Methods for identifying functional neoantigens and their use in cancer immunotherapy

Abstract

Disclosed are methods for identifying functional neoantigen peptides and using them in cancer immunotherapy. Candidate HLA-restricted peptides are derived e.g., from tumor mutations, loaded onto paramagnetic nano-artificial antigen-presenting cells (nano-aAPCs) that provide peptide- HLA (signal 1) and costimulatory ligands (signal 2), incubated with T cells, magnetically enriched, expanded in culture, and characterized for antigen specificity, phenotype, and function to select therapeutic peptides. The workflow supports peptide cocktails across Class I / IIHLA presentation for heterozygous subjects, and a wide binding¬ affinity range. Resulting T-cell products are enriched for central / effector memory and polyfunctional responses. The methods enable vaccines, polymeric aAPC therapeutics, adoptive cell therapies, and TCR- based biologies (e.g., bispecific T-cell engagers).

Description

[0001] NEX-017PC 107578-5017

[0002] METHODS FOR IDENTIFYING FUNCTIONAL NEO ANTIGENS AND THEIR USE IN CANCER IMMUNOTHERAPY

[0003] BACKGROUND

[0004] Cancer immunotherapy, including vaccination and cell therapy approaches, can target tumor associated antigens (TAAs) or neoantigens that are specific for the subject’s tumor. See, generally, Chakraborty, C. et al. The landscape of neoantigens and its clinical applications: From immunobiology to cancer vaccines. Current Research in Biotechnology Vol. 7 (2024). While known TAAs that are strongly immunogenic can be selected for immunotherapy, T cells specific for these antigens are often rare, since these T cells are largely lost through central tolerance mechanisms. Further, TAAs will also be expressed in some healthy tissues and cells, and therefore targeting TAAs may not be entirely tumor specific. On the other hand, neoantigen epitopes (e.g., based on somatic mutations) are highly specific for the subject’s cancer since these mutations are not present in non- malignant tissue. However, many somatic mutations will not produce functional T cell epitopes or will produce epitopes that are poorly immunogenic. Accordingly, techniques are desired for identifying and predicting functional neoantigen epitopes and peptides, as well as methods for their use in cancer immunotherapy.

[0005] DESCRIPTION OF THE FIGURES

[0006] FIG. 1 summarizes a process, starting with a given mutation, for predicting HLA- restricted neoantigen peptides.

[0007] FIG. 2 summarizes NSCLC antigens presented by seven nano-aAPC cocktails, and shows the level of antigen specificity for cell products expanded from healthy donor PBMCs.

[0008] FIG. 3 shows the T cell phenotype of the expanded cells across the seven nano-aAPC cocktails for the lung cancer antigens.

[0009] FIG. 4 shows the phenotype of antigen-specific T cells for select neoantigens, showing that the cells are largely central and effector memory T cells.

[0010] DBl / 149941487.1 1 NEX-017PC 107578-5017

[0011] FIG. 5 correlates the predicted IC50 for TP53 antigens with the % Specificity of the expanded cells.

[0012] FIG. 6 correlates the predicted IC50 for DEUP1 and SQLE antigens with the % Specificity of the expanded cells.

[0013] FIG. 7 summarizes the melanoma antigens presented by seven nano-aAPC cocktails, and shows the level of antigen specificity for cell products expanded from healthy donor PBMCs.

[0014] FIG. 8 shows the T cell phenotype of the expanded cells across the seven nano-aAPC cocktails for the melanoma neoantigens.

[0015] FIG. 9 shows the phenotype of antigen-specific cells for select neoantigens, showing that the cells are largely central and effector memory T cells.

[0016] FIG. 10 correlates the predicted IC50 for p401-4 and p401-3 antigens with the % Specificity of the expanded cells.

[0017] FIG. 11 shows the % Specificity across several leokopacks for various neoantigens.

[0018] FIG. 12 shows that the process according to this disclosure can be run with nano- aAPC cocktails presenting two HLA subtypes simultaneously to the same T cell population.

[0019] DETAILED DESCRIPTION

[0020] The present disclosure, in the various aspects and embodiments, provides methods for screening neoantigen peptides, to identify those that induce T lymphocyte (“T cell”) expansion. The present disclosure further provides methods for generating functional T cell compositions comprising T cells specific for one or more neoantigen epitopes, and optionally one or more tumor associated antigens (TAAs), and methods for treating cancer by adoptive cell therapy. In certain aspects, the present disclosure provides cancer therapy relevant neoantigen epitopes and peptides that can raise functional T cell responses, which find use in cancer immunotherapy or ex vivo production of adoptive cell therapies. Other aspects and embodiments will be apparent from the following detailed description.

[0021] DBl / 149941487.1 2 NEX-017PC 107578-5017

[0022] In an aspect, the present disclosure provides a method for screening neoantigen peptides. The method comprises providing one or more mutations identified for a subject’s cancer, and obtaining candidate peptide antigens predicted to bind a Class I or Class II human leukocyte antigen (HLA) of the subject. Each candidate peptide antigen comprises at least one mutation identified for the subject’s cancer. The method further comprises loading candidate peptide antigens onto paramagnetic nano-artificial antigen presenting cells (nano- aAPCs), incubating one or more of the loaded nano-aAPCs with source T cells (e.g., from a healthy donor or from the subject), passing the nano-aAPCs and the T cells over a magnetic column, recovering cells associated with the bound fraction, and expanding the cells in the bound fraction in culture. The expanded cells are characterized for their antigen specificity, as well as other features, such as phenotype and functional properties. One or more candidate peptide antigens may be selected for immunotherapy based on these analyses.

[0023] Several candidate peptide antigens can be designed based on sequence analysis and publicly available software tools, and tested according to this disclosure, for example, by loading onto paramagnetic nano-aAPCs and testing individually or in combinations (i.e., enriching and expanding T cells with a nano-aAPC “cocktail”). In embodiments, at least five candidate peptide antigens are prepared and loaded onto aAPCs, or at least ten candidate peptide antigens are prepared and loaded onto aAPCs, or at least 15 candidate peptide antigens are prepared and loaded onto aAPCs, or at least 20 candidate peptide antigens are prepared and loaded onto aAPCs, and these may be tested individually or as nano-aAPC cocktails. In embodiments, at least 100 candidate peptide antigens, or at least 200 candidate peptide antigens, or at least 400 candidate peptide antigens are prepared and loaded onto aAPCs, and these may be tested individually or as nano-aAPC cocktails (i.e., a mixture of aAPCs presenting different peptide antigens). aAPC cocktails may present different peptide antigens for the same HLA ligand, or may present peptides for different HLA ligands (including combinations of class I HLA, such as HLA-A, HLA-B, or HLA-C; or combinations of class II HLA, such as HLA-DR, HLA-DP, and HLA-DQ). In embodiments, the aAPC cocktails present two ligands for a particular HLA, e.g., matched for a heterozygous subject.

[0024] DBl / 149941487.1 3 NEX-017PC 107578-5017

[0025] As used herein, the term “target peptide antigen(s)” or “target antigens” refers to peptide antigens employed ex vivo to enrich and expand a T cell population, for example in connection with the aAPC platform described herein. The aAPCs are employed to activate, enrich, and expand T lymphocytes specific for the target antigens from donor lymphocytes or lymphocytes from an intended recipient. Thus, the term “specific for the target peptide antigen” means that the T cell is antigen experienced with the target antigen. In various embodiments, the target peptide antigens are neoantigen peptides (comprising one or more somatic mutations) or are tumor or cancer associated antigens.

[0026] “Tumor-associated antigens” (TAA) or “cancer-associated antigens” (which are used interchangeably) include tumor or cancer antigens expressed by the tumor or malignant cells from which they are derived, and include shared tumor antigens expressed in many tumors but not in normal adult tissues (oncofetal antigens), and antigens expressed also by the normal tissue from which the tumor arose. Tumor associated antigens can be, for example, embryonic antigens, antigens with abnormal post-translational modifications, differentiation antigens, antigens that are more highly expressed in cancer cells (as compared to non-cancer cells of the same tissue), and oncoviral proteins. TAA generally do not comprise somatic mutations that characterize the tumor or cancer.

[0027] As used herein, the term “neoantigen” or tumor-specific antigen refers to antigens containing one or more mutations characteristic of a cancer or tumor. In embodiments, the one or more mutations are selected from mutations encoding an amino acid substitution, an insertion and / or deletion (e.g., an INDEL), a frameshift, a fusion protein, and a premature stop codon. In embodiments, the one or more mutations are point mutations encoding an amino acid substitution. As shown herein, such peptide antigens can induce strong responses using donor CD8+ cells, or may induce essentially no response. The present disclosure provides a means to differentiate neoantigens and tumor-specific antigens that induce expansion of functional CTLs, from those do not.

[0028] In embodiments, a subject’s tumor is genetically analyzed (e.g., using next generation sequencing), and tumor antigens are predicted from the subject’s unique tumor mutation signature. In some embodiments, one or more cancer driving mutations are identified. Driving mutations are mutations in key genes involved in pathways regulating

[0029] DBl / 149941487.1 4 NEX-017PC 107578-5017 three main cellular processes (1) cell fate, (2) cell survival, and (3) genome maintenance. See, Vogelstein et al., Science 339, 1546-58 (2013). Sequencing data can provide information about both shared as well as personalized targets for cancer immunotherapy. In principle, mutant proteins are foreign to the immune system and are putative tumor-specific antigens. Indeed, sequencing efforts have defined hundred if not thousands of potentially relevant immune targets. Limited studies suggest that T cell responses against these neoepitopes can be found in cancer patients or induced by cancer vaccines. However, the frequency of such responses against a particular cancer and the extent to which such responses are shared between patients are not well known. One of the main reasons for our limited understanding of tumor-specific immune responses is the limited means for validating potential immunologically relevant targets.

