEQYF JH124_34 0 0.056275 0.020964 0 0.075131 0 CASSTGTGFGEQYF JH124_52 0 0 0 0 0 0 CASSEGGSDEQYF JH124_52 0 0 0 0 0 0 CASSLDPRSSYNSPLHF JH124_52 0 0 0 0 0 0 CASTAGTVHSNQPQHF JH124_58 0 0 0 0 0 0 JH124_47_ JH124_62_ JH124_02_ JH124_26_ JH124_37_ JH124_57_ JH124_25_ condition percent percent percent percent percent percent percent CASSGQGGVSEKLFF JH124_04 0 0 0 0 0 0 0 CASSLTGGYTGELFF JH124_07 0.105005 0 0.252845 0 0.035063 0.062933 0 CASSLRSSSETQYF JH124_07 0 0 0 0 0 0 0 CASSLQAGSSYNEQFF JH124_63 0 0 0 0 0 0 0 CASSPSGGSLYNEQFF JH124_63 0 0 0 0 0 0 0 CASSGGISNTEAFF JH124_35 0 0 0 0 0 0 0 CATSTLPSGGEGYEQYF JH124_54 0 0 0 0.055835 0 0.062933 0 CASSGTTYGYTF JH124_09 0 0 0 0 0 0 0 CSASRGLSGYTF JH124_53 0 0 0 0 0.175316 0 0 LGPGTFTSYEQYF JH124_28 0 0 0 0 0 0 0 CASSTGTGFGEQYF JH124_57 0 0 0 0 0 0.755192 0 CASSEGGSDEQYF JH124_25 0 0 0 0 0 0 1.070579 CASSLDPRSSYNSPLHF JH124_33 0 0 0 0 0 0 0 CASTAGTVHSNQPQHF JH124_55 0 0 0 0 0 0 0 CASRDTRELNTEAFF JH124_59 0 0 0 0 0 0 0 CASSLGDLRGFTEAFF JH124_59 0 0 0 0 0 0 0 CATSDLVARDEQFF JH124_18 0 0 0 0 0 0 0 CASSYRGTGGGGYTF JH124_08 0 0 0 0 0 0 0.237906 CASSLQGGMGNQPQHF JH124_30 0 0 0 0 0 0.062933 0.039651 CASSLGLGDEQFF JH124_30 0 0 0 0 0 0 0 CASFRQVNYGYTF JH124_30 0 0 0 0 0 0 0 CASSFRQGSYEQYF JH124_46 0 0 0 0 0 0 0 CASSPDIQYF JH124_51 0 0 0 0 0 0 0 CASSETGWGAFF JH124_34 0 0.470588 0 0.055835 0 0.062933 0 CASRRHSNQPQHF JH124_52 0 0 0 0 0 0 0 CASSLVGLAGNEQFF JH124_52 0 0 0 0 0 0 0 CASSPQDQHVYEQYF JH124_52 0 0 0 0 0 0 0 CASTLRVDTEAFF JH124_58 0 0 0 0 0 0 0 JH124_25_ JH124_33_ JH124_38_ JH124_60_ JH124_03_ JH124_24_ condition percent percent percent percent percent percent CASSGQGGVSEKLFF JH124_04 0 0 0 0 0 0 CASSLTGGYTGELFF JH124_07 0 0.128287 0.035112 0.0899526 0 0.103128 CASSLRSSSETQYF JH124_07 0 0 0 0 0 0 CASSLQAGSSYNEQFF JH124_63 0 0 0 0 0 0 CASSPSGGSLYNEQFF JH124_63 0 0 0 0 0 0 CASSGGISNTEAFF JH124_35 0 0 0 0 0 0 CATSTLPSGGEGYEQYF JH124_54 0 0 0 0 0 0 CASSGTTYGYTF JH124_09 0 0 0 0 0 0 CSASRGLSGYTF JH124_53 0 0 0 0 0 0 LGPGTFTSYEQYF JH124_28 0 0 0 0 0 0 CASSTGTGFGEQYF JH124_57 0 0 0 0 0 0 CASSEGGSDEQYF JH124_25 1.070579 0 0 0 0 0.068752 CASSLDPRSSYNSPLHF JH124_33 0 0.705581 0 0 0 0 CASTAGTVHSNQPQHF JH124_55 0 0 0 0 0 0 CASRDTRELNTEAFF JH124_59 0 0 0 0 0 0 CASSLGDLRGFTEAFF JH124_59 0 0 0 0 0 0 CATSDLVARDEQFF JH124_18 0 0 0 0 0 0 CASSYRGTGGGGYTF JH124_08 0.237906 0 0.070225 0 0 0 CASSLQGGMGNQPQHF JH124_30 0.039651 0 0 0 0.122624 0 CASSLGLGDEQFF JH124_30 0 0 0 0 0 0 CASFRQVNYGYTF JH124_30 0 0 0 0 0 0 CASSFRQGSYEQYF JH124_46 0 0 0 0 0 0 CASSPDIQYF JH124_51 0 0 0 0 0 0 CASSETGWGAFF JH124_34 0 0 0 0.268577 0 0 