CN117500512A - T cell receptor for RAS derived recurrent neoantigen and method for identifying same - Google Patents

Abstract

A method of treating cancer in a subject is disclosed. The method comprises administering to the subject a therapeutically effective amount of a population of T cells, wherein at least 10% of the T cells in the population have the CDR3 amino acid sequence as set forth in SEQ ID No. 6 and the β chain of the CDR has the CDR3 amino acid sequence as set forth in SEQ ID No. 7.

Description

T cell receptor for RAS derived recurrent neoantigen and method for identifying same

RELATED APPLICATIONS

The present application claims priority from U.S. provisional patent application No. 63/223,114 filed on 7/19 and from israel patent application No. 282814 filed on 29/4/2021, both of which are incorporated herein by reference in their entireties.

Statement of sequence Listing

An ASCll file including 9,217 bytes, filed concurrently with the present application, and having a 26 th day file name 91651Sequence Listing.txt created at 2022, 4 th month, is incorporated herein by reference.

Technical field and background of the invention

The invention, in some embodiments thereof, relates to the identification of T cell receptors that bind to repeatedly mutated neopeptides, more particularly Ras-derived mutated neopeptides.

In recent years, immunotherapy has raised new promise in oncology because of its remarkable ability to induce long-term tumor regression of metastatic cancers. This feature is shared in immunotherapeutic patterns, including checkpoint blockade of TIL and Adoptive Cell Transfer (ACT). The final common pathway for both treatments is believed to be the specific recognition of tumor antigens by cytotoxic T lymphocytes. In particular, with the advancement of sequencing capability, a thorough analysis of successful cases of immunotherapy revealed a central role for mutation-derived antigens (termed neoantigens) in mediating anti-tumor immune responses.

The neoantigen is a cell surface peptide/Human Leukocyte Antigen (HLA) complex, wherein the peptide component, i.e., the neopeptide, is an altered degradation product of the mutein. Expression is limited by diseased tissue and is not affected by immune tolerance, and the neoantigen can elicit a specific anti-tumor response when the TCR participates, thus being an ideal therapeutic target.

Most of the new antigens identified from patients receiving treatment are derived from private, non-recurrent mutations and, therefore, although effective, cannot be generalized outside of individual patients. Hot spot neoantigens, i.e. neoantigens that occur in a large group of cancer patients, are apparently formed only at the intersections of recurrent oncogenic mutations and common HLA alleles. The high level of antigen challenge is due to two main reasons. First, hot spot neoantigens may pave the way for "off-the-shelf cell therapy, vaccine and patient screening strategies. Tumor cells expressing validated mutation/HLA combinations should be suitable for immunotherapy. Even in the absence of a priori immune recognition, predetermined TCRs from other patients or even healthy donors can be used to redirect autologous T cells against the hot spot neoantigens that are ignored. In addition, pre-treatment undetectable neoantigen-specific T cells showed significant expansion after mutation-based vaccines. Second, as a therapeutic target, hot spot neoantigens may be superior to private neoantigens. This is because immunotherapy against subcloning mutations of heterogeneous tumors may yield immune escape, while hot spot neoantigens derived from cloned oncogenic mutations are expected to exist more homogenously within the tumor.

Several hot spot neoantigens discovered in recent years are derived from major oncogenes such as BRAF, NRAS and p53. However, the clinical relevance of this neoantigen was directly demonstrated by successful ACT treatment of patients with metastatic colon cancer with autologous TIL against newly discovered HLA-C08:02/KRAS. G12D hot spot neoantigen. These recent successes have motivated a renewed effort toward the discovery of hot spot neoantigens. P 53-centered screening showed that 8% of screened patients had natural TIL reactivity to the derived neoantigen and in some cases hot spot neoantigen occurred. Other work has focused on identifying T cells targeting specific hot spots from peripheral blood of patients or healthy donors, thereby expanding the pool of known KRAS and other oncogene derived neoantigens against HLA-class I and HLA-class II.

To date, the discovery of new antigens has been almost entirely T cell-centered. In these methods, candidate neopeptides are expressed artificially in Antigen Presenting Cells (APCs), either as pulsed synthetic peptides or by minigene overexpression. APCs are then co-incubated with T cells (most commonly TIL) and their response profile is interpreted to indirectly identify the neoantigen. Further characterization and validation relies largely on computer-simulated binding predictions, such that the identified neoantigens are limited to only those antigens that are predicted to bind and be immunogenic in the subject patient. Furthermore, irrelevant neoantigens, i.e. antigens compiled from existing libraries in tumor evolution, will still be identified as long as they were once immunogenic.

The causal role of RAS proteins in cancer has long been recognized, with activating mutations occurring in one third of human cancers. Three major subtypes, KRAS, NRAS and HRAS, share one and the same 86 amino acid long N-terminus. In this same sequence, three mutational hot spots were identified: positions 12, 13 and 61. In pan-carcinoma, KRAS is the most mutated RAS subtype (85% of RAS mutations). However, NRAS mutations predominate in the most successful immunotherapeutic target melanoma to date. In particular, nras.61 is the location of the second most mutation in melanoma, occurring in up to 20% of patients. NRAS mutant melanoma has a poorer prognosis than non-NRAS mutant melanoma. Various attempts to develop RAS-targeted therapies have not resulted in effective, specifically approved methods for the treatment of NRAS mutant melanoma.

Background art includes WO2020/234875, WO/2020/037302, WO/2019/075112; WO/2020/180648; WO/2019/226939; WO/2019/168984; WO/2019/226941, WO/2019/133853; WO/2019/036688; WO/2018/227030, WO/2019/050994; WO/2018/208856; WO/2018/195357; WO/2018/098362; WO/2019/104203 and WO/2017/106638.

Disclosure of Invention

According to one aspect of the invention there is provided a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a population of T cells, wherein at least 10% of the T cells in the population have the CDR3 amino acid sequence as shown in SEQ ID No. 6 and the β chain of the CDR has the CDR3 amino acid sequence as shown in SEQ ID No. 7, thereby treating cancer in the subject.

According to one aspect of the present invention, there is provided a method of treating cancer in a subject, comprising:

(a) Ascertaining an HLA profile of the subject;

(b) Determining whether the object expresses nras.q61k or nras.q61r; and

(c) When the subject is identified as HLA-A 01/nras.q61k or HLA-A 01/nras.q61r, the subject is treated with a therapeutically effective amount of a population of T cells expressing a TCR, wherein the α chain of the TCR has a CDR3 amino acid sequence as set forth in SEQ ID NO:6 and the β chain of the TCR has a CDR3 amino acid sequence as set forth in SEQ ID NO:7, thereby treating the cancer.

According to one aspect of the invention there is provided a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a population of T cells, wherein the alpha chain of the TCR of the T cells of the population has the CDR3 amino acid sequence as shown in SEQ ID No. 2 and the beta chain of the CDR has the CDR3 amino acid sequence as shown in SEQ ID No. 17, thereby treating cancer in the subject.

According to one aspect of the invention there is provided a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a population of T cells, wherein the alpha chain of the TCR of the T cells of the population has the CDR3 amino acid sequence as shown in SEQ ID No. 16 and the beta chain of the CDR has the CDR3 amino acid sequence as shown in SEQ ID No. 3, thereby treating cancer in the subject.

According to one aspect of the invention there is provided an isolated population of T cells genetically modified to express a T Cell Receptor (TCR), wherein the α chain of the TCR has a CDR3 amino acid sequence as shown in SEQ ID No. 6 and the β chain of the CDR has a CDR3 amino acid sequence as shown in SEQ ID No. 7.

According to one aspect of the invention there is provided an isolated population of T cells wherein at least 10% of the T cells in the population have the CDR3 amino acid sequence shown in SEQ ID NO. 6 for the alpha chain and the CDR3 amino acid sequence shown in SEQ ID NO. 7 for the beta chain.

According to one aspect of the invention there is provided an isolated population of T cells wherein the alpha chain of the TCR of the T cells of the population has the CDR3 amino acid sequence as shown in SEQ ID NO:2 and the beta chain of the TCR has the CDR3 amino acid sequence as shown in SEQ ID NO: 17.

According to one aspect of the invention there is provided an isolated population of T cells wherein the alpha chain of the TCR of the T cells of the population has the CDR3 amino acid sequence as shown in SEQ ID NO:16 and the beta chain of the TCR has the CDR3 amino acid sequence as shown in SEQ ID NO: 3.

According to one aspect of the present invention there is provided a method of selecting a neoantigen for presentation by a recurrent HLA that can be targeted in the treatment of cancer immunotherapy, the method comprising:

(a) Analyzing the frequency of occurrence of cancer-associated muteins in the context of a single HLA allele in tumor cells of a plurality of cancer patients; and

(b) Determining the binding affinity of a peptide of 8-14 amino acids in length derived from a cancer-associated mutein to a single HLA allele, wherein the peptide comprises a mutation compared to the wild type protein;

wherein a candidate peptide that binds with an affinity above a first predetermined level to an HLA allele that occurs more frequently than a second predetermined level is selected as a neoantigen that can be presented by a candidate HLA targeted in the cancer immunotherapy treatment;

(c) Confirming (corroboraning) that the candidate peptide is presented by an HLA allele in an in vitro expression system of antigen presenting cells; and

(d) Confirmation that the confirmed peptide was presented by HLA alleles in tumor cells.

According to an embodiment of the invention, at least 10% of the T cells in the population have the CDR3 amino acid sequence as shown in SEQ ID NO. 2 and the beta chain of the CDR has the CDR3 amino acid sequence as shown in SEQ ID NO. 17.

According to an embodiment of the invention, at least 10% of the T cells in the population have the CDR3 amino acid sequence as shown in SEQ ID NO. 16 and the beta chain of the CDR has the CDR3 amino acid sequence as shown in SEQ ID NO. 3.

According to an embodiment of the invention, the TCR binds to a peptide of sequence shown in SEQ ID No. 1 or SEQ ID No. 34 in a complex with an HLA-A.times.01:01 allele in a subject.

According to an embodiment of the invention, the T cells are autologous to the subject.

According to an embodiment of the invention, the T cells are non-autologous to the subject.

According to an embodiment of the invention, the T cells are genetically modified to express T cell receptors.

According to an embodiment of the invention, the T cells comprise cd8+ T cells.

According to an embodiment of the invention, the cancer is selected from the group consisting of melanoma (melanoma), colon cancer, breast cancer, thyroid cancer, gastric cancer, colorectal cancer, leukemia cancer, bladder cancer, lung cancer, ovarian cancer, breast cancer and prostate cancer.

According to an embodiment of the invention, the cancer is melanoma.

According to an embodiment of the invention, the method further comprises treating the subject with a checkpoint inhibitor.

According to an embodiment of the invention, the isolated T cell population is genetically modified to express a TCR.

According to an embodiment of the invention, the T cells are cd8+ T cells.

According to an embodiment of the invention, the isolated T cell population is used for the treatment of cancer.

According to an embodiment of the invention, the determining comprises using a predictive algorithm to predict the binding affinity.

According to an embodiment of the invention, the prediction algorithm comprises NetMHCpan.

According to an embodiment of the present invention, confirmation is performed using targeted mass spectrometry.

According to an embodiment of the invention, the HLA comprises HLA class I.

According to an embodiment of the invention, the plurality of patients includes patients resistant to at least one cancer therapy.

According to an embodiment of the invention, the method further comprises analyzing the proportion of tumor cells containing the cancer-associated mutein prior to step (c), wherein a candidate peptide that binds to HLA alleles having an affinity above a first predetermined level and a frequency above a second predetermined level is selected as a candidate HLA-presented neoantigen, said candidate peptide being comprised in the proportion of tumor cells above the predetermined level, said candidate peptide being targeted in the treatment of cancer immunotherapy.

According to an embodiment of the invention, the in vitro expression system comprises an extended peptide expressing 25-27 amino acids or 45-47 amino acids, wherein the extended peptide comprises the amino acid sequence of a cancer-associated mutein.

According to an embodiment of the invention, the antigen presenting cells comprise B cells.

According to an embodiment of the invention, the cancer-related mutein is a member of the RAS family.

According to an embodiment of the invention, the member is selected from the group consisting of NRAS, KRAS and HRAS.

According to an embodiment of the present invention, the member is an NRAS.

According to an embodiment of the invention, the cancer-associated mutein is a RAF kinase.

According to an embodiment of the invention, the RAF kinase is B-RAF.

According to an embodiment of the present invention, cancer patients include melanoma patients, thyroid cancer patients, pheochromocytoma patients, seminoma patients, gastric adenocarcinoma patients, cholangiocarcinoma patients, pancreatic cancer patients, colorectal adenocarcinoma, leukemia patients, bladder urothelial carcinoma patients, endometrial carcinoma patients, thymus epithelial tumor patients, non-small cell lung carcinoma patients, sarcoma patients, ovarian cancer patients, and prostate cancer patients.

Unless defined otherwise, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, these materials, methods, and examples are illustrative only and are not meant to be necessarily limiting.