[0030] Although central tolerance abrogates T cell responses against self-proteins, oncogenic mutations induce neo-epitopes against which T cell responses can form. Mutation catalogues derived from whole exome sequencing provide a starting point for identifying such neo-epitopes. Using HLA binding prediction algorithms (Srivastava, PLoS One 4, e6094 (2009), it has been predicted that each cancer may have around 7-10 neo-epitopes. A different study estimated hundreds of tumor neo-epitopes. Such algorithms, however, may have low accuracy in predicting T cell responses, and only 10% of predicted HLA-binding epitopes are expected to bind in the context of HLA (Lundegaard C, Immunology 130, 309- 18 (2010)). Thus, predicted epitopes must be validated for the existence of T cell responses against those potential neo-epitopes.

[0031] An exemplary workflow for identifying HLA-restricted neoantigen peptides is shown in FIG. 1. In embodiments, cancer-specific peptide antigens are predicted, and which comprise at least one mutation. For example, Class I HLA peptides can be in the range of 8 to 15 amino acids in length, or 9 to 13 amino acids in length, or 9 to 11 amino acids in length. For Class II, peptide antigens may be in the range of 12 to 20 amino acids in length. In embodiments, corresponding wild-type epitopes can also be evaluated in parallel. In embodiments, the peptide antigen comprising the mutation(s) has a predicted binding to HLA (the subject’s HLA) that is distinct from (e.g., greater than) the wild-type antigen (e.g.,

[0032] DBl / 149941487.1 5 NEX-017PC 107578-5017 in binding affinity). In embodiments, the mutation is predicted to be exposed when the peptide antigen is bound to HLA, based on in silica analysis.

[0033] As described herein, publicly available tools can be used to estimate binding affinity of a candidate peptide antigen for the subject’s HLA (e g., HLA-A, HLA-B, HLA-C, HLA- DR, HLA-DP, HLA-DQ). Conventionally, this is a primary property used to predict the immunogenicity of antigen peptides. However, according to this disclosure, neoantigen peptides of varying affinities show strong properties for expanding T cells, and indeed some antigen peptides that are predicted to strongly bind HLA do not exhibit any T cell expansion properties or exhibit minimal ability to expand antigen-specific T cells. Accordingly, in embodiments, the candidate peptide antigens have predicted affinities for the HLA of from about 1 nM to about 10,000 nM. For example, in embodiments, at least one candidate peptide antigen has a predicted affinity for the HLA of less than about 100 nM, or less than about 50 nM. In addition, at least one candidate peptide antigen may have a predicted affinity for the HLA of greater than about 100 nM, or greater than about 200 nM, or greater than about 300 nM, or greater than about 500 nM, or greater than about 1000 nM. In embodiments, the HLA is Class I (e g., HLA-A, HLA-B, or HLA-C) or is Class II (e g., HLA-DR, HLA-DP, or HLA- DQ).

[0034] Exemplary tools for predicting HLA-restricted neoantigen peptides include NetMHCpan 4.1 (DTU Health Tech), which predicts binding of peptides to any MHC molecule of known sequence using artificial neural networks (ANNs). This tool is trained on a combination of more than 850,000 quantitative Binding Affinity (BA) and Mass- Spectrometry Eluted Ligands (EL) peptides.

[0035] In embodiments, the neoantigen peptides are further modified (e.g., by amino acid substitution with respect to non-mutated positions) for increased properties to stimulate T cell activation and expansion. Such “heteroclitic” peptides can be prepared according to known methods. Adegoke AO and Grant MD, Enhancing human immunodeficiency virusspecific CD8+ T cell responses with heteroclitic peptides. Front. Immunol., 22 July 2015; Cavalluzzo, B., et al. Identification and characterization of heteroclitic peptides in TCR- binding positions with improved HLA-binding efficacy. J Transl Med 19, 89 (2021).

[0036] DBl / 149941487.1 6 NEX-017PC 107578-5017

[0037] Accordingly, heteroclitic peptides can be prepared from the neoantigens or TAA according to this disclosure, and screened for their properties to activate and expand functional T cells.

[0038] According to this aspect of the disclosure, neoantigen epitopes and peptides can be characterized from a variety of cancers, including hematological cancers and solid tumors from various tissues. In embodiments, the cancer is a hematological cancer, such as a leukemia, a lymphoma, or a myeloma. For example, the hematological malignancy may be selected from acute myeloid leukemia, chronic myelogenous leukemia, childhood acute leukemia, non-Hodgkin’s lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome, malignant cutaneous T-cells, mycosis fungoids, non- MF cutaneous T-cell lymphoma, lymphomatoid papulosis, and T-cell rich cutaneous lymphoid hyperplasia. In embodiments, the cancer is a solid tumor, such as a solid tumor selected from melanoma, colon cancer, duodenal cancer, prostate cancer, breast cancer, ovarian cancer, ductal cancer, hepatic cancer, pancreatic cancer, renal cancer, endometrial cancer, testicular cancer, stomach cancer, dysplastic oral mucosa, polyposis, head and neck cancer, invasive oral cancer, non-small cell lung carcinoma, small-cell lung cancer, mesothelioma, transitional and squamous cell urinary carcinoma, brain cancer, neuroblastoma, and glioma.

[0039] In embodiments, the one or more mutations are associated with an oncogene or a gene encoding a tumor suppressor. In embodiments, the one or more mutations are selected from mutations encoding an amino acid substitution, an insertion and / or deletion (e.g., an INDEL), a frameshift, a fusion protein, and a premature stop codon. In embodiments, the one or more mutations are point mutations encoding an amino acid substitution. As shown herein, such peptide antigens can induce strong responses using donor cells, or may induce essentially no response. The present disclosure provides a means to distinguish such peptide antigens.

[0040] In embodiments, the neoantigen peptides are HLA-restricted for the subject’s HLA, that is, the neoantigen peptides are designed to bind an HLA of the subject, which can be a Class I HLA selected from HLA-A, HLA-B, HLA-C, and HLA-E. In embodiments, at least one HLA is HLA-A, optionally with one or more of HLA-B and HLA-C. In some embodiments, the subject is homozygous for the selected HLA (e g., HLA-A), and the nano-

[0041] DBl / 149941487.1 7 NEX-017PC 107578-5017 aAPCs present a matching HLA ligand. In embodiments, the subject is heterozygous for the selected HLA (e.g., HLA-A), and the nano-aAPC present one or both of the subject’s HLA (e.g., as a cocktail of two nano-aAPC populations each presenting one of the HLA ligands). In these embodiments, candidate peptide antigens for each HLA of a heterozygous subject can be characterized simultaneously. In embodiments, at least one HLA is HLA-A2 (e.g., HLA-A*02:01), HLA-A3 (e.g., HLA-A*03:01), HLA-A11 (e.g., HLA-A*ll:01), or HLA- A24 (e.g., HLA-A*24:02). The prevalence of HLA variants in global populations is well known.

[0042] In embodiments, the neoantigen peptides are HLA-restricted for the subject’s HLA, and the neoantigen peptides are designed to bind a Class II HLA selected from HLA-DR, HLA-DP, and HLA-DQ. In embodiments, at least one HLA is HLA-DR, optionally with one or more of HLA-DP and HLA-DQ. In some embodiments, the subject is homozygous for the selected HLA (e.g., HLA-DR), and the nano-aAPCs present a matching HLA ligand. In embodiments, the subject is heterozygous for the selected HLA (e.g., HLA-DR), and the nano-aAPC present one or both of the subject’s HLA (e.g., as a cocktail of two nano-aAPC populations each presenting one of the HLA ligands). In these embodiments, candidate peptide antigens for each HLA of a heterozygous subject can be characterized simultaneously. The prevalence of HLA variants in global populations is well known.

[0043] The candidate peptides are loaded onto paramagnetic nano-sized artificial Antigen Presenting Cells (nano-aAPCs). Paramagnetic materials have a small, positive susceptibility to magnetic fields. These materials are attracted by a magnetic field and the material does not retain the magnetic properties when the external field is removed. Exemplary paramagnetic materials include, without limitation, magnesium, molybdenum, lithium, tantalum, and iron oxide. Paramagnetic beads suitable for magnetic enrichment are commercially available (DYNABEADS™, MACS MICROBEADS™, Miltenyi Biotec). In some embodiments, the paramagnetic aAPC particle is an iron dextran bead (e.g., dextran- coated iron-oxide bead). Exemplary paramagnetic aAPCs are described in US Patent No. 10,435,668 and US Patent No. 10,987,412, which are hereby incorporated by reference in their entireties. In embodiments, the paramagnetic nano-aAPCs are constructed from

[0044] DBl / 149941487.1 8 NEX-017PC 107578-5017 paramagnetic beads that have a mean diameter in the range of 50 to 200 nm (e.g., from about 75 to 150 nm).