CASRRHSNQPQHF JH124_52 0 0 0 0 0 0 CASSLVGLAGNEQFF JH124_52 0 0 0 0 0 0 CASSPQDQHVYEQYF JH124_52 0 0 0 0 0 0 CASTLRVDTEAFF JH124_58 0 0 0 0 0 0 JH124_48_ JH124_43_ JH124_55_ JH124_59_ JH124_18_ JH124_44_ condition percent percent percent percent percent percent CASSGQGGVSEKLFF JH124_04 0.067935 0.049044 0.027226 0.116212 0 0 CASSLTGGYTGELFF JH124_07 0 0 0 0 0 0 CASSLRSSSETQYF JH124_07 0 0 0 0 0 0 CASSLQAGSSYNEQFF JH124_63 0 0 0 0 0 0 CASSPSGGSLYNEQFF JH124_63 0 0 0 0 0 0 CASSGGISNTEAFF JH124_35 0 0 0 0 0 0 CATSTLPSGGEGYEQYF JH124_54 0 0 0 0 0 0 CASSGTTYGYTF JH124_09 0 0 0 0 0 0 CSASRGLSGYTF JH124_53 0 0 0 0 0 0 LGPGTFTSYEQYF JH124_28 0 0 0 0 0 0 CASSTGTGFGEQYF JH124_57 0 0 0 0 0 0 CASSEGGSDEQYF JH124_25 0 0 0 0 0 0 CASSLDPRSSYNSPLHF JH124_33 0 0 0 0 0 0 CASTAGTVHSNQPQHF JH124_55 0 0.049044 4.546692 0 0 0 CASRDTRELNTEAFF JH124_59 0 0 0 3.166764 0 0 CASSLGDLRGFTEAFF JH124_59 0 0 0 2.93434 0 0 CATSDLVARDEQFF JH124_18 0 0 0 0 1.067236 0 CASSYRGTGGGGYTF JH124_08 0 0 0 0 0 0 CASSLQGGMGNQPQHF JH124_30 0 0 0 0 0 0 CASSLGLGDEQFF JH124_30 0 0 0 0 0 0 CASFRQVNYGYTF JH124_30 0 0 0 0 0 0 CASSFRQGSYEQYF JH124_46 0 0 0 0 0 0 CASSPDIQYF JH124_51 0 0 0 0 0 0 CASSETGWGAFF JH124_34 0.271739 0.049044 0 0.029053 0.106724 0 CASRRHSNQPQHF JH124_52 0 0 0 0 0 0 CASSLVGLAGNEQFF JH124_52 0 0 0 0 0 0 CASSPQDQHVYEQYF JH124_52 0 0 0 0 0 0 CASTLRVDTEAFF JH124_58 0 0 0 0 0 0 JH124_56_ JH124_08_ JH124_29_ JH124_30_ JH124_46_ JH124_20_ condition percent percent percent percent percent percent CASSGQGGVSEKLFF JH124_04 0 0 0 0 0 0 CASSLTGGYTGELFF JH124_07 0.055177 0 0.098814 0 0 0 CASSLRSSSETQYF JH124_07 0 0 0 0 0 0 CASSLQAGSSYNEQFF JH124_63 0 0 0 0 0 0 CASSPSGGSLYNEQFF JH124_63 0 0 0 0 0 0 CASSGGISNTEAFF JH124_35 0 0 0 0 0 0 CATSTLPSGGEGYEQYF JH124_54 0 0 0 0 0 0 CASSGTTYGYTF JH124_09 0 0 0 0 0 0 CSASRGLSGYTF JH124_53 0 0 0 0 0 0 LGPGTFTSYEQYF JH124_28 0 0 0 0 0 0 CASSTGTGFGEQYF JH124_57 0 0 0 0 0 0 CASSEGGSDEQYF JH124_25 0 0 0 0 0 0 CASSLDPRSSYNSPLHF JH124_33 0 0 0 0 0 0 CASTAGTVHSNQPQHF JH124_55 0 0 0 0 0 0 CASRDTRELNTEAFF JH124_59 0 0 0 0 0 0 CASSLGDLRGFTEAFF JH124_59 0 0 0 0 0 0 CATSDLVARDEQFF JH124_18 0 0 0 0 0 0 CASSYRGTGGGGYTF JH124_08 0.018392 6.757391 0 0 0 0 CASSLQGGMGNQPQHF JH124_30 0 0 0 2.060222 0 0 CASSLGLGDEQFF JH124_30 0 0 0 1.003698 0 0 CASFRQVNYGYTF JH124_30 0 0 0 0.633914 0 0 CASSFRQGSYEQYF JH124_46 0 0 0 0 2.004008 0 CASSPDIQYF JH124_51 0 0 0 0 0 0 CASSETGWGAFF JH124_34 0 0.046926 0 0.052826 0 0 CASRRHSNQPQHF JH124_52 0 0 0 0 0 0 CASSLVGLAGNEQFF JH124_52 0 0 0 0 0 0 CASSPQDQHVYEQYF JH124_52 0 0 0 0 0 0 CASTLRVDTEAFF JH124_58 0 0 0 0 0 0 JH124_23_ JH124_51_ JH124_34_ JH124_52_ JH124_58_ condition percent percent percent percent percent CASSGQGGVSEKLFF JH124_04 0 0 0 0 0 CASSLTGGYTGELFF JH124_07 0.177305 0 0.039479 0.02764 0 CASSLRSSSETQYF JH124_07 0 0 0 0 0 CASSLQAGSSYNEQFF JH124_63 0 0 0 0 0 CASSPSGGSLYNEQFF JH124_63 0 0 0 0 0 CASSGGISNTEAFF JH124_35 0 0 0 0 0 CATSTLPSGGEGYEQYF JH124_54 0 0 0 0 0 CASSGTTYGYTF JH124_09 0 0 0 0 0 CSASRGLSGYTF JH124_53 0 0 0 0 0 LGPGTFTSYEQYF JH124_28 0 0 0 0 0 CASSTGTGFGEQYF JH124_57 0 0 0 0 0 CASSEGGSDEQYF JH124_25 0 0 0 0 0 CASSLDPRSSYNSPLHF JH124_33 0 0 0 0 0 CASTAGTVHSNQPQHF JH124_55 0 0 0 0 0 CASRDTRELNTEAFF JH124_59 0 0 0 0 0 CASSLGDLRGFTEAFF JH124_59 0 0 0 0 0 CATSDLVARDEQFF JH124_18 0 0 0 0 0 CASSYRGTGGGGYTF JH124_08 0 0 0 0.055279 0 CASSLQGGMGNQPQHF JH124_30 0 0.222816 0 0.055279 0 CASSLGLGDEQFF JH124_30 0 0 0 0 0 CASFRQVNYGYTF JH124_30 0 0 0 0 0 CASSFRQGSYEQYF JH124_46 0 0 0 0 0 CASSPDIQYF JH124_51 0 0.668449 0 0 0 CASSETGWGAFF JH124_34 0 0.089127 3.039874 0.082919 0.076628 CASRRHSNQPQHF JH124_52 0 0 0 4.339414 0 CASSLVGLAGNEQFF JH124_52 0 0.311943 0 1.768933 0 CASSPQDQHVYEQYF JH124_52 0 0 0 0.801548 0 CASTLRVDTEAFF JH124_58 0 0 0 0 3.218391 JH124_PBMC_2w_ JH124_PBMC_10w_ JH124_PBMC4w_ JH124_PBMC_ JH124_Tumor condition percent percent percent PreRx_percent percent CASSGQGGVSEKLFF JH124_04 0 0 0 0 0 CASSLTGGYTGELFF JH124_07 0.0259175 0.002950636 0.015061148 0.0032341 0.520833 CASSLRSSSETQYF JH124_07 0 0 0 0 0 CASSLQAGSSYNEQFF JH124_63 0 0 0 0 0 CASSPSGGSLYNEQFF JH124_63 0 0 0 0 0 CASSGGISNTEAFF JH124_35 0 0 0 0 0 CATSTLPSGGEGYEQYF JH124_54 0 0 0 0 0 CASSGTTYGYTF JH124_09 0.020734 0.019916792 0.015061148 0.014553452 0 CSASRGLSGYTF JH124_53 0 0 0 0 0 LGPGTFTSYEQYF JH124_28 0 0 0.004518344 0.004851151 0 CASSTGTGFGEQYF JH124_57 0 0 0 0 0 CASSEGGSDEQYF JH124_25 0 0 0 0 0 CASSLDPRSSYNSPLHF JH124_33 0 0 0 0 0 CASTAGTVHSNQPQHF JH124_55 0 0 0 0 0 CASRDTRELNTEAFF JH124_59 0 0 0 0 0 CASSLGDLRGFTEAFF JH124_59 0 0 0 0 0 CATSDLVARDEQFF JH124_18 0 0 0 0 0 CASSYRGTGGGGYTF JH124_08 0 0 0 0 0 CASSLQGGMGNQPQHF JH124_30 0 0.008114249 0.010542804 0.0032341 0 CASSLGLGDEQFF JH124_30 0 0.001475318 0 0.0032341 0 CASFRQVNYGYTF JH124_30 0 0 0 0 0 CASSFRQGSYEQYF JH124_46 0 0 0 0 0 CASSPDIQYF JH124_51 0 0 0 0 0 CASSETGWGAFF JH124_34 0 0 0 0 0 CASRRHSNQPQHF JH124_52 0 0 0 0 0 CASSLVGLAGNEQFF JH124_52 0 0 0 0 0 CASSPQDQHVYEQYF JH124_52 0 0 0 0 0 CASTLRVDTEAFF JH124_58 0 0 0 0 0

Other Embodiments

[0207] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0208] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

[0209] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.