Brief description of the drawings

Some embodiments of the invention are described herein, by way of example, with reference to the accompanying drawings. Referring now in specific detail to the drawings, it is emphasized that the details shown are exemplary and are for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how the embodiments of the present invention may be embodied.

In the drawings:

FIGS. 1A-C: libraries of potent neoantigen-specific TCRs.

Healthy donor T cells were electroporated with TCR alpha and beta chains transcribed in vitro. (A) Flow cytometry of A.times.01/ILDTAGKEEY (SEQ ID NO: 1) -tetramer stained cells. Negative control-cells without mRNA electroporation ("non-electroporated", EN) (B-C) electroporated T cells were incubated overnight with peptide pulsed IHW01161 cells. (B) IFN gamma ELISA;10 μm peptide; one-way anova with Tukey correction for multiple comparisons; error bars represent SEM of biological triplicate. (C) 4-1BB peptide titration assay; two-factor anova with Sidak correction for multiple comparisons.

Fig. 2A-B: the neoantigen-specific TCR has reactivity and cytotoxic capacity for tumor cells endogenously expressing the neoantigen.

Healthy donor (D3) T-cells electroporated with in vitro transcribed TCR alpha and beta chains and co-incubated with tumor derived cell lines. T cell negative control: donor cells that do not contain mRNA electroporation ("non-electroporated", EN). (A) After a 1:1 co-incubation overnight, the percentage of T cells expressing activation marker 4-1 BB; 108T-a 01:01+/NRAS wild type; MM150414-a 01:01-/nras.q61k; two-way anova with Sidak correction for multiple comparisons. (B) cleaved caspase-3 killing assay: after a 3:1 ratio of effector to target co-incubation for 3 hours, the percentage of tumor cells expressing cleaved caspase-3; one-way analysis of variance with Tukey correction was used for multiple comparisons. Error bars represent SEM of biological triplicate.

Fig. 3A-F: examination of the 17TIL library revealed four TCR clusters similar to N135.1: n17.3.2, N17.5, N17.6 and N17.7. The most common TCRs in this list, N17.3.2 and N17.5, proved to be potent and neoantigen specific. (A) Both the alpha and beta chains of the similarity cluster are enriched in tetramer+ subsets, and alpha/beta pairing is confirmed by single cell TCR sequencing. Representative data from batch TCR sequencing copy # 1. (B) sequence comparison of TCR variable regions. Note the edit distance of up to four amino acids and the similarity of the V/J genes. (C) TCRs are plotted against their probabilities of production of alpha (X axis) and beta chains (Y axis). N135.1 similarity clusters are circled. NH1 (NRAS mixed TCR # 1) binds the most likely alpha/beta within the similarity cluster; it is generated by a chain exchange between N135.1 and N17.5. NH2 is the NH1's counterpart, mixed with NA17.5 and NB135.1. (D) schematic representation of the exchange strand of NA135.1 with NB17.5 to form NH 1-. (E-F) electroporation of donor (D3) T cells with in vitro transcribed TCRs to express NH1 and NH2.T cell negative control: donor cells that do not contain mRNA electroporation ("non-electroporated", EN). (E) Flow cytometry analysis of CD8 and tetramer stained cells. (F) IFNg ELISA was performed after a 1:1 co-incubation of one night with IHW01161 presenting cells pulsed with wild-type or mutant peptides. IHW01161 without pulsed peptide (DMSO only) served as a negative control. Error bars represent SEM of biological triplicate; two-way anova with Tukey correction was used for multiple comparisons.

Fig. 4: tandem mass spectrometry of ILDTAGKEEY (SEQ ID NO: 1) identified in HLA peptide group science of 17T. method-FTMS; HCD, score-91.55, m/z-569.79.

Detailed Description

The present invention, in some embodiments thereof, relates to T cell receptors that bind to recurrent mutated novel peptides.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by the examples. The invention is capable of other embodiments or of being practiced or of being carried out in various ways.

Immunotherapy has therapeutic potential for metastatic cancers, particularly in melanoma. The anti-tumor effect is typically mediated by T cell recognition of new antigens; i.e., HLA-presented peptides with mutations. With few exceptions, the identified neoantigens from responders originate from private mutations and therefore cannot be generalized outside of individual patients. By definition, "recurrent neoantigen" refers to an antigen shared among a patient population. Naturally, these are antigens derived from common driving mutations and present on common HLA alleles. Furthermore, due to the clonality of the driving mutations, they are expected to exist uniformly within the tumor and between metastases. Recurrent neoantigens should therefore be of great clinical value, as they can be used to develop effective, tumor-specific, "off-the-shelf" therapies.

Previously, the present inventors have discovered a novel method of identifying recurrent neoantigens that incorporates bioinformatic analysis that takes into account the presence of recurrent mutations and patient HLA allotypes and binding predictions (WO 2020/234875). Using this approach, they predicted that HLA-A 01:01/NRAS.Q61K hotspot neoantigen was a powerful immunogenic target associated with thousands of patients.

The inventors have now found a method of simplifying this method whereby the number of potential candidate neoantigens is further reduced using an in vitro system prior to in vivo validation. This step is performed after the above described bioinformatic analysis and binding prediction analysis and allows for a faster and more efficient identification process. Since in vivo procedures can use expensive reagents (e.g., incorporating isotopically labeled candidates into tumor samples for mass spectrometry), narrowing the list of potential candidates prior to the validation step has a significant impact on the efficiency of the procedure.

Thus, according to a first aspect of the present invention there is provided a method of selecting recurrent HLA presenting neoantigens for therapeutic purposes of cancer immunotherapy, the method comprising:

(a) Analyzing the frequency of occurrence of cancer-associated muteins in the context of a single HLA allele in tumor cells of a plurality of cancer patients; and

(b) Determining the binding affinity of a peptide of 8-14 amino acids in length derived from a cancer-associated mutein to a single HLA allele, wherein the peptide comprises a mutation compared to the wild type protein;

wherein a candidate peptide that binds with an affinity above a first predetermined level to an HLA allele that occurs more frequently than a second predetermined level is selected as a neoantigen that can be presented by a candidate HLA targeted in the cancer immunotherapy treatment;

(c) Confirming that the candidate peptide is presented by the HLA allele in an in vitro expression system of antigen presenting cells; and

(d) Confirmation that the validated peptide is presented by HLA alleles in tumor cells of at least one cancer patient.

As used herein, the term "neoantigen" is an epitope with at least one alteration that makes it different from the corresponding wild-type, parent antigen, e.g., by mutation in or post-translational modification specific for tumor cells. The novel antigen may comprise a polypeptide sequence or a nucleotide sequence. Mutations may include frameshift or non-frameshift indels, missense or nonsense substitutions, splice site changes, genomic rearrangements or gene fusions, or any genomic or expression change that produces a neoORF. Mutations may also include splice variants. Tumor cell specific post-translational modifications can include aberrant phosphorylation. Tumor cell specific post-translational modifications may also include splice antigens produced by the proteasome.

In one embodiment, the neoantigen is a short peptide that binds to class I or class II MHC receptors, thereby forming a ternary complex that can be recognized by T cells that carry a matching T cell receptor that binds to the MHC/peptide complex with appropriate affinity. Peptides that bind to MHC class I molecules are typically about 8-14 amino acids in length. T cell epitopes bound to MHC class II molecules are typically about 12-30 amino acids in length. In the case of peptides binding to MHC class II molecules, the same peptide and the corresponding T cell epitope may share a common core fragment, but their total lengths are different due to the different lengths of the flanking sequences upstream of the amino terminus and downstream of the carboxy terminus of the core sequence, respectively. T cell epitopes can be classified as antigens if they elicit an immune response.

The proteins derived from the novel antigens comprise cancer-related modifications. Exemplary modifications include, but are not limited to, cancer-related mutations and cancer-related phosphorylation patterns.

The term "mutation" refers to a change or difference (nucleotide substitution, addition or deletion) in a nucleic acid sequence as compared to a reference. "somatic mutations" can occur in any cell of the body other than germ cells (sperm and ovum) and therefore are not inherited to children. These changes may (but are not always) result in cancer or other diseases. Preferably, the mutation is a non-synonymous mutation. The term "nonsubstantial mutation" refers to a mutation, preferably a nucleotide substitution, which does result in an amino acid change, such as an amino acid substitution in a translation product.

According to the present invention, the term "mutation" includes point mutation, indel, fusion, chromosome disruption and RNA editing.

According to a specific embodiment, the mutation is a point mutation-i.e. a single amino acid substitution.

According to the present invention, the term "Indel" describes a specific class of mutations, defined as mutations that result in co-localized insertions and deletions of nucleotides as well as a net increase or loss of nucleotides. In the coding region of the genome, unless the length of the insertion deletion is a multiple of 3, they generate frame shift mutations. Insertion deletions can be compared to point mutations; point mutations are a form of substitution that replaces one of the nucleotides when indels insert and delete nucleotides from the sequence. In one embodiment, the insertion deletion is a frameshift deletion mutation. In another embodiment, the indels are frameshift insertion mutations.

Fusion can result in a hybrid gene formed from two previously isolated genes. It may be the result of a translocation, an intermediate deletion or a chromosomal inversion. Typically, the fusion gene is an oncogene. An oncogene may result in a gene product that has a new or different function than the two fusion partners. Alternatively, the protooncogene is fused to a strong promoter, thereby exerting an oncogenic function through upregulation by the strong promoter of the upstream fusion partner. Oncogenic fusion transcripts may also be caused by trans-splicing or read-through events.

According to the present invention, the term "chromosome disruption" refers to a genetic phenomenon in which specific regions of the genome are disrupted and then stitched together by a single destructive event.

According to the present invention, the term "RNA editing" refers to a molecular process in which the information content of an RNA molecule is changed by chemical changes in the base composition. RNA editing includes nucleoside modifications such as cytidine (C) to uridine (U) and adenosine (a) to inosine (I) deamination, as well as non-template nucleotide additions and insertions. RNA editing in mRNA effectively alters the amino acid sequence of the encoded protein to a different amino acid sequence than predicted by the genomic DNA sequence.

Preferably, the mutation is a non-synonymous mutation, preferably a non-synonymous mutation of a protein expressed in a tumor or cancer cell.

In a specific embodiment, the protein that expresses the cancer-related modification pattern is expressed in a melanoma cell, a lung cancer cell, a kidney cancer cell, or a head and neck squamous cell carcinoma cell.

Preferably, the protein expressing the cancer-related modification pattern is expressed in melanoma cells.

Preferably, the protein expressing a cancer-related modification pattern is a human protein.

Examples of proteins that may express cancer-related modification patterns include proteins of RAS family members, such as neuroblastoma RAS virus (V-RAS) oncogene homolog (NRAS; uniProtKB-P01111), kirsten rat sarcoma virus oncogene homolog (KRAS; uniProtKB-P01116), and Harvey rat sarcoma virus oncogene homolog (HRAS, uniProtKB-P01112).

NRAS variants of particular concern include Q61K, Q61R, Q L and Q61H.

Another example of a cancer-associated mutein is RAF kinase-such as B-RAF UniProtKB-P15056.

Specific B-RAF variants include V600E, V600M, G466E, H725Y, K E and V600G.