[0045] The nano-aAPCs present an antigen presenting complex (e.g., an HLA ligand). Antigen presenting complexes comprise an antigen binding cleft, and can be MHC class I or Class II, which can be linked or tethered to provide dimeric or multimeric MHC. In some embodiments, the MHC are monomeric, but their close association on the nano-particle is sufficient for avidity and activation. In some embodiments, the MHC are dimeric. Dimeric MHC class I ligands can be constructed by fusion to immunoglobulin heavy chain sequences (e.g., HLA-Ig), which are then associated through one or more disulfide bonds (with or without associated light chains). MHC multimers can be created by direct tethering through peptide or chemical linkers, or can be multimeric via association with streptavidin through biotin moieties. HLA-Ig fusion constructs for MHC Class I and II are disclosed in US 6,458,345 and US 6,015,884, which are hereby incorporated by reference in their entireties.

[0046] In various embodiments, the HLA is an HLA-Ig dimer (e.g., HLA Class I), and the immunoglobulin heavy chain sequence is not full length, but comprises an immunoglobulin (Ig) hinge region, and one or more of CHI, CH2, and / or CH3 domains. The Ig sequence may or may not comprise a variable region, but where variable region sequences are present, the variable region may be full or partial. The complex may further comprise immunoglobulin light chains. MHC class I ligands (e.g., HLA-Ig dimers) lacking variable chain sequences (and lacking any light chain) may be employed with site-directed conjugation to particles, as described in US Patent No. 10,632,193, which is hereby incorporated by reference in its entirety.

[0047] Accordingly, in embodiments, MHC class I molecular complexes comprise at least two fusion proteins. A first fusion protein comprises a first MHC class I a chain and a first immunoglobulin heavy chain (or portion thereof comprising the hinge region), and a second fusion protein comprises a second MHC class I a chain and a second immunoglobulin heavy chain (or portion thereof comprising the hinge region). The first and second immunoglobulin heavy chains associate to form the MHC class I molecular complex, which comprises two MHC class I peptide-binding clefts. The immunoglobulin heavy chain can be the heavy

[0048] DBl / 149941487.1 9 NEX-017PC 107578-5017 chain of an IgM, IgD, IgGl, IgG3, IgG2p, IgG2a, IgG4, IgE, or IgA. In some embodiments, an IgG heavy chain is used to form MHC class I molecular complexes. If multivalent MHC class I molecular complexes are desired, IgM or IgA heavy chains can be used to provide pentavalent or tetravalent molecules, respectively. The term MHC as used herein, can be replaced by HLA in each instance.

[0049] In some embodiments, the HLA ligand is modified with cysteines that form a disulfide bond across the alpha helices that make up the peptide binding groove, to increase complex stability with bound peptide. Such Cysteines in some embodiments are substituted at positions 84 and 139 of the extracellular domain. These modifications bridge the F-pocket, where the C-terminus of the peptide binds.

[0050] In various embodiments, the recombinant HLA class I ligand is associated with a P2 microglobulin protein. The amino acid sequence of P2 microglobulin is provided herein as SEQ ID NO: 8. In various embodiments, derivatives of P2 microglobulin may be employed, for example, having from 1 to 10 or from 1 to 5 amino acid modifications independently selected from substitutions, deletions, and insertions.

[0051] Exemplary MHC class II molecular complexes are described in U.S. Pat. Nos. 6,458,354, 6,015,884, 6,140,113, and 6,448,071, which are hereby incorporated by reference in their entireties. MHC class II molecular complexes can comprise at least four fusion proteins. Two first fusion proteins comprise (i) an immunoglobulin heavy chain (or portion thereof comprising the hinge region) and (ii) an extracellular domain of an MHC class lip chain. Two second fusion proteins comprise (i) an immunoglobulin K or light chain (or portion thereof) and (ii) an extracellular domain of an MHC class Ila chain. The two first and the two second fusion proteins associate to form the MHC class II molecular complex. The extracellular domain of the MHC class 110 chain of each first fusion protein and the extracellular domain of the MHC class Ila chain of each second fusion protein form an MHC class II peptide binding cleft. The immunoglobulin heavy chain can be the heavy chain of an IgM, IgD, IgG3, IgGl, IgG2p, IgG2a, IgG4, IgE, or IgA. In some embodiments, an IgGl heavy chain is used to form divalent molecular complexes comprising two antigen binding clefts.

[0052] DBl / 149941487.1 10 NEX-017PC 107578-5017

[0053] The nano-aAPCs may also contain a “Signal 2”, such as an agonistic anti-CD28 ligand. Signal 2 is generally a T cell costimulatory molecule. T cell costimulatory molecules contribute to the activation of antigen-specific T cells. Such molecules include, but are not limited to, molecules that specifically bind to CD28 (including antibodies), CD80 (B7-1), CD86 (B7-2), B7-H3, 4-1BB, 4-1BBL, CD27, CD30, CD134 (OX-40L), B7h (B7RP-1), CD40, LIGHT, antibodies that specifically bind to HVEM, antibodies that specifically bind to CD40L, and antibodies that specifically bind to 0X40. In some embodiments, the costimulatory molecule (signal 2) is an agonistic antibody (e g., a monoclonal antibody) or portion thereof, such as F(ab’)2, Fab, scFv, or single chain antibody, or other antigen binding fragment. In some embodiments, the antibody is a humanized monoclonal antibody or portion thereof having antigen-binding activity, or is a fully human antibody or portion thereof having antigen-binding activity. Combinations of co-stimulatory ligands that may be employed (on the same or separate nanoparticles) include anti-CD28 / anti-CD27 and anti- CD28 / anti-41BB. The ratios of these co-stimulatory ligands can be varied to effect expansion.

[0054] Exemplary signal 1 (HLA-Ig) and signal 2 (anti-CD28) ligands are described in US Patent No. 11,712,477, which is hereby incorporated by reference in its entirety, and which describes ligands having a free sulfhydryl (e.g., unpaired cysteine), such that the constant region may be coupled to nanoparticle supports having the appropriate chemical functionality. In such embodiments, signal 1 is provided by peptide-HLA-Ig complexes, and signal 2 is provided by anti-CD28. An exemplary anti-CD28 monoclonal antibody is 9.3 mAb (Tan et al., J. Exp. Med. 1993 177:165), which may be humanized in certain embodiments and / or conjugated to the bead as a fully intact antibody or an antigen-binding fragment thereof. In embodiments, the signal 1 and signal 2 can be conjugated to the same or different particles, or signal 2 can be added in soluble form. See US 2023 / 0332131, which is hereby incorporated by reference in its entirety.

[0055] Precursor T cells to functionally characterize the candidate peptide antigens can be obtained from a subject or from a suitable HLA-matched donor. Precursor T cells can be obtained from a number of sources that comprise WBCs, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, spleen tissue, buffy coat

[0056] DBl / 149941487.1 11 NEX-017PC 107578-5017 fraction, and tumors. In some embodiments, precursor T cells are obtained from a unit of blood collected from a subject or donor using any number of techniques known to one or skill in the art. For example, precursor T cells from the circulating blood of an individual can be obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells and precursor T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. Leukapheresis is a laboratory procedure in which white blood cells are separated from a sample of blood. If desired, precursor T cells can be isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient.

[0057] In certain embodiments, leukocytes are collected by leukapheresis, and may be subsequently enriched for CD8+ (for generation of CTLs) or CD4+ T cells (for generation of T Helper cells), for example, by depleting the sample of CD4+ cells and / or positively enriching for CD8+ cells, or by depleting the sample of CD8+ cells and / or positively enriching for CD4+ cells. In some embodiments, other cell types are depleted, such as NK cells.

[0058] In embodiments, nano-aAPCs are screened as a cocktail of up to ten peptides per cocktail, or up to seven peptides per cocktail, or up to six peptides per cocktail. In embodiments, the nano-aAPCs (e.g., or cocktail thereof) are incubated with at least about 25 million T cells, or at least 50 million T cells, or at least 100 T cells, or at least about 150 million T cells. In embodiments, the nano-aAPCs (or cocktail thereof) are incubated with about 500 million T cells or less, or about 250 million T cells or less, or about 200 million T cells or less. In embodiments, the T cells are CD8+ (e.g., CD4+ depleted), and used for screening HLA Class I peptides as described.

[0059] The nano-aAPC or cocktail thereof are incubated with the T cells, and then passed over a magnetic column for simultaneous T cell activation and enrichment for antigenspecific T cells. See, US Patent Nos. 10,435,668 and 10,987,412, which are hereby incorporated by reference in their entireties. Magnetic activation may take place for from 2 minutes to 5 hours, or from 5 minutes to 2 hours, followed by expansion in culture. In some

[0060] DBl / 149941487.1 12 NEX-017PC 107578-5017 embodiments, magnetic activation occurs for at least 2 minutes, but less than 30 minutes or less than 15 minutes (e.g., about 2 to about 10 minutes, such as about 5 minutes).

[0061] In embodiments, the bound (i.e., magnetic) fraction is expanded in culture for at least five days, or at least one week, or at least ten days, or at least two weeks. For example, the bound fraction may be expanded in culture for about two weeks (e.g., 10-17 days, such as about 14 days). In embodiments, aAPCs are supplemented in the culture. For example, aAPCs can be supplemented at least once, after about day 3 or day 5 of the culture. In embodiments, the aAPCs are supplemented on about day 7, in the case where the culture duration is at least 10 days (e.g., about 14 days).