Other examples include, but are not limited to: kallikrein 4, papilloma virus binding factor (PBF), melanoma preferential expression antigen (PRAME), wilms tumor-1 (WT 1), hydroxysteroid dehydrogenase-like 1 (HSDL 1), mesothelin, cancer testis antigen (NY-ESO-1), carcinoembryonic antigen (CEA), p53, human epidermal growth factor receptor 2/neuroreceptor tyrosine kinase (Her 2/Neu), cancer-associated epithelial cell adhesion molecule (EpCAM), ovarian cancer and uterine cancer antigen (CA 125), folate receptor a, sperm protein 17, tumor-associated differentially expressed gene-12 (TADG-12), mucin-16 (MUC-16), L1 cell adhesion molecule (L1 CAM), mannan-MUC-1, human endogenous retrovirus K (HERV-K-MEL), north lung cancer antigen-1 (Kita-kyushu lung cancer antigen-1, KK-LC-1), human cancer/antigen (KM-HN-1), cancer testis antigen (LAGE-1), melanoma antigen-A1 (Sp-1), human tumor-antigen-X-4, transient glycoprotein (human tumor-cell adhesion antigen-X-2), transient glycoprotein (MAG-2), transient glycoprotein-4, transient glycoprotein-E-2, transient tumor-surface antigen-X-4 (human tumor-tumor antigen-X-4) and transient glycoprotein-receptor glycoprotein-4 (human tumor antigen-E), ENAH), mammaglobin-a, NY-BR-1, breast cancer antigen (BAGE-1), B melanoma antigen, melanoma antigen-A1 (MAGE-A1), melanoma antigen-A2 (MAGE-A2), mucin K, synovial sarcoma, X breakpoint 2 (SSX-2), paclitaxel resistance-associated gene-3 (TRAG-3), avian myelomatosis virus oncogene (C-myc), cyclin B1, mucin 1 (MUC 1), p62, survivin, lymphocyte common antigen (CD 45), dickkopf WNT signaling pathway inhibitor 1 (DKK 1), telomerase, kirsten rat sarcoma virus oncogene homolog (K-ras), G250, intestinal carboxyesterase, alpha fetoprotein, macrophage colony stimulating factor (M-CSF), prostate Specific Membrane Antigen (PSMA), caspase 5 (CASP-5), cytochrome C oxidase assembly factor 1 homolog (COA-1), O-linked beta-N-acetyltransferase (O-62-d-glucose 62-38d), OGT), osteosarcoma expansion 9, endoplasmic reticulum lectin (OS-9), transforming growth factor beta receptor 2 (TGF-betaRII), murine leukemia glycoprotein 70 (gp 70), calcitonin-related polypeptide alpha (CALCA), programmed cell death 1 ligand 1 (CD 274), murine double minute 2 homolog (mdm-2), alpha-actin 4, extension factor 2, maltase 1 (ME 1), nuclear transcription factor Y subunit C (NFYC), G antigen 1,3 (GAGE-1, 3), melanoma antigen-A6 (MAGE-A6), cancer testis antigen XAGE-1B, prostate six transmembrane epithelial antigen 1 target (STEAP 1), PAP, prostate Specific Antigen (PSA), fibroblast growth factor 5 (FGF 5), heat shock protein hsp70-2, melanoma antigen-A9 (MAGE-A9), arg specific ADP-ribosyl transferase family C (ARTC 1), B-Raf protooncogene (B-RAF), serine/threonine kinase, beta-catenin, cell division cycle 27 homolog (CDC 27) cyclin-dependent kinase 4 (CDK 4), cyclin-dependent kinase 12 (CDK 12), cyclin-dependent kinase inhibitor 2A (CDKN 2A), casein kinase 1 alpha 1 (CSNK 1A 1), fibronectin 1 (FN 1), growth arrest specificity 7 (GAS 7), non-metastatic melanoma glycoprotein B (GPNMB), HAUS Augmin-like complex subunit 3 (HAUS 3), LDLR-fucosyltransferase, T-cell recognized melanoma antigen 2 (MART 2), myostatin (MSTN), melanoma-associated antigen (mutation) 1 (MUM-1-2-3), poly (A) polymerase gamma (neo-PAP), myosin class I, and methods of making and using the same, protein phosphatase 1 regulatory subunit 3B (PPP 1R 3B), peroxiredoxin-5 (PRDXS), receptor tyrosine protein phosphatase K (PTPRK), transforming protein N-Ras (N-Ras), retinoblastoma-related factor 600 (RBAF 600), sirtuin-2 (SIRT 2), SNRPD1, triose phosphate isomerase, albino-1 type protein (OA 1), RAS oncogene family member (RAB 38), tyrosinase-related protein 1-2 (TRP-1-2), melanoma antigen gp75 (gp 75), tyrosinase, melan-A (MART-1), glycoprotein 100 melanoma antigen (gp 100) N-acetylglucosamine transferase V Gene (GnTVF), lymphocyte antigen 6 Complex Gene locus K (LY 6K), melanoma antigen-A10 (MAGE-A10), melanoma antigen-A12 (MAGE-A12), melanoma antigen-C2 (MAGE-C2), melanoma antigen NA88-A, paclitaxel resistance associated protein 3 (TRAG-3), BDZ-binding kinase (pbk), caspase 8 (CASP-8), sarcoma antigen 1 (SAGE), breakpoint cluster region-Aberson oncogene (BCR-ABL), fusion protein in leukemia, dek-can, extension factor Tu GTP-containing binding domain 2 (Elongation Factor Tu GTP Binding Domain Containing, EFTUD 2), ETS variant 6/acute myeloid leukemia fusion protein (ETV 6-AML 1), FMS-like tyrosine kinase-3 internal tandem repeat (FLT 3-ITD), cyclin-A1, fibronectin type III domain protein 3B antibody (FDNC 3B), promyelocytic leukemia/retinoic acid receptor alpha fusion protein (pml-RARalpha), melanoma antigen-C1 (MAGE-C1), membrane protein alternative splice isomer (D393-CD 20), melanoma antigen-A4 (MAGE-A4), or melanoma antigen-A3 (MAGE-A3).

Other examples of proteins that can express cancer-related modification patterns are known in the art and are described, for example, in the following documents: reuschenbach et al, tumor immunization and immunotherapy (Cancer immunol. Immunother) 58:1535-1544 (2009); parmiani et al, J.Nat.cancer institute (J.Nat.cancer Inst.) 94:805-818 (2002); zarour et al, cancer medicine (2003); bright et al, human vaccine and immunotherapy (hum. Vaccine. Immunother.) 10:3297-3305 (2014); wurz et al, tumor medical treatment progression (ter. Adv. Med. Oncol.) 8:4-31 (2016); criscitiello, breast treatment (Breast Care) 7:262-266 (2012); chester et al, J.ImmunotherCancer 3:7 (2015); li et al, molecular medicine report (mol. Med. Report) 1:589-594 (2008); liu et al, J.Hematol.Oncol.3:7 (2010); bertino et al, international biomedical research (biomed. Res. Int.) 731469 (2015); and Suri et al, J.gastrointestinal oncology journal (World J. Gastrointest. Oncol.) 7:492-502 (2015).

In one embodiment, the mutation is a cancer specific somatic mutation.

Methods for detecting sequence changes are well known in the art and include, but are not limited to, DNA sequencing, electrophoresis, enzyme-based mismatch detection assays, and hybridization assays, such as PCR, RT-PCR, rnase protection, in situ hybridization, primer extension, southern blotting, northern blotting, and dot blot analysis.

Sequence changes in a particular gene can also be determined at the protein level using, for example, chromatography, electrophoresis methods, immunodetection assays (e.g., ELISA and western blot analysis), and immunohistochemistry.

In one embodiment, the step of identifying cancer specific somatic mutations or identifying sequence differences comprises using Next Generation Sequencing (NGS).

In one embodiment, the step of identifying cancer specific somatic mutations or identifying sequence differences comprises sequencing genomic DNA and/or RNA of the tumor sample.

To reveal cancer specific somatic mutations or sequence differences, it is preferred to compare sequence information obtained from a tumor sample with a reference, such as sequence information obtained from nucleic acid (e.g., DNA or RNA) sequencing of normal non-cancerous cells (e.g., germ cells) that may be obtained from a patient or from a different individual. In one embodiment, the normal genomic germline DNA is obtained from Peripheral Blood Mononuclear Cells (PBMCs).

The term "genome" relates to the total amount of genetic information in the chromosome of an organism or cell.

The term "exome" refers to a portion of the genome of an organism formed by exons, which are the coding portions of expressed genes. The exome provides a genetic blueprint for the synthesis of proteins and other functional gene products. It is the most functionally relevant part of the genome and therefore it is most likely to contribute to the phenotype of an organism. It is estimated that the exome of the human genome accounts for 1.5% of the total genome (Ng, P C et al, journal of public science library genetics (PLoS Gen.) 4 (8): 1-15,2008).

The term "transcriptome" refers to a collection of all RNA molecules produced in a cell or cell population, including mRNA, rRNA, tRNA and other non-coding RNAs. Transcriptome in the context of the present invention refers to the collection of all RNA molecules produced in one cell, a population of cells, preferably a population of cancer cells, or all cells of a given individual at a certain point in time.

According to the present invention, a "reference" may be used to correlate and compare results obtained from tumor samples in the methods of the present invention. In general, a "reference" may be obtained from a patient or one or more different individuals, preferably healthy individuals, in particular individuals of the same species, based on one or more normal samples, in particular samples not affected by a cancer disease. The "reference" may be determined empirically by testing a sufficient number of normal samples.

According to the invention, mutations may be determined using any suitable sequencing method, preferably Next Generation Sequencing (NGS) techniques. Third generation sequencing methods may replace NGS technology in the future to expedite the sequencing steps of the method. For purposes of clarity: in the context of the present invention, the term "next generation sequencing" or "NGS" refers to all new high throughput sequencing technologies that read nucleic acid templates in parallel randomly along the entire genome by dividing the entire genome into small fragments, in contrast to the "conventional" sequencing method known as Sanger chemistry.

Methods for identifying disease-specific phosphorylation patterns are known in the art and include, for example, amino acid Stable Isotope Labeling (SILAC), RRPA, and phosphate-specific western blotting in cell culture.

In a preferred embodiment, the HLA allele is a class I HLA allele. In specific embodiments, the HLA class I allele is an HLA-A allele or an HLA-B allele. In a preferred embodiment, the HLA allele is a class II HLA allele. The sequences of class I and class II HLA alleles can be found in the IPD-EVIGT/HLA database. Exemplary HLA alleles include, but are not limited to: a.01:01, A.02:01, A.02:03, A.02:04, A.02:07, A.03:01, A.24:02, A.29:02, A.31:01, A.68:02, B.35:01, B.44:02, B.44:03, B.51:01, B.54:01 or B.57:01. In specific embodiments, the HLA allele is HLA-a 01:01.

The subject-specific HLA allele or HLA genotype of a subject can be determined by any method known in the art. In a specific embodiment, the HLA genotype is determined by any of the methods described in international patent application number PCT/US2014/068746 published as WO2015085147 at 2015, 6, 11. Briefly, the method includes determining a polymorphic genotype, which may include generating an alignment of reads extracted from a sequencing dataset with a reference set of genes comprising allelic variants of the polymorphic gene, determining a first posterior probability or posterior probability derivative score for each allelic variant in the alignment, identifying the allelic variant having the greatest first posterior probability or posterior probability derivative score as the first allelic variant, identifying one or more overlapping reads that are aligned with the first allelic variant and one or more other allelic variants, determining a second posterior probability or posterior probability derivative score for the one or more other allelic variants using a weighting factor, identifying the second allelic variant by selecting the allelic variant having the greatest second posterior probability or posterior probability derivative score, the first and second allelic variants defining the genotype of the polymorphic gene, and providing an output of the first and second allelic variants.

Preferably, a cancer-associated mutein is selected that has a high frequency (e.g., at least greater than 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000 or more) in a predetermined number of cancer patients in the context of an individual HLA allele.

The cancer patient group may have the same cancer type, melanoma, or may be part of a pan-cancer group with multiple different cancer types.

The cancer patient group may include a melanoma patient, a thyroid cancer patient, a pheochromocytoma patient, a seminoma patient, a gastric adenocarcinoma patient, a cholangiocarcinoma patient, a pancreatic cancer patient, a colorectal adenocarcinoma, a leukemia patient, a bladder urothelial cancer patient, an endometrial cancer patient, a thymus epithelial tumor patient, a non-small cell lung cancer patient, a sarcoma patient, an ovarian cancer patient, and a prostate cancer patient, or any combination of the above cancer patients.

In one embodiment, the cancer patient group includes only melanoma cancer patients.

In another embodiment, the cancer patient group includes those patients who have been shown to be resistant to a particular therapy.

It should be appreciated that HLA status may have a high frequency in the group and/or a high frequency of specific mutations in the group. Preferably, the frequency of HLA status is high (e.g., greater than 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%), and the frequency of specific mutations in the group is also high (e.g., greater than 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%).

In a specific embodiment, the proportion of tumor cells containing the cancer-associated mutein (i.e., clonality) is also considered during the initial screening stage. Thus, candidate peptides that bind to HLA alleles with an affinity above a first predetermined level and with a frequency above a second predetermined level are selected as candidate HLA presentation neoantigens, said candidate peptides being comprised in a proportion of tumor cells above a predetermined level, which candidate peptides can be targeted in cancer immunotherapy treatment.

In one embodiment, the measure of clonality is the Variant Allele Frequency (VAF).

In one embodiment, the variant allele frequency is the sum of the detection sequences (particularly reads) covering the mutation site and carrying the mutation divided by the sum of all detection sequences (particularly reads) covering the mutation site. In one embodiment, the variant allele frequency is the sum of the mutant nucleotides at the mutation site divided by the sum of all nucleotides determined at the mutation site. In one embodiment, to determine the fraction used to determine the proportion of tumor cells containing the cancer-associated mutant protein, the fraction of the expression level of the modification-related protein is multiplied by the fraction of the frequency of modification proteins in the modification-related protein.

In general, the value of the variant allele frequency can be from 0 to 1, where a variant allele frequency of 0 indicates that there are no sequence reads with a substitution allele at that position and a variant allele frequency of 1 indicates that all sequence reads have a substitution allele at that position. In other embodiments, other ranges and/or values of variant allele frequencies may be used.

Thus, for example, in one embodiment, a protein is considered mutated when the average VAF in a tumor sample is between 0.1 and 0.55. In another embodiment, a protein is considered mutated when the median VAF is between 0.1 and 0.55 in a tumor sample.

Once the HLA allele and the particular mutein are selected, peptides of 8-14 amino acids in length (which contain the selected mutation) derived from the selected cancer-associated mutein can be analyzed for binding affinity to the selected HLA allele.

Methods for assaying binding affinity of peptides to HLA alleles are known in the art.

In one embodiment, a predictive algorithm of HLA binding may be used to predict binding affinity. Such predictive algorithms include, but are not limited to: netMHC, netMHC II, netMHCpan, IEDB analytical resources (URL immuneepitope (-) org), RANKPEP, PREDEP, SVMHC, epipredict, HLABinding, etc. (see, e.g., journal of immunology (J Immunol Methods) 2011; 374:1-4).

Using this prediction, a list of candidate neoantigens can be generated that bind to HLA with an affinity above a predetermined amount. According to a specific embodiment, only candidate peptides that bind at% rank.ltoreq.0.5 (default parameters of NetMHCpan) are selected, or the corresponding levels are selected using different predictive algorithms. According to another embodiment, candidate peptides are selected whose binding can be characterized as% rank.ltoreq.2 (default parameters of NetMHCpan) or corresponding levels using different predictive algorithms.

It will be appreciated that if the HLA allele frequency and/or mutation frequency is high, the binding affinity may be lower than the above levels.

Thus, it will be appreciated that the combination of the three parameters of frequency of HLA alleles, frequency of occurrence of mutations and binding affinity together determine the selection of candidate neoantigens, not just based on a single parameter. Thus, the predetermined amount for any one parameter is not a fixed amount, but is variable, and may vary depending on the level of the other two parameters.

After candidate neoantigens are determined using the above procedure, the candidate neoantigens are tested in an in vitro system to further narrow down the list of possible candidates.

In vitro systems typically comprise antigen presenting cells genetically modified to express candidate neoantigens and their cognate HLAs.

Antigen Presenting Cells (APCs) are cells that present peptide fragments of protein antigens associated with their HLA (MHC) molecules on their cell surfaces.

Examples of APCs include, but are not limited to, dendritic cells, macrophages, langerhans cells, and B cells.

According to a specific embodiment, the APC is a dendritic cell or B cell. Most preferred are B cells.

In one embodiment, the APCs are immortalized, i.e., transformed cell lines, such as Epstein Barr Virus (EBV) transformed B cells.

In one embodiment, APCs are genetically modified to express specific HLA alleles that are predicted to bind candidate neoantigens.

HLA-deficient B cells (e.g., B721.221) can be used in order for the system to "clear" irrelevant HLA.

An exemplary method for deleting/inactivating endogenous class I or class II genes in antigen presenting cells expressing non-associated HLA alleles is CRISPR-Cas9 mediated genome editing.

Candidate peptides are typically expressed in cells using a 25-27mer minigene system (minigene system) and/or a 45-47mer minigene system.

In this way, various lengths of the elongate peptide around the identified mutation are expressed in the cell. Upon expression, the endogenous proteasome system of the cell digests the extended peptide into a shorter length peptide, such that the neoantigen appears on the cell surface in the context of HLA.

Minigenes comprise nucleic acid molecules which encode extended neoantigens. In any of the above embodiments, the expression construct may comprise tandem minigenes, wherein each minigene comprises a nucleic acid molecule encoding a neoantigen. For example, a tandem minigene may comprise a nucleic acid molecule encoding 2 to about 20 neoantigens, or 2 to about 10 neoantigens, or 2 to about 5 neoantigens.

The use of in vitro systems ensures that the neoantigen is expressed at a much higher level than endogenous, and thus it is easier to identify specific peptides presented by HLA using mass spectrometry and other similar techniques including, but not limited to, thin layer chromatography, electrophoresis, in particular capillary electrophoresis, solid phase extraction (CSPE), reversed phase high performance liquid chromatography, amino acid analysis after acid hydrolysis and Fast Atom Bombardment (FAB) mass spectrometry, as well as MALDI and ESI-Q-TOF mass spectrometry.

In one embodiment, analysis may be performed using liquid chromatography and tandem mass spectrometry (LC-MS/MS) and/or HPLC-see, e.g., kalaora et al, tumor target (oncotarget), month 2016, 2; 7 (5): 5110-5117, the contents of which are incorporated herein by reference.

After confirming that candidate neoantigens are presented by the cognate HLA, the candidate peptide and HLA pairs are validated using tumor cells (e.g., fresh tumor cells, PDX, or tumor cell lines). According to a specific embodiment, the candidate peptide and HLA pair is demonstrated in an in vivo system using tumor cells from cancer patients known to have associated HLA status.

The presentation of neoantigens by specific HLA's was confirmed in tumor cells using methods known in the art. For example, peptides can be acid eluted from tumor cells or from tumor immunoprecipitated HLA molecules and then identified using mass spectrometry. During the identification process, the incorporated heavy peptide (candidate neoantigen) can be introduced into the system as a control. This also helps to quantify neoantigens in tumor samples.

The reactivity of the selected neoantigens can then be assessed as described further below.

In one embodiment, the candidate peptides are loaded onto the APC under conditions that allow them to be presented on the APC surface.

For presentation on the surface of APCs, they must cross the APC cell membrane and load onto newly synthesized HLA class I or class II receptors. The HLA-peptide complexes formed are transported to the cell membrane where they are readily recognized by T cells.

In one embodiment, the peptide is incubated with the APCs in a medium (e.g., RPMI) that maintains the APCs in a live state for 12-48 hours, 12-24 hours, 6-48 hours, or 8-48 hours. The concentration of peptide during the loading phase is preferably between 10 μm and 50 μm, more preferably between 10 μm and 30 μm.

Next, activation of cd4+ or cd8+ T cells may be assayed. Methods for detecting specific T cell activation include detecting proliferation of T cells, production of cytokines (e.g., lymphokines, interferon gamma, tnfα), or production of cytolytic activity. For cd4+ T cells, a preferred method of detecting specific T cell activation is to detect proliferation of T cells. For cd8+ T cells, a preferred method of detecting specific T cell activation is to detect the production of cytolytic activity.

According to a specific embodiment, to determine the reactivity of peptides, an ELISPOT assay can be performed in which CD8+ CTL responses, which can be assessed by measuring IFN-gamma production by antigen specific effector cells, are quantified by measuring the number of Spot Forming Units (SFU) under a stereo microscope (Rinsland et al, (2000) J.Immunol.methods (J Immunol Methods): 240 (1-2): 143-155). In this assay, antigen Presenting Cells (APC) are immobilized on the plastic surface of a microtiter well and are labeled with different effectors: target proportion is added to effector T cells. The antigen presenting cells are preferably B cells or dendritic cells. Binding of APCs to antigen-specific effector cells triggers cytokine production by effector cells, including IFN-gamma (Murali-Krishna et al, (1998) experimental medicine and biological progress (Adv Exp Med biol.): 452:123-142). In one embodiment, the subject-specific T cells are used in an ELISPOT assay. The amount of soluble ifnγ secreted from TIL can also be measured by ELISA assay (e.g., biolegend).

Another method of determining the reactivity of peptides is by direct measurement of cell lysis, as measured by classical assays of CTL activity (i.e., chromium release assays) (Walker et al, (1987) Nature: 328:345-348); scheibenbogen et al, (2000) journal of immunological methods (J Immunol Methods): 244 (1-2): 81-89). Effector Cytotoxic T Lymphocytes (CTLs) target class I MHC with antigenic peptides and signal the target for apoptosis. If used before adding CTL ⁵¹ Chromium-labeled targets, released into the supernatant ⁵¹ The amount of Cr is proportional to the number of targets killed. Antigen-specific lysis is calculated by comparing lysis of target cells expressing disease or control antigens in the presence or absence of patient effector cells, and is typically expressed as% specific lysis. The percent specific cytotoxicity was calculated by (specific release-spontaneous release)/(maximum release-spontaneous release), and may be 20% to 85% for positive assays. The percent specific cytotoxicity is typically determined at several ratios of effector (CTL) to target cells (E: T). Furthermore, standard lysis assays are qualitative, and must rely on Limiting Dilution Analysis (LDA) for quantitative results, whereas LDA often underestimates the true level of CTL responses. Although CTLs can kill many targets in vivo, in vitro, the assay requires C The number of TL is equal to or greater than the number of detectable killed targets. In one embodiment, CTL responses are measured by a chromium release assay, monitoring the ability of T cells (effector cells) to lyse radiolabeled HLA-matched "target cells" expressing the appropriate antigen-MHC complex.

As described above, the inventors previously predicted that HLA-A 01/NRAS.Q61K hotspot neoantigen is a powerful immunogenic target, associated with thousands of patients per year-WO 2020/234875. The present inventors have now identified a T cell receptor that is highly reactive to such HLA-presented neoantigens.

In particular, the TCR has been shown to identify ILDTAG at concentrations as low as 1nMKEEY (SEQ ID NO: 1) and ILDTAGREEY (SEQ ID NO: 34) and induces 4-1BB upregulation upon co-incubation with related cancer cell lines, such as melanoma cell lines and lung adenocarcinoma cell lines.

Thus, according to a first aspect of the present invention there is provided a method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a population of T cells, wherein at least 10% of the T cells in the population have the CDR3 amino acid sequence as shown in SEQ ID No. 6 and the β chain of the CDR has the CDR3 amino acid sequence as shown in SEQ ID No. 7, thereby treating cancer in the subject.

Examples of cancers include, but are not limited to, melanoma, colon cancer, breast cancer, thyroid cancer, gastric cancer, colorectal cancer, leukemia, bladder cancer, lung cancer, ovarian cancer, breast cancer, and prostate cancer.

In one embodiment, the cancer is a RAS-related cancer, such as an oncogene homolog (NRAS; uniProtKB-P01111) -related cancer-e.g., Q61K, Q R NRAS.

In one embodiment, the cancer is a metastatic cancer.

T cells for use in treating the cancers described herein may be derived from a subject suffering from cancer (adoptive cell therapy-ACT). In one embodiment, the T cells are derived from peripheral blood lymphocytes of the subject.

In one embodiment, the T cell is an autologous T cell.

In another embodiment, the T cell is a non-autologous T cell.

ACT refers to the transfer of cells (most commonly immune-derived cells) back into the same patient or a new recipient host, with the aim of transferring immune functions and features into the new host. The use of autologous cells, if possible, may help the recipient minimize the GVHD problem. Adoptive transfer of autologous Tumor Infiltrating Lymphocytes (TIL) (Besser et al, (2010) clinical cancer research (Clin. Cancer Res) 16 (9) 2646-55; dudley et al, (2002) Science 298 (5594): 850-4; and Dudley et al, (2005) journal of clinical oncology (Journal of Clinical Oncology) 23 (10): 2346-57) or genetically redirected peripheral Blood mononuclear cells (Johnson et al, (2009) Blood (Blood) 114 (3): 535-46; and Morgan et al, (2006) Science) 314 (5796) 126-9) have been used to successfully treat patients with advanced solid tumors, including melanoma and colorectal cancer, as well as patients with hematological malignancies expressing CD19 (Kalos et al, (2011) journal of scientific conversion medicine (Science Translational Medicine) 3 (95): 95ra 73). In one embodiment, the TCR is selected for administration to a subject based on binding to a neoantigen identified herein. In one embodiment, T cells are expanded using methods known in the art. Expanded T cells expressing the tumor-specific TCR can be returned to the subject. In another embodiment, PBMCs are transduced or transfected with a polynucleotide for expressing a TCR and administered to a subject. T cells expressing the neoantigen-specific TCR are expanded and administered back to the subject.

The T cell population expressing T cell receptors on its surface binds to at least one peptide epitope having the sequence shown in SEQ ID NOs 1 and 34 and has antigen specificity for the corresponding peptide. In one embodiment, the T cells have antigen specificity for both of the peptide epitopes shown in SEQ ID NOS 1 and 34.

As used herein, the phrase "antigen-specific" refers to a TCR that can specifically bind and immunospecifically recognize a mutated target, such as mutated NRAS or BRAF, with high affinity. For example, when co-cultured with target cells as follows: (a) Antigen negative HLA-A 01:01 pulsed with low concentration of mutant target peptide ⁺ Target cellsThe mutant target peptide has the sequence set forth in SEQ ID No. 1 or 34 (e.g., about 0.05ng/mL to about 5ng/mL, 0.05ng/mL, 0.1ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, or a range defined by any two of the foregoing values) or (b) antigen negative HLA-A 01:01 ⁺ Target cells into which a nucleic acid encoding a polypeptide having the sequence as set forth in SEQ ID NO:1 or 34 such that the target cell expresses the mutant target, a TCR can be considered "antigen-specific" for the mutant target if the T cell expressing the TCR secretes IFN- γ of at least about 200pg/mL or more (e.g., 200pg/mL or more, 300pg/mL or more, 400pg/mL or more, 500pg/mL or more, 600pg/mL or more, 700pg/mL or more, 1000pg/mL or more, 5,000pg/mL or more, 7,000pg/mL or more, 10,000pg/mL or more, 20,000pg/mL or more, or a range defined by any two of the foregoing values). Cells expressing TCRs of the invention were pulsed with higher concentrations of mutant target peptides at antigen negative HLA-A 01:01 ⁺ IFN-gamma may also be secreted when the target cells are co-cultured.