[0062] The aAPC nanoparticles supplemented in the culture can be made of any material, and may comprise, for example, metals such as iron, nickel, cobalt, or alloy of rare earth metal. Paramagnetic materials also include magnesium, molybdenum, lithium, tantalum, and iron oxide. Paramagnetic beads suitable for enrichment of materials (including cells) are commercially available, and include iron dextran beads, such as dextran-coated iron oxide beads. In aspects of the invention where magnetic properties are not required, nanoparticles can also be made of nonmetal or organic (e.g., polymeric) materials such as cellulose, ceramics, glass, nylon, polystyrene, rubber, plastic, or latex. In exemplary material for preparation of nanoparticles is poly(lactic-co-glycolic acid) (PLGA) or PLA and copolymers thereof, which may be employed in connection with these embodiments. Other materials including polymers and co-polymers that may be employed include those described in US Patent No. 10,632,193, which is hereby incorporated by reference in its entirety. In embodiments, the culture is supplemented with soluble anti-CD28.

[0063] In embodiments, the cells are expanded in culture in the presence of growth factors that affect proliferation and / or differentiation of T cells. Examples of T cell growth factors include cytokines (e.g., interleukins, interferons) and superantigens. If desired, cytokines can be present in molecular complexes comprising fusion proteins or provided in soluble form or immobilized. In embodiments, cytokines are selected from MIP-ip, IL-ip, IL-2, IL -4, IL- 6, IL-7, IL-10, IL-12, IL-15, IL-21, IFN-y, and CXCL10. In some embodiments, the growth

[0064] DBl / 149941487.1 13 NEX-017PC 107578-5017 factors include 3, 4, 5, or 6 from MIP-10, IL-10, IL-2, IL-4, IL-6, IL-7, IL-10, IL-15, IL-21, and INF-y.

[0065] In embodiments, the supplemented growth factors comprise or consist essentially of IL-2. IL-4, IL-6, INF-y, and IL- IB, to preferentially expand antigen-specific T cells and / or to induce the desired memory phenotype. See US Patent No. 11,007,222, which is hereby incorporated by reference in its entirety.

[0066] In embodiments, IL-2 is present at the start of culture at 10 to 200 International Units (IU) per ml, such as from about 20 to about 100 lU / ml, or about 20 to about 60 lU / ml. In some embodiments, IL-2 is present at the start of culture at about 2 to about 25 ng / ml, or at about 2 to about 15 ng / ml, such as from about 5 to about 15 ng / ml.

[0067] In embodiments, IL-4 is present at the start of culture at 0.2 to 25 International Units (IU) per ml, such as from about 0.5 to about 10 lU / ml, or from about 0.5 to about 5 lU / ml. In some embodiments, IL-4 is present at the start of culture at about 0.2 to about 2 ng / ml, such as from about 0.2 to about 1 ng / ml (e.g., about 0.5 ng / ml).

[0068] In embodiments, IL-6 may be present at the start of culture at 10 to 200 International Units (IU) per ml, such as from about 25 to about 100 TU / ml, such as from 25 to 75 lU / ml. In some embodiments, IL-6 is present at the start of culture at about 0.2 to about 10 ng / ml, such as from about 0.2 to about 5 ng / ml (e.g., about 0.2 to 1 ng / ml, or about 0.5 to 2 ng / ml).

[0069] In embodiments, Interferon gamma (INF-y) may be present at the start of culture at from 10 to 200 International Units (IU) per ml, such as from about 20 to about 100 lU / ml, such as from 20 to 60 lU / ml. In some embodiments, INF-y is present at the start of culture at about 0.5 to about 20 ng / ml, such as from about 0.5 to about 10 ng / ml, or from about 0.5 to about 5 ng / ml, or from about 1 to about 10 ng / ml (e.g., from 1 to 5 ng / ml).

[0070] IL- 10 may be present at the start of culture at 5 to 100 International Units (IU) per ml, such as from about 10 to about 50 lU / ml, such as from about 10 to about 30 lU / ml. In some embodiments, IL-10 is present at the start of culture at about 0.1 to 5 ng / ml, or at about 0.2 to about 5 ng / ml, such as from about 0.2 to about 2 ng / ml, or from about 0.2 to about 1 ng / ml.

[0071] DBl / 149941487.1 14 NEX-017PC 107578-5017

[0072] In embodiments, the growth factors are added on day 1 and again between days 5 and 9 for a culture of more than one week. For example, the growth factors may be supplemented on day 7 for a culture of at least 10 days (e.g., about 14 days).

[0073] The expanded cell product is characterized for antigen specificity against the target peptide antigens, using, for example, staining with target peptide-HLA reagents (e.g., HLA- Ig dimers, or MHC multimers, e.g., tetramers). In embodiments, candidate peptide antigens are selected that have a % Specificity (% T cells specific for the given target peptide antigen) in the culture of at least about 0.5%, or at least about 1%, or at least about 2%, or at least about 4%, or at least about 5%, or at least about 10%. These numbers assume that background specificity is subtracted, that is, by staining with an irrelevant HLA-peptide reagent to determine background.

[0074] The expanded T cells can further be characterized for phenotype, where high percentage of central and effector memory phenotypes, as well as stem cell memory phenotype, are preferred. Further, the antigen-specific component (e.g., for each target peptide antigen) can be characterized for phenotype. In embodiments, the following phenotypes are quantified: naive, central memory, effector memory, and terminally differentiated memory cells, and optionally T memory stem cell.

[0075] A naive T cell has differentiated in bone marrow, and has successfully undergone the positive and negative processes of central selection in the thymus. A naive T cell is considered mature and, unlike activated or memory T cells, has not encountered its cognate antigen. Naive T cells can be characterized by the surface expression of L-selectin (CD62L) and the absence of activation surface markers. In the naive state, T cells are generally quiescent and non-dividing. In accordance with this disclosure, naive T cells are defined as CD62L+ and CD45RA+.

[0076] Memory T cells include T memory stem cells (TSCM), central memory and effector memory T cells. Memory T cells have previously responded to their cognate antigen. At a second encounter with the cognate antigen, memory T cells can reproduce to mount a faster and stronger immune response. Memory T cells include at least T memory stem cells, effector memory T cells, and central memory T cells. Memory T cell subtypes are long-lived

[0077] DBl / 149941487.1 15 NEX-017PC 107578-5017 and can quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen.

[0078] T memory stem cells (TSCM) are defined herein as CD45RA+ and as having at least the following surface markers: CD62L+, CD45RA+, and CD95+. In some embodiments, the T memory stem cells disclosed herein may have one or more of the following additional surface markers: CD28+, CD27+, CXCR3+ CDl la+, IL-2R0+, CD58+, and CD57-. In some embodiments, the T memory stem cells comprise cells that are CD62L+, CD45RA+, CD28+, CD27+, and CD95+. In some embodiments, the T memory stem cells comprise cells that are CD62L+, CD45RA+, CD95+ and CXCR3+. In some embodiments, the T memory stem cells comprise cells that are CD62L+, CD45RA+, CD95+ and CDl la+. In some embodiments, the T memory stem cells comprise cells that are CD62L+, CD45RA+, CD95+ and IL-2RP+. In some embodiments, the T memory stem cells comprise cells that are CD62L+, CD45RA+, CD95+ and CD58+. In some embodiments, the T memory stem cells comprise cells that are CD62L+, CD45RA+, CD95+ and CD57-. This memory subpopulation has the stem cell-like capacity for self-renewal, as well as the multipotent capacity to reconstitute the memory and effector T cell subpopulations. TSCM cells typically represent a small fraction of circulating T lymphocytes (e.g., >5%), and have the ability to proliferate rapidly and release inflammatory cytokines in response to antigen re-exposure. Accordingly, TSC cells are a subset of the memory T cell subpopulation.

[0079] In accordance with this disclosure, central memory T cells (TCM cells) are defined herein as CD62L+ and CD45RA-. This memory subpopulation is commonly found in the lymph nodes and in the peripheral circulation. Effector memory T cells (TEM cells) are defined herein as CD62L- and CD45RA-. These memory T cells lack lymph node-homing receptors and are thus found in the peripheral circulation and tissues.

[0080] TEMRA stands for terminally differentiated effector memory cells re-expressing CD45RA (TEMRA). These cells do not have the capacity to divide, and are CD62L- and CD45RA+.

[0081] In embodiments, a high percentage of central and effector memory T cells are preferred. In embodiments, the T cells in the expanded culture (and / or the T cells specific

[0082] DBl / 149941487.1 16 NEX-017PC 107578-5017 for a given target peptide antigen) are at least 60% central and effector memory T cells, or at least 70% central and effector memory T cells at the end of the culture. The desired phenotype comprises less than about 10% or less than about 5% terminally differentiated memory T cells (e.g., TEMRA cells). Further, the desired T cells contain no more than about 10% naive cells, or in some embodiments, no more than about 5% naive cells, or no more than about 3% naive cells.

[0083] The expanded cells will generally be at least 90% T cells, or at least 95% T cells, or at least 98%, or at least 99% T cells. For purposes of this disclosure, T cells are characterized by CD3+ cells. In embodiments, the T cells are generally CD8+ or CD4-. As used herein, the terms “CD8+” and “CD4-” are interchangeable unless stated otherwise.

[0084] In embodiments, the expanded cells are characterized for their polyfunctional nature upon activation. The presence of polyfunctional CD8+ T cells correlates with response to cancer vaccine therapy with peptide neoantigens. Ott PA, et al., An immunogenic personal neoantigen vaccine for patients with melanoma. Nature 547(7662):217-221 (2017). For example, neoantigen peptides are desired where, upon activation, the antigen-specific T cells are positive for two or more of: intracellular staining for IL-2, which is a marker for proliferation and memory; IFN-y production, which activates other T cells, and induces memory and upregulation of MHC); production of TNF-a, a pro-inflammatory marker; and CD107A, which is a marker for granzyme release and cytotoxic activity. In various embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of the antigen-specific T cells display at least three of these markers. In various embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of the antigen-specific T cells display all four of these markers. In some embodiments, polyfunctionality is assessed or quantified using target killing assays, which assess the ability of CD8+ cytotoxic T cells to lyse target cells presenting the peptide antigen in complex with MHC.