Alternatively or additionally, when co-cultured with target cells as follows: (a) Antigen negative HLA-A 01:01 pulsed with low concentration of mutant target peptide ⁺ Target cells, or (b) antigen negative HLA-A 01:01 ⁺ Target cells into which a nucleotide sequence encoding a mutant target peptide has been introduced such that the target cells express the mutant target, a TCR can be considered "antigen-specific" for the mutant target if the T cell expressing the TCR secretes at least twice the amount of IFN- γ as expressed by the negative control. The negative control can be, for example, (i) a T cell expressing a TCR co-cultured with a target cell as follows: (a) Antigen negative HLA-A 01:01 pulsed with the same concentration of unrelated peptide (e.g., some other peptide of a different sequence than the mutated target peptide) ⁺ Target cells, or (b) antigen negative HLA-A 01:01 ⁺ Target cells into which a nucleotide sequence encoding an unrelated peptide has been introduced such that the target cells express the unrelated peptide, or (ii) non-transduced T cells (e.g., derived from PBMCs that do not express a TCR) co-cultured with target cells that are: (a) Antigen negative HLA-A 01:01 pulsed with the same concentration of mutant target peptide ⁺ Target cells, or (b) antigen negative HLA-A 01:01 ⁺ A target cell into which a nucleotide sequence encoding a mutant target is introduced such that the target cell expresses the mutant target. IFN-gamma secretion can be measured by methods known in the art, such as an enzyme-linked immunosorbent assay (ELISA).

Alternatively or additionally, when co-cultured with target cells as follows: (a) Antigen negative HLA-A 01:01 pulsed with low concentration of mutant target peptide ⁺ Target cells or (b) antigen negative HLA-A 01:01 ⁺ Target cells into which a nucleotide sequence encoding a mutant target is introduced such that the target cells express the mutant target, a TCR can be considered "antigen-specific" for the mutant target if the T cells expressing the TCR secrete at least twice the amount of IFN- γ as expressed by the negative control. The concentration of peptide and negative control may be as described herein with respect to other aspects of the invention. The number of IFN-gamma secreting cells can be measured by methods known in the art, such as ELISPOT.

Methods of engineering T cells to express recombinant T cell receptors for cancer treatment are disclosed in the following documents: ping et al, protein Cell, month 3 2018, 9 (3): 254-266.

The present invention provides T cells expressing TCRs comprising two polypeptides (i.e., polypeptide chains), such as the alpha chain of the TCR, the beta chain of the TCR, the gamma chain of the TCR, the delta chain of the TCR, or a combination thereof. The polypeptides of the TCRs of the invention may comprise any amino acid sequence, provided that the TCRs have antigen specificity for a mutated target (e.g., mutated NRAS).

In one embodiment of the invention, a TCR comprises two polypeptide chains, each comprising a variable region comprising Complementarity Determining Regions (CDRs) 1, CDR2 and CDR3 of the TCR.

Like the CDRs, the TCRs disclosed herein also include V and J regions. Specific combinations of V and J regions are listed in table 1 below.

The sequence of the CDR3 region of an exemplary beta chain of a T cell receptor that can be used according to this aspect of the invention is given in SEQ ID NO. 7.

The sequence of the CDR3 region of an exemplary alpha chain of a T cell receptor that can be used according to this aspect of the invention is given in SEQ ID NO. 6.

The present invention relates to a population of T cells, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the T cells in the population express a T cell receptor having a β chain CDR3 amino acid sequence as set forth in SEQ ID No. 7 and an α chain CDR3 amino acid sequence as set forth in SEQ ID No. 6.

In one embodiment, at least 95% of the T cells in the population express T cell receptors having the beta chain CDR3 amino acid sequence shown in SEQ ID NO:7 and the alpha chain CDR3 amino acid sequence shown in SEQ ID NO: 6.

The inventors further contemplate CAR-T cells, i.e. T cells expressing chimeric antibodies having the CDR3 amino acid sequences shown in SEQ ID nos. 6 and 7.

It will be appreciated that the sequence of the CDR3 region may comprise at least one or even two amino acid substitutions and retain binding activity.

In one embodiment, the amino acid substitution is a conservative substitution.

The term "conservative substitution" as used herein refers to the substitution of a naturally occurring or non-naturally occurring amino group or a peptidomimetic having similar steric properties for an amino acid found in a natural sequence in a peptide. When the side chain of the natural amino acid to be substituted is polar or hydrophobic, conservative substitutions should be with naturally occurring amino acids, non-naturally occurring amino acids, or with peptide mimetic moieties that are also polar or hydrophobic (except for having the same steric properties as the side chain of the amino acid being substituted).

Since naturally occurring amino acids are generally grouped by their nature, conservative substitutions of naturally occurring amino acids can be readily ascertained, while considering that substitution of charged amino acids with sterically similar uncharged amino acids is considered to be a conservative substitution in accordance with the present invention.

Amino acid analogs (synthetic amino acids) well known in the art may also be used in order to create conservative substitutions by non-naturally occurring amino acids. Peptide mimics of naturally occurring amino acids are described in detail in the literature known to those skilled in the art.

When conservative substitutions are affected, the substituted amino acid should have the same or similar functional groups in the side chain as the original amino acid.

As used herein, the phrase "non-conservative substitution" refers to the substitution of an amino acid present in a parent sequence with another naturally or non-naturally occurring amino acid having different electrochemical and/or spatial properties. Thus, the side chain of the substituted amino acid may be significantly larger (or smaller) than the side chain of the substituted natural amino acid and/or may have a functional group with significantly different electronic properties than the substituted amino acid. Examples of non-conservative substitutions of this type include substitution of phenylalanine or cyclohexylmethylglycine for alanine, substitution of isoleucine for glycine, or substitution of-NH-CH [ (-CH) ₂ ) ₅ -COOH]-CO-substituted aspartic acid. Those non-conservative substitutions that fall within the scope of the present invention are those that still constitute peptides with antibacterial properties.

According to a specific embodiment, the TCR receptor comprises an alpha chain comprising the CDR3 region as shown in SEQ ID NO. 6 and a beta chain comprising the CDR3 region as shown in SEQ ID NO. 7.

According to another embodiment, the TCR receptor comprises an alpha chain comprising a CDR3 region as shown in SEQ ID NO. 6 and a beta chain comprising a CDR3 region as shown in SEQ ID NO. 3, 5, 7, 9, 11, 13, 15 or 17.

According to another embodiment, the TCR receptor comprises a beta chain comprising a CDR3 region as shown in SEQ ID NO 7 and an alpha chain comprising a CDR3 region as shown in SEQ ID NO 2, 4, 6, 8, 10, 12, 14 or 16.

According to a specific embodiment, the TCR receptor comprises an alpha chain comprising a CDR3 region as shown in SEQ ID NO. 2 and a beta chain comprising a CDR3 region as shown in SEQ ID NO. 17.

According to a specific embodiment, the TCR receptor comprises an alpha chain comprising the CDR3 region as shown in SEQ ID NO. 16 and a beta chain comprising the CDR3 region as shown in SEQ ID NO. 3.

Isolated antibodies and/or diabodies comprising at least one CDR sequence as described herein are also contemplated.

TCRs (and antibodies) of the invention may comprise synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexane carboxylic acid, norleucine, α -amino-N-decanoic acid, homoserine, S-acetamidomethyl-cysteine, trans-3-and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β -phenylserine, β -hydroxyphenylalanine, phenylglycine, α -naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid monoamide, N ' -benzyl-N ' -methyl-lysine, N ' -dibenzyl-lysine, 6-hydroxylysine, ornithine, α -aminocyclopentane carboxylic acid, α -aminocyclohexane carboxylic acid, α -aminocycloheptane carboxylic acid, α - (2-amino-2-norbornane) -carboxylic acid, α, γ -diaminobutyric acid, α, β -diaminopropionic acid, homophenylalanine and α -tert-butylglycine.

The TCRs (and antibodies), including functional variants thereof, of the invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, via, for example, a disulfide bridge (disulfide bridge), or converted to an acid addition salt and/or optionally dimerized or polymerized, or conjugated.

The TCRs (and antibodies) of the invention may be obtained by methods known in the art, for example de novo synthesis. Furthermore, TCRs can be recombinantly produced using standard recombinant methods using the nucleic acids described herein. See, e.g., green and Sambrook, molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), 4 th edition, cold spring harbor Press, cold spring harbor, new York (2012). Alternatively, TCRs, polypeptides and/or proteins described herein (including functional variants thereof) can be synthesized commercially by companies such as Synpep (dublin, california), peptide Technologies corp (gaisephsburg, maryland) and Multiple Peptide Systems (san diego, california). In this regard, TCRs of the present invention may be synthetic, recombinant, isolated and/or purified. Included within the scope of the invention are conjugates, such as bioconjugates comprising any of the inventive TCR conjugates, and methods of synthesizing conjugates in general are known in the art.

The tumor-reactive T cell population expressing the subject-specific TCR can be combined with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition comprising a personalized cell population of tumor-reactive T cells. Preferably, the carrier is a pharmaceutically acceptable carrier. With respect to the pharmaceutical composition, the carrier may be any carrier conventionally used for cellular administration. Such pharmaceutically acceptable carriers are well known to those skilled in the art and are readily available to the public. Preferably, the pharmaceutically acceptable carrier is one that does not have deleterious side effects or toxicity under the conditions of use. Suitable pharmaceutically acceptable carriers for the injected cells may include any isotonic carrier, for example, physiological saline (about 0.90% w/v aqueous NaCl, about 300mOsm/L aqueous NaCl, or about 9.0g NaCl/liter water), normosol R electrolyte solution (Abbott, chicago, ill.), PLASMA-LYTE A (Baxter, dirofield, ill.), about 5% dextrose in water, or ringer's lactate. In one embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

T cells may be administered by any suitable route known in the art. Preferably, the T cells are administered as an intra-arterial or intravenous infusion, which preferably lasts about 30 to 60 minutes. Other examples of routes of administration include intraperitoneal, intrathecal and intralymphatic. T cells may also be administered by injection. T cells can be introduced at the tumor site.

T cells described herein are autologous or non-autologous to the subject.

For the purposes of the present invention, the dose administered (e.g., the number of cells in a cell population of the invention that expresses a subject-specific TCR) should be sufficient to effect, for example, a therapeutic or prophylactic response in the subject within a reasonable time frame. For example, the number of cells should be sufficient to bind to the cancer antigen, or detect, treat, or prevent cancer, for about 2 hours or more, e.g., 12 to 24 hours or more, from the time of administration. In some embodiments, the time period may be even longer. The number of cells will be determined by, for example, the efficacy of the particular cell and the condition of the subject (e.g., human) and the weight of the subject (e.g., human) to be treated.

Numerous assays for determining the number of cells administered from a cell population of the invention expressing a subject-specific TCR are known in the art. For the purposes of the present invention, an assay may be used to determine the initial amount administered to a mammal, which comprises comparing the extent to which target cells are lysed or the extent of secretion of one or more cytokines such as IFN-gamma and IL-2 after a given number of such cells have been administered to a subject. The extent to which target cells are lysed, or the extent to which cytokines such as IFN-gamma and IL-2 are secreted, after administration of a certain number of cells, can be determined by methods known in the art. Secretion of cytokines such as IL-2 may also provide an indicator of the quality (e.g., phenotype and/or effectiveness) of a cell preparation.

The number of cells administered from a cell population of the invention that expresses a subject-specific TCR can also be determined by the presence, nature, and extent of any adverse side effects that may accompany administration of the particular cell population.

The subject that can be treated is typically a mammalian subject-e.g., a human.

In one embodiment, the subject has been pre-selected based on whether the subject expresses nras.q61k or nras.q61r mutations and has been identified as having an HLA-A x 01:01 allotype.

The subject-specific HLA allele or HLA genotype of a subject can be determined by any method known in the art. In a specific embodiment, the HLA genotype is determined by any of the methods described in international patent application number PCT/US2014/068746 published as WO2015085147 at 2015, 6, 11. Briefly, the method includes determining a polymorphic genotype, which may include generating an alignment of reads extracted from a sequencing dataset with a reference set of genes comprising allelic variants of the polymorphic gene, determining a first posterior probability or posterior probability derivative score for each allelic variant in the alignment, identifying the allelic variant having the greatest first posterior probability or posterior probability derivative score as the first allelic variant, identifying one or more overlapping reads that are aligned with the first allelic variant and one or more other allelic variants, determining a second posterior probability or posterior probability derivative score for the one or more other allelic variants using a weighting factor, identifying the second allelic variant by selecting the allelic variant having the greatest second posterior probability or posterior probability derivative score, the first and second allelic variants defining the genotype of the polymorphic gene, and providing an output of the first and second allelic variants.

The T cell populations disclosed herein can be used in combination with at least one other therapeutic agent. Examples of therapeutic agents that may be used in conjunction with the T cells disclosed herein include, but are not limited to: immunomodulatory cytokines including, but not limited to, IL-2, IL-15, IL-7, IL-21, GM-CSF and any other cytokine capable of further enhancing an immune response; immunomodulatory antibodies, including, but not limited to, anti-CTLA 4, anti-CD 40, anti-41 BB, anti-OX 40, anti-PD 1, and anti-PDL 1; and immunomodulatory drugs including, but not limited to, lenalidomide (revlimit).

In addition, the T cell populations disclosed herein can be administered in combination with chemotherapy for cancer treatment in a regimen that does not inhibit the immune system, including but not limited to low doses of cyclophosphamide and paclitaxel. The vaccine may also be administered in combination with therapeutic antibodies for cancer, including but not limited to anti-HER 2/neu (herceptin) and anti-CD 20 (rituximab).

The T cell population may be administered in combination with drugs for treating a particular type of infection, including but not limited to antiviral drugs, antiretroviral drugs, antimalarial drugs, etc., for treating chronic infections

In one embodiment, the agent of this aspect of the invention is administered with an immune checkpoint inhibitor.

As used herein, the phrase "immune checkpoint inhibitor" refers to a compound capable of inhibiting the function of an immune checkpoint protein. Inhibition includes reduced function and complete blockage. In particular, the immune checkpoint protein is a human immune checkpoint protein. Thus, the immune checkpoint protein inhibitor is preferably an inhibitor of a human immune checkpoint protein. Immune checkpoint proteins are described in the art (see, e.g., pardoll,2012, cancer Nature Rev. Cancer) 12:252-264. Immune checkpoint this name includes experiments that stimulate antigen receptor-triggered T lymphocyte responses by inhibiting immune checkpoint proteins in vitro or in vivo, e.g., mice deficient in expression of immune checkpoint proteins exhibit enhanced antigen-specific T lymphocyte responses or signs of autoimmunity (e.g., disclosed in Waterhouse et al, 1995, science 270:985-988; nishimura et al, 1999, immunization (immunoy) 11:141-151). It may also include agents that demonstrate inhibition of antigen receptor triggered cd4+ or cd8+ T cell responses due to intentional stimulation of immune checkpoint proteins in vitro or in vivo (e.g., zhu et al, 2005, natural immunology (Nature immunol.) 6:1245-1252).

Preferred inhibitors of immune checkpoint proteins are antibodies that specifically recognize immune checkpoint proteins. Many CTLA-4, PD1, PDL-1, PD-L2, LAG-3, BTLA, B7H3, B7H4, tim3 and KIR inhibitors are known, and similar to these known immune checkpoint protein inhibitors, alternative immune checkpoint inhibitors may be developed in the future (soon). For example, yiprimer (ipilimumab) is a fully human CTLA-4 blocking antibody, currently sold under the name YIrvoy (Bristol-Myers Squibb). The second CTLA-4H is Tiximumab (see Ribas et al, 2013, J.Clin. Oncol.) 31:616-22. Examples of PD-1 inhibitors include, but are not limited to: humanized antibodies that block human PD-1, such as palbociclizumab (lambrolizumab) (e.g., disclosed in WO2008/156712; hamid et al, new England J. Med.) 369:134-144 2013, hPD A and its humanized derivatives h409A11, h409A16 and h409A 17), or Pidilizumab (disclosed in Rosenblatt et al, 2011, immunotherapy J. Immunothether.) 34:409-18), and fully human antibodies, such as Nafiumab (previously referred to as MDX-1106 or BMS-936558, topalian et al 2012, new England J. Med.) 366:2443-2454, disclosed in U.S. Pat. No. 8,008,449 B2. Other PD-1 inhibitors may include presentation of soluble PD-1 ligands, including but not limited to PD-L2 Fc fusion proteins, also known as B7-DC-Ig or AMP-244 (disclosed in Mkrtichyan M et al, J immunol. J. Immunol. No..) 189:2338-47 2012) and other PD-1 inhibitors currently being studied and/or developed for use in therapy. Furthermore, immune checkpoint inhibitors may include, but are not limited to: humanized or fully human antibodies blocking PD-L, such as MEDI-4736 (disclosed in WO2011066389A 1), MPDL3280A (disclosed in U.S. Pat. No. 8,217,149 B2) and MIH1 (Affymetrix available via eBioscience (16.5983.82)) and other PD-L1 inhibitors currently under investigation. According to the invention, the immune checkpoint inhibitor is preferably selected from CTLA-4, PD-1 or PD-L1R, e.g. from CTLA-4, PD-1 or PD-L1R (moprimma, tiximab, larvicizumab), nivolumab, pilizumab, AMP-244, medi-4736, MPDL3280A, MIH 1) known as described above. Known inhibitors of these immune checkpoint proteins may be used as such or analogues, in particular chimeric, humanized or human forms of antibodies may be used.

As used herein, the term "about" refers to ± 10%.

The terms "including (comprises, comprising, includes, including)", "having (having)" and their cognates mean "including but not limited to".

The term "consisting of … …" is intended to be "including and limited to".

The term "consisting essentially of … …" means that the composition, method, or structure can include additional ingredients, steps, and/or portions, provided that the additional ingredients, steps, and/or portions do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "compound" or "at least one compound (at least one compound)" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description of the range format is merely for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges as well as individual values within the range. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within the range, e.g., 1, 2, 3, 4, 5, and 6. Regardless of the breadth of the range, is applicable.

Whenever numerical ranges are indicated herein, it is intended to include any reference number (fractional or integer) within the indicated range. The expressions "a range between the first indicator number and the second indicator number" and "a range from the first indicator number to the second indicator number" are used interchangeably herein and are meant to include the first indicator number and the second indicator number and all numbers and integers therebetween.

As used herein, the term "method" refers to means, techniques, and procedures for accomplishing a given task including, but not limited to, those means, techniques, and procedures known to, or readily developed from, practitioners of the chemical, pharmacological, biological, biochemical, and medical arts.

As used herein, the term "treating" includes cancelling, substantially inhibiting, slowing or reversing the progression of a disorder, substantially ameliorating the clinical or aesthetic symptoms of the disorder. In one embodiment, the method is used to substantially prevent the appearance of clinical or aesthetic symptoms of a disorder.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered as essential features of such embodiments unless the embodiment is not operable in the context of the absence of such elements.

Various embodiments and aspects of the invention as described above and as claimed in the claims section below are experimentally supported in the following examples.

Examples

Reference is now made to the following examples, which together with the above description illustrate some embodiments of the invention in a non-limiting manner.

Materials and methods

Data driven analysis for recurrent neoantigen candidate prioritization

HLA/mutation co-occurrence analysis using patient data

TCGA temporary cohort data was used to accumulate a total of 8038 patients ^37,61,62 . High resolution HLA-I typing of 9176 TCGA patients previously disclosed was also obtained. The crossover of these two sources resulted in a total of 6840 patients of mutations and HLA annotation groups (cohort), of which 368 are melanoma patients. Mutation and HLA data from 11033 patients with cancer (including 221 melanoma patients) were used and treated as previously described. Samples of HLA types are annotated based on their ras.q61 status. NRAS (ENST 00000369535), KRAS (ENST 00000256078) and HRAS (ENST 00000451590) Ensembl V101 at 31 in 8 in 2020, and their protein coding sequence coordinates are obtained from Gencode V35 at 30 in 8 in 2020. The previously disclosed MELA-AU dataset was obtained, containing 69 melanoma patients. For each of these three data sets we calculated a list of pan-carcinomas and melanomas for all HLA-I alleles that occurred with the ras.q61 mutation. For each HLA/mutation combination we calculated its frequency in melanoma and carcinoma. For the TCGA dataset, HLA-I frequencies between ras.q61 mutations and the general population (fisher exact test with FDR correction) were compared.

NetMHCpan prediction

We used netMHCpan 4.0 to predict ras.q61 derived novel peptides. Using the protein sequence of NRAS we extracted a 27 amino acid long stretch flanking position 61. This sequence is conserved among RAS variants; thus, it is representative of NRAS, KRAS and HRAS. Four mutant variants were constructed by substituting glutamine 61 with arginine, lysine, leucine or histidine. All five versions were packaged in FASTA format and taken as input to netMHCpan along with a compiled list of HLA alleles that occurred simultaneously with the ras.q61 mutation in the analyzed patient database. The algorithm is set up to scan for binding peptides of 8-14 amino acids in length, where peptides ranked at 0.5.ltoreq.rank.ltoreq.2 are considered weak binders, and those peptides with a% rank.ltoreq.0.5 are considered strong binders. The output was filtered, leaving only peptides spanning position 61. By enumerating predicted novel peptides, each HLA/mutation combination was assigned a "best% Rank" score.

Tumor tissue, cell line, TIL and PBMC

Metastatic melanoma cell lines 17T, 35T, 108T and their cognate TIL products were established. As previously described ⁶ The established TIL was amplified via a rapid amplification protocol (REP).

Commercial melanoma cell line SK-MEL-30 (ACC-151) was purchased from DSMZ. Other tumor cell lines: hut78, hs940T and calu6, and HB95 (W6/32) and HB145 (IVA 12) hybridoma cells were purchased from ATCC. Healthy donor leukocyte preparations for study were purchased from MDA (national blood service center in israel). PBMCs were extracted from these samples by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare), stored frozen in aliquots in FCS/10% DMSO and thawed as required for the experiment.

Hybridoma cells HB95 and HB145 were used to purify the pan-HLA-I and pan-HLA-ll antibodies to prepare HLA affinity columns. All cell lines were tested regularly and found to be negative for mycoplasma contamination (EZ-PCR mycoplasma kit, biological Industries).

Melanoma cell lines were cultured in complete RPMI medium consisting of RPMI supplemented with 10% FCS, 2mM L-glutamine, 2.5% HEPES, 100IU/ml penicillin and 0.1mg/ml streptomycin. B-LCL is cultured in B cell medium consisting of complete RPMI medium supplemented with 1mM sodium pyruvate. TIL and PBMC were cultured in T cell culture medium consisting of human serum AB supplemented with 10% heat inactivation, 2mM glutamate, 100U/ml penicillin, 0.1mg/ml streptomycin and 5.5X10 ^-5 RPMI of Mbeta-mercaptoethanolComposition is prepared. Prior to use in downstream assays, TIL was thawed and recovered in T cell medium containing 6000IU/ml IL-2 (Proleukin) for three days. At 7 days prior to the experiment, PBMC were thawed and T cells were selected in T cell medium containing 300IU/ml IL-2 and 50ng/ml OKT3 anti-CD 3 antibody (# 317304, bioleged). On days 3-4 after thawing, PBMC were subcultured in T cell medium supplemented with 300IU/ml IL-2. T cell culture medium was used for co-incubation in caspase-3 killing and neoantigen induced amplification assays. Complete RPMI medium was used for one night for TIL/B-LCL and PBMC/B-LCL co-incubation.

Structural modeling of HLA-A.times.01:01 in Complex ILDTAGKEEY (SEQ ID NO: 1)

De-peptide (PDB: 6at 9) was used as a receptor for ALK tyrosine kinase ¹ The crystal structure of the complexed HLA-A 01:01 models the structure of HLA-A 01:01 complexed with ras.61 derived peptides. The crystallographically bound peptide was mutated manually to produce ILDTAGKEEY (SEQ ID NO: 1) and ILDTAGQEEY (SEQ ID NO: 35) peptides that were complexed with HLA receptors. The HLA structure is truncated to the peptide binding domain (chain a, residues 1 to 180). The resulting peptide-HLA structure serves as the starting conformation for peptide docking and molecular dynamics simulation.

Using freely accessible network server interfaces FlexPepdock ² 、ClusPro ³ And DINC ⁴ Peptide docking was performed. Furthermore, the GROMACS2018.3.5 version and the GROMOS 54a7 are used to combine the atomic force field ⁶ Molecular dynamics simulation was performed. Placing the composite in a rhombic dodecahedron tank, wherein the minimum distance between the solute and the tank wall is And solvated with SPC water. The charge of the system was neutralized by adding a 5na+ counterion. By minimizing the elimination of steric hindrance, up to 5,000 steps are performed using the steepest descent algorithm. The system was equilibrated at constant volume and temperature (NVT ensemble), with all protein and peptide heavy atoms limited to 100ps at 10K, followed by further 100ps without limitation at 300K. At 300K, byThe system pressure was balanced by modeling 300ps at constant atmospheric pressure (NPT ensemble). In all balancing steps, the LINCS algorithm is used ⁷ Positional restrictions are imposed on protein residues. The final coordinates generated by the equilibrium were used to start (ILDTAGKEEY (SEQ ID NO: 1) and ILDTAGQEEY (SEQ ID NO: 35)) 5 independent production runs of the system, each run for 500ns in the NPT system. Temperature coupling using speed rescaling (velocity rescaling thermostat) ⁸ And a time constant of 0.1ps to keep the temperature constant at 300K, and Parrinello-Rahman potentiostat was used ⁹ And a time constant of 0.2ps keeps the pressure of the system constant at 1 bar. The motion of the system is integrated using a time step of 2 fs. Using a particle grid Ewald ¹⁰ The method calculates long-range static electricity, while the short-range cut-off value of the vdW and coulomb interactions is set to 1.0nm. In order to focus on the binding conformation, only the N-and C-termini of RAS peptides were analyzed in the HLA B-and F-pockets, respectively>Internal conformation. The distance between the following atoms was measured: HLA Tyr171 side chain hydroxyl Oxygen (OH) and peptide Ile1 backbone amide nitrogen (N), and HLA Tyr123 side chain phenolic Carbon (CZ) and peptide Tyr10 side chain gamma Carbon (CG). Using PyMOL version 1.3 ¹¹ The molecular structure is visualized. Conformations from docking and from the binding mimetic framework aggregate through HLA-peptide hydrogen bonding interactions. Using MDTraj ¹³ Wernet-Nilsson Standard implemented in edition 1.9.2 ¹² Hydrogen bonding was detected. The cluster centroid is identified as the simulated framework in which the corresponding hydrogen bond fingerprint has the lowest Manhattan distance (Manhattan/Cityblock distance) to all cluster members.