[0085] Based on these analyses, candidate neoantigen peptides are selected for the subject’s immunotherapy. For example, the neoantigen peptides can be selected for use as a cancer vaccine (e g., as a component of a peptide or RNA or dendritic cell cancer vaccine), as known

[0086] DBl / 149941487.1 17 NEX-017PC 107578-5017 in the art. In embodiments, the neoantigen peptides can be loaded onto polymeric aAPCs for administration to activate and expand antigen-specific T cells in vivo. See, US Patent No. 10,632,193, which is hereby incorporated by reference in its entirety. In some embodiments, one or several neoantigen peptides are selected, and combined with one or more HLA- restricted peptides derived from TAAs. In embodiments, the one or more selected neoantigen peptides (optionally with one or more HLA-restricted peptides derived from TAAs) are used to prepare an adoptive cell therapy product for the subject.

[0087] In an aspect, the disclosure provides methods for personalized cancer immunotherapy. The methods are accomplished using the aAPCs to identify HLA-restricted neoantigen peptides (as described), followed by administration of the appropriate peptide- loaded aAPC to the patient, or followed by enrichment and expansion of the antigen specific T cells ex vivo for adoptive cell therapy.

[0088] In still other embodiments, cognate TCR specific for the desired peptide HLA is cloned, and expressed in a cell (e.g., T cell) for administration to a subject, or used to prepare a biologic therapy. For example, in embodiments, the TCR (identified according to this disclosure) is conjugated to or complexed with a T cell engaging molecule to thereby prepare a bispecific T cell engager (BiTE). T cell-targeting ligands and BiTE constructs are described in WO 2024 / 148216, which is hereby incorporated by reference in its entirety. BiTE molecules allow for a targeted immunotherapy and engagement with a patient’s own T cells to treat hematologic malignancies and solid tumors. In embodiments, the BiTE can engage with T cells through binding to a T-cell receptor or component thereof, such as CD3, and engage a target cell through the cloned TCR (identified according to this disclosure). In embodiments, the BiTE activates T cells through binding to CD3. That is, the domain that binds CD3 is an agonist or partial agonist for CD3. Concurrent binding to both the T cell target (e g., CD3) and the tumor antigen (e.g., MHC presenting the target neoantigen peptide) results in lysing of the tumor cell.

[0089] According to embodiments, the BiTE comprises at least a first domain that binds a T cell surface antigen or receptor, and a second domain that comprises at least the extracellular domains (or portions thereof) of the alpha and beta chains of the cloned TCR

[0090] DBl / 149941487.1 18 NEX-017PC 107578-5017

[0091] (e.g., recognizing the MHC-neoantigen peptide). In embodiments, the alpha and beta chains (or portions thereof) are fused to the N-terminus of heavy and light chains of an IgG antibody (or portions thereof) to assemble a molecular complex, as described in US 6,458,354, which is hereby incorporated by reference in its entirety. In embodiments, the heavy and light chain variable regions are present, or alternatively are partially or fully deleted. In embodiments, the BiTE can be constructed such that one set of heavy and light chains are fused to the TCR sequences, and the second heavy and light chain pair constitute a full or partial agonist for CD3. In still other embodiments, the BiTE comprises a dimerized TCR, and one or more single chain antibodies (scFv or VHH) that is a full or partial agonist for CD3 is fused to an Fc domain (e.g., at the C-terminus). In embodiments, the TCR recognizes a neoantigen disclosed herein, including but not limited to a TP53 neoantigen peptide.

[0092] In embodiments, the cloned TCR is used to create a drug conjugate. In embodiments, the cloned TCR alpha and beta chains (extracellular domains or portions thereof) can optionally be fused to antibody (e.g., IgG) heavy and light chains as described above, with conjugation of chemotherapeutic agent (or other agent that induces apoptosis of cancer cells), according to known techniques in the field of antibody drug conjugates.

[0093] In an aspect, the present disclosure provides a method for preparing a T cell composition suitable for adoptive cell therapy for the subject (e.g., having a genetically analyzed cancer as described). The method comprises selecting at least one neoantigen peptide according to this disclosure, and loading the neoantigen peptide onto paramagnetic nano-artificial antigen presenting cells (nano-aAPCs). In embodiments, an aAPC cocktail is created, where aAPC populations are separately loaded with a neoantigen peptide and one or more TAA peptides. The aAPC or cocktail thereof are incubated with T cells from the subject or a healthy donor, passed over a magnetic column (as described), and cells in the bound fraction are recovered and expanded in culture as already described. The resulting cell product can be infused into the subject, once or a plurality of times. See US Patent No. 11,007,222, which is hereby incorporated by reference in its entirety.

[0094] In embodiments, the nano-aAPC are employed as a cocktail to prepare the adoptive cell product, and the cocktail presents at least two peptide antigens, or at least three peptide

[0095] DBl / 149941487.1 19 NEX-017PC 107578-5017 antigens, or at least five peptide antigens, or at least seven peptide antigens. In embodiments, the peptide antigens have predicted affinities for the HLA presented by the aAPC of from about 1 nM to about 10,000 nM. In embodiments, at least one peptide antigen has a predicted affinity for the HLA presented by the aAPC of less than about 100 nM, or less than about 50 nM. In addition, in embodiments, at least one peptide antigen has a predicted affinity for the HLA presented by the aAPC of greater than about 100 nM, or greater than about 200 nM, or greater than about 300 nM, or greater than about 500 nM, or greater than about 1000 nM.

[0096] In embodiments, the T cells in the resulting composition are at least 60% central and effector memory T cells, or at least 70% central and effector memory T cells at the end of the culture. In embodiments, the expanded cell composition has a specificity for each peptide antigen of at least about 0.5%, or at least about 1%, or at least about 2%, or at least about 4%, or at least about 5%.

[0097] In embodiments, the cell composition comprises at least about 106T cells specific for the target peptide antigens, or at least about 107T cells specific for the target peptide antigens, or at least about 108, at least about 109, or at least about 1010T cells specific for the target peptide antigens. In embodiments, the T cells are CD8+ CTLs to provide robust destruction of target cells. In some embodiments, the cell composition contains from 1 x 107to 1 x 109T cells specific for the target antigens, or in some embodiments from 5 x 107to 5 x 108T cells specific for the target antigens. For example, the composition can comprise from about 5 x 105to about 5 x 106cells per ml, in a volume of from 50 to 200 ml. In certain embodiments, the volume of the composition is <100 ml (e.g., from 50 to 100 ml). The cells of the composition in various embodiments are at least 70% viable or at least about 80% or about 90% viable, and provided in a sterile medium, which may be a cryoprotectant medium (e.g., 10% DMSO). The medium can be an aqueous medium suitable for intravenous infusion, e.g., including water and electrolytes. An exemplary medium is PLASMALYTE.

[0098] A variety of tumor-associated antigens are known in the art, and may be used according to this disclosure in combination with one or more neoantigen peptides. For example, oncofetal and embryonic antigens include carcinoembryonic antigen and alphafetoprotein (usually only highly expressed in developing embryos but frequently highly

[0099] DBl / 149941487.1 20 NEX-017PC 107578-5017 expressed by tumors of the liver and colon, respectively), MAGE-1 and MAGE-3 (expressed in melanoma, breast cancer, and glioma), placental alkaline phosphatase sialyl-Lewis X (expressed in adenocarcinoma), CA-125 and CA-19 (expressed in gastrointestinal, hepatic, and gynecological tumors), TAG-72 (expressed in colorectal tumors), epithelial glycoprotein 2 (expressed in many carcinomas), pancreatic oncofetal antigen, 5T4 (expressed in gastriccarcinoma), alphafetoprotein receptor (expressed in multiple tumor types, particularly mammary tumors), and M2A (expressed in germ cell neoplasia).

[0100] Tumor-associated differentiation antigens include tyrosinase (expressed in melanoma) and particular surface immunoglobulins (expressed in lymphomas).

[0101] Mutated oncogene or tumor-suppressor gene products include Ras and p53, both of which are expressed in many tumor types, Her-2 / neu (expressed in breast and gynecological cancers), EGF-R, estrogen receptor, progesterone receptor, retinoblastoma gene product, myc (associated with lung cancer), and MAGE-1 and MAGE-3 (associated with melanoma, lung, and other cancers). Other tumor-specific antigens that can be employed include fusion protein antigens including BCR-ABL, which is expressed in chromic myeloid leukemia. Oncoviral proteins can also be employed, and these include HPV type 16, E6, and E7, which are found in cervical carcinoma.

[0102] Tissue-specific antigens that can be employed include melanotransferrin and MUC1 (expressed in pancreatic and breast cancers); CD 10 (previously known as common acute lymphoblastic leukemia antigen, or CALLA) or surface immunoglobulin (expressed in B cell leukemias and lymphomas); the a chain of the IL-2 receptor, T cell receptor, CD45R, CD4+ / CD8+ (expressed in T cell leukemias and lymphomas); prostate specific antigen and prostatic acid-phosphatase (expressed in prostate carcinoma); GP100, MelanA / Mart-1, tyrosinase, gp75 / brown, BAGE, and S-100 (expressed in melanoma); cytokeratins (expressed in various carcinomas); and CD 19, CD20, and CD37 (expressed in lymphoma).