RAS.Q61 mutant status and HLA typing of tumor tissue

For 17T and 108T, ras.q61 status and HLA typing were both extracted from whole exome sequencing; using PolySolver software ¹⁴ HLA typing is determined. MM121224 and MM150414, HLA and mutation data are provided by their contributors. For 135T and quick frozen tumors, RAS.Q61 status was informed by its contributor, while genomic DNA extracted using the QIAGEN DNeasy Blood and Tissue kit (# 69504) was used for HLA typing (GenDx SBTexcellerator HLA-A reagent) Kit #4100234 or MX6-1NGS typing kit, # 7371464). From "TRON cell line Inlet" ^14,15 Mutations and HLA data were extracted for the cell lines SK-MEL-30, MZ2-MEL, huT78, hs940T and calu 6. HLA typing of EBV-transformed B cells by IHWG cells and DNA pool ¹⁶ Providing.

cDNA sequencing

Total RNA was extracted from melanoma cell lines 17T, 135T, SK-MEL-30, MM121224, MZ2-MEL, huT78, calu6 and Hs940T using the RNeasy Mini kit (# 74104, QIAGEN) according to the manufacturer's protocol and eluted with 30. Mu.l of distilled water treated with diethyl pyrocarbonate (DEPC). The iScript reverse transcription supermix for the RT-qPCR kit (# 1708841, biorad) was used according to the manufacturer's protocol, using a total of 500ng RNA for single stranded complementary DNA (cDNA) synthesis. The NRAS and KRAS regions containing position 61 were amplified by PCR using the following primers:

NRAS forward: 5'-TTGGAGCAGGTGGTGTTGGG-3', (SEQ ID NO: 36);

NRAS reverse: 5'-GTATCAACTGTCCTTGTTGGC 3' (SEQ ID NO: 37);

KRAS forward: 5' -TAAACTTGTGGTAGTTGGAGCTGGT-3, (SEQ ID NO: 38); and

KRAS reverse: 5'-TTCTTTGCTGATTTTTTTCAA-3' (SEQ ID NO: 39).

Mu.l of cDNA was used for PCR reactions and mixed with 2 XKAPA HIFI (#KM2605, KAPA Biosystems) to a final volume of 25. Mu.l using standard PCR procedures with the following parameters: one cycle at 95℃for 3 minutes; at 98 ℃,20 seconds and 35 cycles; an annealing temperature of 58 ℃ is maintained for 30 seconds; and maintained at 72℃for 1 minute. The PCR products were separated on a 1% agarose Gel and then purified by Wizard SV Gel and PCR Clean-Up System (#A9281, promega), followed by Sanger sequencing using a 3730DNA Analyzer (ABI). The sequencing primer is identical to the PCR primer. Sequencing results were analyzed using snapge software (version 4.3.2).

Establishment of double transfectants 721.221

DNA sequences encoding HLA-A 01:01 and 25-mer minigenes (RAS.Q61K and RAS.Q61R) were designed with flanking Xbal and Notl restriction sites at the 5 'and 3' ends, respectively. These designs purchased synthetic dsDNA from Twist bioscience or IDT, optimized for human expression by the respective company's platform. PCR amplified templates were cloned restrictively into lentiviral vectors pCDH-CMV-MCS-EF 1. Alpha. -Neo (SBI, #CD514B-1) and pCDH-CMV-MCS-EF 1. Alpha. -GreenPuro (SBI, #CD513B-1) using XbaI/NotI (NEB, #R0145L and #R0189L). HLA and minigene inserts were ligated into neomycin and puromycin vectors using T4 DNA ligase (NEB, #M0202L) to generate pCDH-Neo-A.times.01:01, pCDH-Puro-GFP-mRAS.Q61K and pCDH-Puro-GFP-mRAS.Q61R, respectively. Lentiviral particles were generated using sequence verified cloning plasmids by co-transfection with the envelope and packaging of plasmids PMD2.G and psPAX2 into HEK293T cells using Lipofectamine 2000 (Invitrogen, # 11668027) as per manufacturer's instructions. The harvested virus containing supernatant was filtered, aliquoted and stored at-80 ℃. Cells of HLA-I deficient human B-LCL 721.221 were progressively infected, first expressing HLA-A 01:01, and then infected with a minigene virus (RAS.Q61K or RAS.Q61R). 48-72 hours post infection, selection was started with 800. Mu.g/ml neomycin (G418) or 1. Mu.g/ml puromycin. Infected cells were propagated continuously under selection. About two weeks after infection, high-efficiency overexpression of HLA-A 01:01 was verified by flow cytometry using monoclonal antibody W6/32. After confirming HLA expression, 721.221a x 01:01 cells were infected as described above and selected to induce minigene overexpression. 721.221a.01:01; ras.q61k and 721.221a.01:01; ras.q61r cells were grown in medium containing both selection agents for about two weeks, then propagated in medium without antibiotic for two more weeks, and then HLA-peptide histology amplified.

HLA peptide group science

Preparation and purification of membrane HLA molecules

Collection from 2X 10 ⁸ Cell pellet consisting of individual cells and lysed on ice using lysis buffer containing 0.25% sodium deoxycholate, 0.2mM iodoacetamide, 1mM EDTA, 1:200 protease inhibitor Cocktail (Sigma-Aldrich, P8340), 1mM PMSF and 1% octyl-beta-D glucopyranoside in PBS. The samples were then incubated at 4℃for 1h. The lysate was centrifuged at 48,000g for 60 min at 4℃and then passed through a pre-clarification column (pre-washing column) containing protein-aserod beads.

HLA-I molecules were isolated by hybridization with protein-A Sepharose beads (Thermo-Fisher Scientific, supra) ¹⁷ _, ¹⁸ Covalently bound pan-HLA-I antibodies (W6/32 antibodies purified from HB95 hybridoma cells) were immunoaffinity purified from the clarified lysate. The affinity column was washed with 400mM NaCl, 20mM Tris HCl pH8.0, followed by 20mM Tris HCl, pH 8.0. The HLA peptide and HLA molecule were then eluted with 1% trifluoroacetic acid, followed by separation of the peptide from the protein by binding the eluted fraction (fraction) to Sep-Pak (Waters). Eluting the peptide with 0.1% trifluoroacetic acid containing 28% acetonitrile ¹⁷ 。

Identification of eluted HLA peptides

Liquid chromatography:

cell line 17T, SK-MEL-30 and MM121224: HLA peptides were dried by vacuum centrifugation, dissolved with 0.1% formic acid and separated (resolve) for 180 min with a gradient of 7-40% acetonitrile containing 0.1% formic acid on a capillary column packed with Repro sil C18-Aqua (Dr. Maisch, gmbH, ammerberuch-Entringen, germany) pressure as described above, 0.15. Mu.L/min ¹⁹ . For cell lines 17T, SK-MEL-30 and MM121224, chromatographic analysis was performed using the UltiMate 3000RSLC nano-capillary UHPLC system (Thermo Fisher Scientific) which was used by electrospray in combination with tandem mass spectrometry on Q-exact-Plus. The HLA peptide was eluted using a linear gradient of 5% to 28% acetonitrile containing 0.1% formic acid at a flow rate of 0.15 μl/min for more than 2 hours.

Cell line 721.221a x 01:01; mRAS.Q61K, 721.221A.01:01; mRAS. Q61R, 135T, MZ2-MEL, huT78, hs940T and 4 tumor samples (Mela-183, mela-49, MM-1369 and MM-1319): HLA peptides were dried by vacuum centrifugation and dissolved in 97:3 water in acetonitrile 0.1% formic acid. An anti-phase symmetric C18 trapping column (inner diameter 180 μm, length 20mm, particle size 5 μm; waters) was used. Peptides were isolated using T3HSS nanorods (75. Mu.m inner diameter, 250mm length, 1.8. Mu.m particle size; waters) at 0.35. Mu.L/min with a gradient of 5-28% acetonitrile containing 0.1% formic acid for 120 min. Chromatography was performed using nanoAcquity (Waters), by electrospray in combination with tandem mass spectrometry on Q-exact-Plus (Thermo Fisher Scientific).

Mass spectrometry: cell line 721.221a x 01:01; mRAS.Q61K, 721.221A.01:01; mRAS. Q61R and 17T operate in discovery mode. Cell lines were analyzed using absolute targeted mass spectrometry: aliquots of SK-MEL-30, MM121224 and 135T, 721.221a x 01:01; mRAS.Q61K, 721.221A.01:01; mNAS.Q61R, 17T, MZ2-MEL, huT78, hs940T samples and 4 tumor samples (Mela-183, mela-49, MM-1369 and MM-1319), specifically looked for ILDTAGKEEY (SEQ ID NO: 1) or ILDTAGREEY (SEQ ID NO: 34), and peptides could also be quantified using heavy peptide incorporation. Synthetic heavy isotope labelled ILDTAGKEEY (SEQ ID NO: 1), spiked with heavy lysine (13C 6;15N 4) or ILDTAGREEY (SEQ ID NO: 34) spiked with heavy arginine (13C 6;15N 4) was purchased from JPT in >95% purity.

Discovery mode: data were obtained using the data-dependent "top-10" method, with peptide fragmentation by higher energy collision dissociation. The full-scan mass spectrogram is acquired at a resolution of 70,000 at 200m/z, with a target value of 3×10 ⁶ And (3) ions. Ion accumulation to Automatic Gain Control (AGC) target value 10 ⁵ The maximum implantation time is typically 100 milliseconds. The peptide matching option is set to be preferred. The normalized collision energy was set to 25% and the MS/MS resolution at 200m/z was 17,500. Fragmented m/z values were dynamically excluded from further selection within 20 seconds. Using MaxQuant (1.5.8.3 edition) ²⁰ MS data were analyzed with FDR 0.05. Peptide identification is based on UniProt database ²¹ Human section (2017, month 4) and a custom reference database containing WES identified 17T mutant sequences.

Absolute targeting mode of SK-MEL-30, MM 121224: 0.1pmoL heavy peptide was added to the peptide set sample injected into the mass spectrometer. Analysis was then performed using the PRM method. The inclusion list is imported into the MS/MS acquisition method. The instrument switches between full MS and MS/MS acquisition to fragment ions in the containment list. A full-scan mass spectrum with a resolution of 70,000 was obtained with a mass to charge ratio (m/z) of 350-1,400AMU. The mass of the segment is accumulated to the AGC target 105 with a maximum injection time of 400 ms and a window of 1.8m/z. Analysis reuse MaxQuant software (version 1.5.8.3) ²⁰ And Andromeda search engine ²² . Peptide identification is based on UniProt database ²¹ Person chapter and bag (month 4 2017)Custom reference database containing ILDTAGKEEY (SEQ ID NO: 1) neoantigen. The following parameters were used: precursor ion mass and secondary mass spectrometry error (fragment mass tolerance) 20ppm, false Discovery Rate (FDR) of SK-MEL-30 of 0.05, false discovery rate of MM121224 of 0.3, and variable modification of oxidation (Met), acetylation (protein N-terminus) and heavy lysine (13C 6;15N 2).

721.221a.01:01; mRAS.Q61K, 721.221A.01:01; mRAS. Q61R, 17T, 135T, MZ2-MEL, huT78, hs940T and 4 tumor samples (Mela-183, mela-49, MM-1369 and MM-1319): absolute targeting mode:

the nanoUPLC was connected online to a quadrupole orbitrap mass spectrometer (Q exact Plus, thermo Scientific) using a Flexlon nanospray device (Proxeon) via a nano ESI emitter (10 μm tip; new Objective; volbot, mass.). Data were collected in Parallel Reaction Monitoring (PRM), with one MS1 scan per 10 PRM scans. The MS1 scan range was set to 300-1800m/z, resolution to 70,000, agc to 3e6, and the maximum injection time to 120 MS. The PRM channel acquisition resolution was 35,000, the maximum injection time was 200 milliseconds, AGC was 2e5, NCE was 27, and isolation was 1.7m/z.

Peptide quantification:

the raw PRM data is imported into Skyline ²³ . Absolute quantification is obtained by summing the extracted ion chromatograms of all fragment ions of each peptide and outputting the ratio of the total signal of the native peptide to the re-labelled internal standard of the added sample, multiplied by the amount of internal standard.