[0103] Tumor-associated antigens also include altered glycolipid and glycoprotein antigens, such as neuraminic acid-containing glycosphingolipids (e g., GM2 and GD2, expressed in melanomas and some brain tumors); blood group antigens, particularly T and sialylated Tn antigens, which can be aberrantly expressed in carcinomas; and mucins, such as CA-125 and

[0104] DBl / 149941487.1 21 NEX-017PC 107578-5017

[0105] CA-19-9 (expressed on ovarian carcinomas) or the underglycosyl ated MUC-1 (expressed on breast and pancreatic carcinomas).

[0106] For example, in some embodiments, one or more target antigens are associated with bladder cancer, such as one or more of NY-ESO-1, MAGE-A10, and MUC-1 antigens. In some embodiments, one or more target antigens are associated with brain cancer, and may include one or more of NY-ESO-1, Survivin, and CMV antigens. In some embodiments, one or more target antigens are associated with breast cancer, and may include one or more of MUC-1, Survivin, WT-1, HER-2, and CEA antigens. In some embodiments, one or more target antigens are associated with cervical cancer, and may include HPV antigen. In some embodiments, one or more target antigens are associated with colorectal cancer, and may include one or more of NY-ESO-1, Survivin, WT-1, MUC-1, and CEA antigens. In some embodiments, one or more target antigens are associated with esophageal cancer, and may include NY-ESO-1 antigen. In some embodiments, one or more target antigens may be associated with head and neck cancer, and may include HPV antigen. In some embodiments, the target antigen is associated with kidney or liver cancer, and may include NY-ESO-1 antigen. In some embodiments, the target antigen is associated with lung cancer, and may include one or more of NY-ESO-1, Survivin, WT-1, MAGE-A10, and MUC-1 antigens. In some embodiments, one or more target antigens is associated with melanoma, and may include one or more of NY-ESO-1, Survivin, MAGE-A10, MART-1, and GP-100. In some embodiments, one or more peptide antigens are associated with ovarian cancer, and may include one or more of NY-ESO-1, WT-1, and Mesothelin antigen. In some embodiments, one or more target antigens are associated with prostate cancer, and may include one or more of Survivin, hTERT, PSA, PAP, and PSMA antigens. In some embodiments, the target antigen is associated with a sarcoma, and may include NY-ESO-1 antigen. In some embodiments, one or more target antigens are associated with lymphoma, and may include EBV antigen. In some embodiments, one or more target antigens are associated with multiple myeloma, and may include one or more ofNY-ESO-1, WT-1, XBP1-US, XBP1-SP, CD138, CS1 (SLAMF7), and SOX2 antigens. In some embodiments, the target antigens associated with multiple myeloma are two or more of (or three, four, five, or six of) peptide antigens disclosed in US 9,096,681, which is hereby incorporated by reference in its entirety. Exemplary peptides comprising antigenic epitopes include XBP1 unspliced (UN)i85-i93,

[0107] DBl / 149941487.1 22 NEX-017PC 107578-5017

[0108] XBP1-US 184-192, XBP1 spliced (SP)223-231, XBP 1 -SP367-375, CD 138265-273, CD 138260-268, CS 1240-248, CS 1239-247, NY-ESOl i57-i65A, and SOX2118- 127. In some embodiments, the target antigens comprise NY-ESO-1, WT-1, SOX-2, CD138, and CS1. In some embodiments, the target antigens comprise NY-ESO-1, WT-1, SOX-2, CD138, CS1, and XBP1-US and / or XBP1-SP. In some embodiments, the peptide antigens comprise NY-ESO-1, WT-1, and SOX-2.

[0109] In some embodiments, the target peptide antigens include one or more neoantigen peptides, one or more tumor associated antigens, and one or more virus-associated antigens (such as CMV, EBV, influenza, or Adenovirus), to provide an antitumor response while protecting against common pathogens that complicate recovery after HSCT.

[0110] Viral peptide antigens include, but are not limited to, those of adenovirus, herpes simplex virus, papilloma virus, respiratory syncytial virus, poxviruses, HIV, influenza viruses, EBV, hepatitis, and CMV. Particularly useful viral peptide antigens include HIV proteins such as HIV gag proteins (including, but not limited to, membrane anchoring (MA) protein, core capsid (CA) protein and nucleocapsid (NC) protein), HIV polymerase, influenza virus matrix (Ml) protein and influenza virus nucleocapsid (NP) protein, hepatitis B surface antigen (HBsAg), hepatitis B core protein (HBcAg), hepatitis e protein (HBeAg), hepatitis B DNA polymerase, hepatitis C antigens, and the like.

[0111] Patients that have undergone HSCT are at particular risk for infectious disease, given the immunocompromised state. The immunocompromised status of these patients permits reactivation of latent virus in seropositive patients or opportunistic infection in seronegative individuals. For example, post-transplant lymphoproliferative disease (PTLD) occurs in a significant fraction of transplant patients and results from Epstein-Barr virus (EBV) infection. EBV infection is believed to be present in approximately 90% of the adult population in the United States. Active viral replication and infection is kept in check by the immune system, but, as in cases of CMV, individuals immunocompromised by transplantation therapies lose the controlling T cell populations, which permits viral reactivation. This represents a serious impediment to transplant protocols. EBV may also be involved in tumor promotion in a variety of hematological and non-hematological cancers.

[0112] DBl / 149941487.1 23 NEX-017PC 107578-5017

[0113] In certain aspects, the present disclosure provides a cell composition comprising cytotoxic T lymphocytes (CTLs) that are specific for at least one neoantigen peptide, such as a neoantigen peptide selected according to this disclosure. In embodiments, the cell composition is made by a process according to this disclosure (described above).

[0114] In certain aspects, the present disclosure provides a method for treating a subject having cancer, comprising administering the cell composition of this disclosure. The cell composition can be administered to subjects by any appropriate routes, including intravenous infusion, intra-arterial administration, intralymphatic administration, and intratumoral administration.

[0115] In some embodiments, the patient receives or initiates immunotherapy with one or more checkpoint inhibitors, prior to (or optionally after) receiving the cell composition by adoptive transfer. In various embodiments, the checkpoint inhibitor(s) target one or more of CTLA-4 or PD-1 / PD-L1, which may include antibodies against such targets, such as monoclonal antibodies, or portions thereof, or humanized or fully human versions thereof. In some embodiments, the checkpoint inhibitor therapy comprises ipilimumab or Keytruda (pembrolizumab), or comparable monoclonal antibody. In some embodiments, the patient previously received PD1 blockade therapy, and was refractory or only partially responsive to that treatment. In such embodiments, the cell composition described herein can restore a robust T cell response, optionally in combination with a second round of immunotherapy (e.g., anti-CTLA4 or PD-1 blockade therapy).

[0116] In some embodiments, the patient receives about 1 to 5 rounds of adoptive immunotherapy (e.g., one, two, three, four or five rounds). In some embodiments, each administration of adoptive immunotherapy is conducted simultaneously with, or after (e.g., from about 1 day to about 1 week after), a round of checkpoint inhibitor therapy. In some embodiments, adoptive immunotherapy is provided about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 1 week after a checkpoint inhibitor dose. In some embodiments, the patient receives only a single administration of the cell composition.

[0117] DBl / 149941487.1 24 NEX-017PC 107578-5017

[0118] In other aspects, the present disclosure provides HLA-restricted neoantigen peptides that expand functional T cell populations. In embodiments, the neoantigen peptide is HLA- A2 restricted, and consists of an amino acid sequence selected from SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 59, and SEQ ID NO: 60. These neoantigen peptides were designed based on somatic mutations identified in NSCLC. In embodiments, the neoantigen peptide is HLA-A24 restricted, and consists of an amino acid sequence selected from SEQ ID NO: 67, SEQ ID NO: 70, SEQ ID NO: 72, and SEQ ID NO: 80. These neoantigen peptides were designed based on somatic mutations identified in melanoma. In embodiments, the neoantigen peptides are loaded onto aAPCs (e.g., polymeric aAPCs) suitable for administration to subjects having a cancer with the corresponding mutation. In embodiments, the neoantigen is combined with one or more TAA peptides (including but not limited to a peptide antigen from a TAA described herein) to prepare a cancer vaccine, or with each peptide antigen loaded onto an aAPC for administration or expansion of T cells ex vivo.

[0119] In other aspects, the present disclosure provides an aAPC cocktail comprising aAPCs loaded with at least one neoantigen (e.g., characterized according to this disclosure), and aAPCs loaded with at least one peptide derived from tumor associated antigen (TAA). In embodiments, the neoantigen peptide is a neoantigen peptide described herein, and / or the TTA is a TTA described herein. The aAPCs in the cocktail can present the same HLA, or may present two HLAs tailored for a heterozygous subject. In embodiments, the aAPC cocktail includes aAPCs with at least two different HLA ligands selected form HLA-A, HLA-B, HLA-C, and HLA-E.

[0120] As used herein, and unless the context requires otherwise, the term “about” means ±10% of an associated numerical value.

[0121] Other aspects and embodiments of the disclosure will be apparent from the following working examples.

[0122] DBl / 149941487.1 25 NEX-017PC 107578-5017

[0123] EXAMPLES

[0124] Prediction and evaluation of patient relevant neo-epitope peptides using healthy donor PBMC.

[0125] This example describes a process, starting from a given tumor mutation, for predicting and evaluating HLA-restricted peptides spanning a broad range of peptide HLA affinities (1-10,000 nM), to identify those that can expand functional T cell populations. The process enables the screening of patient derived neo-epitope peptides while using blood from healthy donors.

[0126] Patient tumor mutations were identified: 15 mutations from a melanoma patient (Table 1), and 30 mutations from a NSCLC patient (Tables 2 and 3). Multiple HLA-A2- restricted peptides were identified from the melanoma patient (Table 1), and multiple HLA- A24-restricted peptides were identified from the lung cancer patient (Table 3), and which were tested for their ability to expand functional antigen specific T cells across several healthy donors.

[0127] Using publicly available software tools (FIG. 1) a pool of 9-11 amino acid long peptides were predicted, which were then synthesized, and loaded individually on paramagnetic nano-aAPCs. The in silico analysis includes predicting HLA binding affinity, including comparison to the corresponding wild-type sequence. Nano-aAPCs were constructed with dextran-coated, iron oxide nanoparticles with conjugated signal 1 (HLA-Ig dimer) and signal 2 (anti-CD28) ligands as described in the art. See US 10,987,412 and US 10,435,668, which are hereby incorporated by reference in their entireties. These individually loaded aAPCs were then combined into aAPC cocktails with up to six different peptide loaded aAPCs. Once the cocktail was created, leukopacks were obtained that were HLA A2 and / or HLA A24 positive. A downscale protocol of an Enrichment and Expansion (E+E) process was employed (see US Patent Nos. 10,987,412 and 11,007,222, which are hereby incorporated by reference in their entireties). Specifically, the leukopacks were depleted of CD4+ T cells, and the E+E process performed using the created cocktails with aliquots containing 150-200 million CD8+ T cells.

[0128] DBl / 149941487.1 26 NEX-017PC 107578-5017

[0129] Briefly, T cells were incubated with peptide loaded nano-aAPC for 1 hour at 4°C, and cell-particle mixtures were subsequently passed through a magnetic enrichment column for simultaneous activation and enrichment. The negative fraction was collected, and the positive fraction (bound fraction) eluted. The bound fraction was cultured with growth factors (IL-2, IL-4, IL-6, IFN-y, IL- 113), which were added on Days 1 and 7. aAPCs were supplemented on Day 7. On day 14 the T cell product was harvested and the expanded T cells characterized for antigen specificity and phenotype.

[0130] FIG. 2 summarizes the NSCLC antigens presented by seven nano-aAPC cocktails, and shows the level of antigen specificity (for each antigen) for the expanded cell product. Percent specificity was determined by staining with peptide-loaded HLA-Ig dimers. FIG. 3 shows the T cell phenotype of the expanded cells across the seven leukopacks. As shown in the bottom row, cells are largely (greater than 80% in all cases) central and effector memory T cells. FIG. 4 shows the phenotype of antigen-specific cells for select antigens, showing that the cells are largely (greater than about 80%) central and effector memory T cells.

[0131] FIG. 5 correlates the predicted IC50 for TP53 antigens with the % Specificity of the expanded cells. FIG. 6 correlates the predicted IC50 for DEUP1 and SQLE antigens with the % Specificity of the expanded cells. These data show that the predicted affinity of the peptide for MHC is not a good predictor for the ability of the peptide to induce antigenspecific T cell expansion.

[0132] FIG. 7 summarizes the melanoma antigens presented by seven nano-aAPC cocktails, and shows the level of antigen specificity for the expanded cell product. FIG. 8 shows the T cell phenotype of the expanded cells across the seven leukopacks. As shown in the bottom row, cells are largely (greater than about 75%) central and effector memory T cells. FIG. 9 shows the phenotype of antigen-specific cells for select antigens, showing that the cells are largely (greater than about 75%) central and effector memory T cells.

[0133] FIG. 10 compares the predicted IC50 for p401-4 and p401-3 antigens with the % Specificity of the expanded cells. These data show that the predicted affinity of the peptide for MHC is not a good predictor for the ability of the peptide to induce antigen-specific T cell expansion. FIG. 11 shows the % Specificity across several Leukopacks for various antigens. FIG. 12 shows that the process can be run with two HLA ligands simultaneously

[0134] DBl / 149941487.1 T1 NEX-017PC 107578-5017

[0135] (HLA-A2 and HLA-A24), which is beneficial for heterozygous patients (thereby preventing immune escape due to HLALoss of Heterozygosity (LOH)).

[0136] The data demonstrate that this process can reproducibly expand neoepitope-specific T cells using healthy donor PBMCs. Notably, some predicted peptides did not induce any T cell expansion, demonstrating that publicly available software tools are not sufficient to predict neoantigen epitopes that induce T cell expansion.

[0137] The identified mutations were examined to determine if they had been previously described, to determine whether some identified functional T cell epitopes are shared neoepitope peptides that can be used across multiple patients. Furthermore, TCRs directed at these epitopes could be used for engineered TCR-based approaches to target neo-epitope peptides. One of those mutations is the TP53 mutation (S— F mutation), which has been reported in melanoma and other cancers, including lung, breast, large intestine, urinary tract, hematopoietic and lymphoid cancers. Others have not been described elsewhere which would classify them as private mutations and peptides. However, with more tumor samples sequenced everyday this conclusion could change.

[0138] In some peptide cocktails, predicted neo-epitope peptides were combined with either known common tumor associated antigens (TAA) (e.g., Mart-1, Her-2neu) or a high affinity viral peptide (EBV / lmp2). In both cases, the addition of such strong / dominant antigen peptides did not hinder the ability to expand neo-epitope peptide specific T cells, which supports the hypothesis that aAPC cocktails can be created spanning a wide range of peptide affinities and containing both neo-epitope specific cocktails and TAA peptides to increase the chance of inducing a broad anti-tumor response, while simultaneously reducing the chance of immune escape due to loss of tumor mutation.

[0139] Further still, aAPC cocktails were created and tested targeting HLA A2 and HLA A24 restricted T cells simultaneously, thereby demonstrating that these T cells can be targeted and expanded at the same time and therefore potentially prevent immune escape due to HLA LOH.

[0140] DBl / 149941487.1 28 NEX-017PC 107578-5017

[0141] Table 1 : Melanoma Mutations

[0142] DB1 / 149941487.1 29 NEX-017PC 107578-5017

[0143] Table 2: NSCLC Mutations

[0144] DBl / 149941487.1 30 NEX-017PC 107578-5017

[0145] Mutation shown in bold / underline type

[0146] Table 3: NSCLC Neo-epitope peptides

[0147] DBl / 149941487.1 31 NEX-017PC 107578-5017

[0148] DB1 / 149941487.1 32

Claims

NEX-017PC 107578-5017CLAIMS:

1. A method for screening neoantigen peptides, comprising: providing one or more mutations identified for a subject’s cancer, and obtaining candidate peptide antigens predicted to bind a human leukocyte antigen (HLA) of the subject, each candidate peptide antigen comprising at least one of said mutations; loading the candidate peptide antigens onto paramagnetic nano-artificial antigen presenting cells (nano-aAPCs); incubating one or more of the loaded nano-aAPCs with T cells from a healthy donor; passing the nano-aAPCs and the T cells over a magnetic column, and recovering cells associated with the bound fraction; expanding the cells in the bound fraction in culture; characterizing the antigen specificity of the expanded cells, and selecting one or more candidate peptide antigens for immunotherapy.

2. The method of claim 1, wherein the HLA is class I and the T cells are CD8+, or the HLA is class II and the T cells are CD4+.

3. The method of claim 1 or 2, wherein at least five candidate peptide antigens are obtained and loaded onto aAPCs, or at least ten candidate peptide antigens are obtained and loaded onto aAPCs, or at least 15 candidate peptide antigens are obtained and loaded onto aAPCs; or at least 20 candidate peptide antigens are obtained and loaded onto aAPCs.

4. The method of claim 1 to 3, wherein the candidate peptide antigens have predicted affinities for the HLA of from about 1 nM to about 10,000 nM.

5. The method of claim 4, wherein at least one candidate peptide antigen has a predicted affinity for the HLA of less than about 100 nM, or less than about 50 nM.

6. The method of claim 4 or 5, wherein at least one candidate peptide antigen has a predicted affinity for the HLA of greater than about 100 nM, or greater than about 200 nM, or greater than about 300 nM, or greater than about 500 nM, or greater than about 1000 nM.DBl / 149941487.1 33NEX-017PC 107578-50177. The method of claim 6, wherein the cancer is a hematological cancer, optionally selected from a leukemia, a lymphoma, or a myeloma.

8. The method of claim 7, wherein the hematological malignancy is selected from acute myeloid leukemia, chronic myelogenous leukemia, childhood acute leukemia, nonHodgkin’s lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome, malignant cutaneous T-cells, mycosis fungoids, non-MF cutaneous T-cell lymphoma, lymphomatoid papulosis, and T-cell rich cutaneous lymphoid hyperplasia.

9. The method of claim 6, wherein the cancer is a solid tumor.

10. The method of claim 9, wherein the solid tumor is selected from melanoma, colon cancer, duodenal cancer, prostate cancer, breast cancer, ovarian cancer, ductal cancer, hepatic cancer, pancreatic cancer, renal cancer, endometrial cancer, testicular cancer, stomach cancer, dysplastic oral mucosa, polyposis, head and neck cancer, invasive oral cancer, nonsmall cell lung carcinoma, small-cell lung cancer, mesothelioma, transitional and squamous cell urinary carcinoma, brain cancer, neuroblastoma, and glioma.

11. The method of any one of claims 1 to 10, wherein the one or more mutations are associated with an oncogene or tumor suppressor.

12. The method of any one of claims 1 to 11, wherein the one or more mutations are selected from mutations encoding an amino acid substitution, an insertion and / or deletion, a frameshift, a fusion protein, and a premature stop codon.

13. The method of claim 12, wherein the one or more mutations are point mutations encoding an amino acid substitution.

14. The method of any one of claims 1 to 13, wherein the Class I HLA includes HLA-A.DBl / 149941487.1 34NEX-017PC 107578-501715. The method of claim 14, wherein the subject is homozygous at HLA-A, and the nano- aAPCs present a matching HLA ligand.

16. The method of claim 14, wherein the subject is heterozygous at HLA-A, and the nano-aAPC present both of the subject’s HLA ligands.

17. The method of any one of claims 14-16, wherein the Class I HLA further comprises one of HLA-B and HLA-C.

18. The method of any one of claims 1 to 17, wherein nano-aAPCs are screened as a cocktail of up to ten peptides per cocktail, or up to seven peptides per cocktail, or up to six peptides per cocktail.

19. The method of any one of claims 1 to 18, wherein the nano-aAPCs are incubated with at least about 25 million T cells, or at least 50 million T cells, or at least 100 T cells, or at least about 150 million T cells.

20. The method of claim 19, wherein the nano-aAPCs are incubated with about 500 million T cells or less, or about 250 million T cells or less, or about 200 million T cells or less.

21. The method of any one of claims 1 to 20, wherein the bound fraction is expanded in culture for at least five days, or at least one week, or at least ten days, or at least two weeks.

22. The method of claim 21, wherein the bound fraction in expanded in culture for about two weeks.

23. The method of any one of claims 1 to 22, wherein aAPCs are supplemented in the culture, optionally on about day 7.DBl / 149941487.1 35NEX-017PC 107578-501724. The method of any one of claims 1 to 22, wherein the cells are expanded in culture in the presence of growth factors.

25. The method of claim 24, wherein the growth factors comprise IL-2. IL-4, IL-6, INF- y, and IL- 113.

26. The method of claim 24 or 25, wherein the growth factors are added on day 1 and again between days 5 and 9, and optionally on day 7.

27. The method of any one of claims 1 to 26, wherein the T cells in the culture are at least 60% central and effector memory T cells, or at least 70% central and effector memory T cells at the end of the culture.

28. The method of any one of claims 1 to 27, wherein the expanded cells are characterized for specificity for each candidate peptide antigen by staining with HLA-Ig peptide dimer.

29. The method of claim 28, wherein candidate peptide antigens are selected that have a % specificity in the culture of at least about 0.5%, or at least about 1%, or at least about 2%, or at least about 4%, or at least about 5%, or at least about 10%.

30. The method of any one of claims 1 to 29, wherein candidate peptide antigens are selected that are shared among subjects.

31. A method for preparing a T cell composition suitable for adoptive cell therapy for subject, comprising: selecting at least one neoantigen peptide, optionally according to any one of claims 1 to 30; loading the neoantigen peptide onto paramagnetic nano-artificial antigen presenting cells (nano-aAPCs), optionally preparing an aAPC cocktail with aAPC loaded with one or more tumor associated antigen (TAA) peptides;DBl / 149941487.1 36NEX-017PC 107578-5017 incubating one or more of the loaded nano-aAPCs with T cells from a subject or a healthy donor; passing the nano-aAPCs and the T cells over a magnetic column, and recovering cells in the bound fraction; and expanding the cells associated with the bound fraction in culture.

32. The method of claim 31, wherein the aAPC cocktail presents at least two peptide antigens, or at least three peptide antigens, or at least five peptide antigens, or at least seven peptide antigens.

33. The method of claim 31 or 32, wherein the peptide antigens have predicted affinities for the HL A presented by the aAPC of from about 1 nM to about 10,000 nM.

34. The method of claim 33, wherein at least one peptide antigen has a predicted affinity for the HLA presented by the aAPC of less than about 100 nM, or less than about 50 nM.

35. The method of claim 33 or 34, wherein at least one peptide antigen has a predicted affinity for the HLA presented by the aAPC of greater than about 100 nM, or greater than about 200 nM, or greater than about 300 nM, or greater than about 500 nM, or greater than about 1000 nM.

36. The method of any one of claims 31 to 35, wherein the subject has a hematological cancer, optionally selected from a leukemia, a lymphoma, or a myeloma.

37. The method of claim 36, wherein the subject has a hematological malignancy selected from acute myeloid leukemia, chronic myelogenous leukemia, childhood acute leukemia, non-Hodgkin’s lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome, malignant cutaneous T-cells, mycosis fungoids, non- MF cutaneous T-cell lymphoma, lymphomatoid papulosis, and T-cell rich cutaneous lymphoid hyperplasia.DBl / 149941487.1 37NEX-017PC 107578-501737. The method of any one of claim 31 to 35, wherein the subject has a solid tumor.

38. The method of claim 37, wherein the solid tumor is selected from melanoma, colon cancer, duodenal cancer, prostate cancer, breast cancer, ovarian cancer, ductal cancer, hepatic cancer, pancreatic cancer, renal cancer, endometrial cancer, testicular cancer, stomach cancer, dysplastic oral mucosa, polyposis, head and neck cancer, invasive oral cancer, nonsmall cell lung carcinoma, small-cell lung cancer, mesothelioma, transitional and squamous cell urinary carcinoma, brain cancer, neuroblastoma, and glioma.

39. The method of any one of claims 31 to 38, wherein the aAPC presents a Class I HLA that includes HLA- A, and optionally one or more of HLA-B and HLA-C.

40. The method of claim 39, wherein the subject is homozygous at HLA- A, and the nano- aAPCs present a matching HLA ligand.

41. The method of claim 40, wherein the subject is heterozygous at HLA- A, and the nano-aAPC present both of the subject’s HLA ligands.

42. The method of any one of claims 31 to 41, wherein the bound fraction is expanded in culture for at least five days, or at least one week, or at least 10 days, or at least two weeks.

43. The method of claim 42, wherein the bound fraction in expanded in culture for about two weeks.

44. The method of any one of claims 31 to 43, wherein aAPCs are supplemented in the culture, optionally on about day 7.

45. The method of any one of claims 31 to 44, wherein the cells are expanded in culture in the presence of growth factors.DBl / 149941487.1 38NEX-017PC 107578-501746. The method of claim 45, wherein the growth factors comprise IL-2. IL-4, IL-6, INF- y, and IL- 113.

47. The method of claim 45 or 46, wherein the growth factors are added on day 1 and again between days 5 and 9, and optionally day 7.

48. The method of any one of claims 31 to 47, wherein the T cells in the culture are at least 60% central and effector memory T cells, or at least 70% central and effector memory T cells at the end of the culture.

49. The method of any one of claims 31 to 48, wherein the expanded cell composition has a specificity for each peptide antigen of at least about 0.5%, or at least about 1%, or at least about 2%, or at least about 4%, or at least about 5%.

50. A method for preparing a cancer immunotherapy, comprising: loading one or more neoantigen peptides onto paramagnetic nano-artificial antigen presenting cells (nano-aAPCs); incubating one or more of the loaded nano-aAPCs with T cells from a subject or a healthy donor; passing the nano-aAPCs and the T cells over a magnetic column, and recovering cells in the bound fraction; expanding the cells associated with the bound fraction in culture; selecting at least one T cell clone that is specific for the neoantigen peptide; cloning the T cell receptor (TCR) of the T cell clone, and preparing an immunotherapy that comprises the cloned TCR or portions thereof.

51. The method of claim 50, wherein the TCR is heterologously expressed in a T cell for administration to a subject.

52. The method of claim 50, wherein the TCR is conjugated to or complexed with a T cell engaging molecule to prepare a bispecific T cell engager (BiTE).DBl / 149941487.1 39NEX-017PC 107578-501753. The method of claim 52, wherein the BiTE comprises at least a first domain that binds a T cell surface antigen or receptor, and a second domain that comprises at least the extracellular domains or portions thereof of the alpha and beta chains of said TCR.

54. The method of claim 53, wherein the alpha and beta chains or portions thereof are fused to heavy and light chains of an IgG antibody.

55. The method of claim 54, wherein one IgG heavy and light chain pair are fused to the TCR alpha and beta chain sequences, and a second IgG heavy and light chain pair constitute a full or partial agonist for CD3.

56. The method of claim 54, wherein both IgG heavy and light chain pairs are genetically fused to TCR alpha and beta chains, and one or more single chain antibodies that is a full or partial agonist for CD3 fused to an Fc domain.

57. The method of claim 50, wherein the cloned TCR is conjugated to a drug, which is optionally a cancer chemotherapeutic agent.

58. A cell composition comprising T lymphocytes that are specific for at least one neoantigen peptide selected according to any one of claims 1 to 30, or made by a process according to any one of claims 31 to 49.

59. A method for treating a subject having cancer, comprising administering the cell composition of claim 58.

60. A peptide antigen consisting of an amino acid sequence selected from SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 59, and SEQ ID NO: 60.

61. A peptide antigen consisting of an amino acid sequence selected from SEQ ID NO: 67, SEQ ID NO: 70, SEQ ID NO: 72, and SEQ ID NO: 80.DBl / 149941487.1 40NEX-017PC 107578-501762. An aAPC cocktail comprising aAPCs loaded with at least one neoantigen, and aAPCs loaded with at least one tumor associated antigen (TAA) peptide.

63. The aAPC cocktail of claim 54, wherein the neoantigen peptide is a peptide antigen of claim 60 or claim 61.DBl / 149941487.1 41