Fluorescence-based in vitro killing assay

In this assay, the loss of fluorescence content is used to quantify target cell death ²⁴ . Gfp+ cells were established by lentiviral infection and antibiotic selection. The day before the experiment, fluorescent target cells were seeded at 70% confluence in 48-well plates (1X 10 for 17T and 135T respectively) ⁵ And 5X 10 ⁴ Individual cells/wells) and allowed to adhere. Homologous TIL was added at effector to target ratios ranging from 0:1 to 4:1 and incubated with melanoma for 16 hours. Each case contained triplicate. After incubation, non-adherent TIL and dead target cells were washed out with PBS. UsingThe fluorescence of the remaining live target cells was quantified by a Tecan-M200 microplate reader (plate reader). The fluorescence reading was focused 3mm above the plate surface. The percentage of specific lysis was calculated as 100× (C-X)/C, where C is fluorescence in the absence of TIL and X is fluorescence in the presence of TIL.

Peptide pulsing on B-LCL

Purity of>95% of the synthetic peptides (ILDTAGKEEY (SEQ ID NO: 1), ILDTAGREEY (SEQ ID NO: 34), ILDTAGQEEY (SEQ ID NO: 35)) were purchased from Genscript and dissolved in DMSO. In an upright 15ml conical tube, the B-LCL carrying HLA-A.times.01:01 was set at 1X 10 ⁶ The individual cells/ml are suspended in complete medium at the desired concentration with the peptide of choice (10 ^-11 To 10 ^-5 ) At 37℃5% CO ₂ Is incubated in a humidified incubator for 2-4 hours. DMSO volumes for all samples were kept at 1%. For no peptide control, DMSO without peptide was added. B cells were washed three times in PBS and then co-incubated with TIL.

Analysis of T cell reactivity by IFN gamma release assay

Release of ifnγ from TIL, as measured in an enzyme-linked immunosorbent assay (ELISA), was used to measure reactivity. TIL and homologous melanoma or BLCL in a 1:1 ratio (10 ⁵ Up to 2X 10 ⁶ Individual cells) were CO-cultured in U-bottom 96-well plates at 37℃with 5% CO ₂ Incubate overnight in a humidified incubator. Soluble ifnγ secreted from TIL was quantified from the co-culture supernatant using a Biolegend Human IFN γ ELISA MAX delay (# 430106) and Tecan-M200 microplate reader. All experiments were performed in triplicate biologically.

Flow cytometry analysis and fluorescence activated cell sorting

As staining and analysis buffer for flow cytometry experiments, PBS1% BSA 2mM EDTA was used unless otherwise indicated. Prior to sorting, cells were passed through a 40 μm cell filter (Corning, # 431750). Single cell suspensions were flow cytometry analyzed in BD LSR II (BD Biosciences) or CytoFlex (Beckman Coulter). BD FACSAria III or BD FACSAria II cell sorter (BD Biosciences) was used for fluorescence activated cell sorting. The cells were incubated with antibody/tetramer at 4℃for 30 min in the dark and then washed. Tetramer and TCR β or anti-CD 8 staining were performed sequentially to avoid possible interference.

Tetramers were obtained from NIH tetramer core facilities and calibrated to minimize background staining: HLA-A 01:01/ILDTAGKEEY (SEQ ID NO: 1) (BV 421-conjugated) was used at 1:8000-10000, A01:01/ILDTAGREEY (SEQ ID NO: 34) (FITC-conjugated) was used at 1:4000. The following antibodies were purchased from Biolegend and used at 1:100 dilution: CD3 (PE/Cy 7, # 300316), CD4 (FITC, # 317407), CD8 (APC #300911 or FITC # 301006), 4-1BB (APC # 309809), mouse TCR β constant region (APC, #109211, bioleged), CD19 (BV 421, # 302234). Staining was performed with CellTrace far red, anti-caspase 3 and ifnγ capture antibodies, as described for the cleavage caspase 3 killing and ifnγ secretion assays.

Size and particle size measurements are used to gate a single peak (single) that is feasible. Where indicated, live/dead staining was performed using propidium iodide (invitrogen#p3566). Sorting experiments gated non-overlapping positive and negative subpopulations.

IFN gamma secretion assay

To evaluate the percentage of A.times.01:01/ILDTAGKEEY (SEQ ID NO: 1) reactive TIL, we used the IFNγ secretion assay (Miltenyi Biotec, # 130-090-762) according to the manufacturer's instructions. In this assay, ifnγ -specific antibodies attach to the cell surface and capture secreted cytokines upon release by the cells. The ifnγ molecules bound to the cell surface were then stained as artificial surface molecules and the cells were analyzed by flow cytometry. This approach is complementary to tetramer staining, as the neoantigen-specific cells are identified based on induced reactivity rather than TCR recognition. IHW01161 cells used as APCs in these experiments were pulsed with either NO peptide (DMSO) or 10 μg/ml mutant (ILDTAGKEEY) (SEQ ID NO: 1) or wild-type peptide (ILDTAGQEEY) (SEQ ID NO: 35). TIL was incubated with APC for 4 hours. Peptide-free and wild-type conditions were used as control protocols and allowed for differentiation of background from neoantigen specific reactivity.

Antigen-induced TIL amplification assay

IHW01161 cells, used as APCs, irradiated with 70Gray, washed with PBSNext, the pulses were then performed with 10. Mu.g/ml of the mutant (ILDTAGKEEY) (SEQ ID NO: 1) or wild-type (ILDTAGQEEY) (SEQ ID NO: 35) peptide as described above. Preparation of 2.5X10 in T cell Medium ⁶ Mu.l of each cell/ml of suspension and 100. Mu.l aliquots were dispensed into wells of a 96 well round bottom tissue culture plate. Mutant and wild type pulsed APCs were 2.5X10 respectively ⁶ The individual cells/ml concentration was resuspended in T cell medium. An additional 100. Mu.l of T cell medium or pulsed APC suspension was added to each well. Additional 1 μg/ml mutant or wild-type peptide was added to the mutant/wild-type wells, respectively. Subsequently, the cells were incubated at 37℃with 5% CO ₂ Is incubated in a humidified incubator for a total of 10 days. On the third day of incubation, half of the medium was replaced with fresh T-cell medium containing 100IU/ml IL-2, 50ng/ml IL-7 (Peprotech, # 200-07) and 50ng/ml IL-15 (Peprotech, # 200-15). On day 7 of incubation, half of the medium was replaced again with fresh T-cell medium containing 200IU/ml IL-2, 50ng/ml IL-7 and 50ng/ml IL-15. On day 10 of incubation, cells were harvested, co-stained with anti-CD 4 antibody and A.times.01:01/ILDTAGKEEY (SEQ ID NO: 1) tetramer, and analyzed by flow cytometry.

Batch TCR sequencing (Bulk TCR sequencing)

As previously described, we performed TCR library preparation on sorted TILs ²⁵ . Briefly, RNA was extracted from TIL library and treated with DNase (RNeasy Micro kit (QIAGEN), RQ1 DNase without RNase activity (Promega)). Reverse transcription was then performed using primers directed to the constant region of the TCR α/β chain (SuperScript III, #18080044, invitrogen). A single stranded oligonucleotide consisting of a universal primer region and a Unique Molecular Identifier (UMI) was ligated to the 3' end of the TCR cDNA transcript (T4 RNA ligase, #0204S, NEB). The library was amplified and split into a library of alpha and beta strands fully through three consecutive PCR steps. The library was cycle sequenced 300 times on the NextSeq Illumina platform and processed using the internal pipeline, mainly the reads were: (1) Clustering the readings according to UMI for accurate frequency assessment; (2) Annotation of V and J germline gene segments according to a predetermined library of IMGTs to which TRDV genes have been manually added ²⁶ The method comprises the steps of carrying out a first treatment on the surface of the And (3) determining their CDR3 sequences at the nucleotide and amino acid levels. The output of the annotation consisted of separate sets of alpha and beta chains and was further filtered to exclude non-efficient sequences and single element sequences. 0.5X10 were collected for each experimental condition ⁶ Individual cells. For tetramer sorting experiments, TIL was stained with HLA-A 01/ILDTAGKEEY (SEQ ID NO: 1) tetramer and anti-CD 4 antibody and sorted into: bulk CD 4-and tetramer+ and tetramer-subsets thereof. Sequencing all three populations; the experiment was a biological triplicate.

Single cell RNA and TCR sequencing of cd8+til

TIL: melanoma co-incubation and tetramer sorting

17TIL/17T and 135TIL/135T were mixed at a ratio of 1:1 at 4X 10 per well ⁶ Individual cells were seeded in 24-well tissue culture plates. After one night of co-incubation, the cells were resuspended by pipetting, washed in PBS and then incubated at 7-10X 10 ⁶ Individual cells/ml were frozen in CryoStor CS10 (# 07930,STEMCELL Technologies). On the day of 10x library preparation, cells were thawed, placed in complete medium supplemented with Benzonase (#e8263, millipore Sigma), washed, and then stained in complete medium. Cells were stained with HLA-A.times.01: ILDTAGKEEY (SEQ ID NO: 1) tetramer, followed by staining for CD4 and CD 8. Propidium iodide was used for live/dead staining.

Samples were separated into complete media and CD8+ tetramer+ (CD8+CD4-tetramer+) was separated from the CD8+ tetramer- (CD8+CD4-tetramer-) population. A large number of cd8+ samples were obtained by staining CD4 and CD8 without tetramer staining and sorting the cd8+cd4-population. Immediately after sorting, cells were transferred to 10x library preparation.

10x library preparation and sequencing

Samples were processed immediately after sorting for single cell library construction as described above. Briefly, the sorted cells were washed and resuspended in PBS containing 0.04% Bovine Serum Albumin (BSA) and counted with trypan blue staining. Single cells were captured in droplets by loading onto a chrome controller at a targeted cell recovery of 10,000 cells per sample. Single cell gene expression and TCR enriched libraries were prepared using the Chromium Single Cell 5'V (D) J v 1.1.1 kit (10X Genomics) according to the manufacturer's protocol. Samples were sequenced on an Illumina NovaSeq, using reading 1 of 26bp, an index of 8-bp i7 and reading 2 of 58-bp for the gene expression library, with paired-end reads of 150bp for the TCR library.

Data processing of scRNA and TCR-seq libraries

Reads from the 10x scRNA expression library were aligned with human genome assembly GRCh38 (hg 38) and quantified using cellrange count (10x Genomics,3.1.0 version). The filtered characteristic barcode matrix contained only cell barcodes for further analysis. Single cell gene expression matrices were introduced into R (version 3.6.1) and analyzed using a semat (version 3.1.1). Cells less than 4,000 or more than 20,000UMIS were detected by filtration. In addition, more than 10% of cells with mitochondrial RNA readings were excluded from subsequent analysis.

Single cell TCR reads were aligned with human genome assembled GRCh38 (hg 38) and assembled into a reconstituted TCR consensus sequence using cellrange vdj (10x Genomics,3.1.0 version). Only high efficiency (production) TCRa and TCR sequences were considered for further analysis. Overall, 95.2% of the cells annotated the TCR sequences, and the 37,934 cells paired tcrαβ sequences (91.3%) were detected for 41,542 cells. Only those chains with conventional paired TCR are retained cells of alpha beta or alpha beta are combined, for downstream analysis (34,966 cells; 84.1%). Cells with the same CDR3 αβ amino acid sequence are defined as belonging to the same TCR clone.

Identification of neoantigen-reactive clones:

TCR cloning frequencies between the categorized tetramer+ and tetramer populations were compared for each patient. Clones were considered tetramer-specific if the following conditions were met: (1) Clone frequency in tetramer+ is greater than or equal to 100 times higher than in tetramer-population, and (2) each TCR chain in tetramer+ is enriched by greater than or equal to 100 times per TCR chain in tetramer-population in at least one repeated batch TCR sequencing experiment, the clone is considered tetramer-specific. Based on the above criteria, 12 and 1 neoantigen-reactive clones were defined as tetrameric specific for 17 and 135TIL, respectively. Tetramer-specific clones consisting of at least 5 cells were retained for downstream neoantigen-specific single cell analysis. N17.5 (7 cells), N17.6 (2 cells) and N17.7 (1 cell) are included based on TCR similarity criteria.

Single cell data integration and clustering

To analyze the gene expression program of the neoantigen-reactive clones, tetramer+ populations (filtering neoantigen-reactive clones) and bulk samples of two patients were integrated. Specifically, first, the logarithmic normalization and the variable feature selection based on the variance-stabilizing transformation are performed on each sample, respectively. Anchor points between the datasets were then identified using findsegregatencnchors, with bulk samples as references, and using inverse PCA in 30 dimensions. The samples were then integrated using a 30-dimensional integradata. The integrated expression matrix is then scaled and centered for each feature. Next, the integrated scaling data was linearly reduced in dimension using PCA (excluding TCR and cell cycle genes). For visualization, UMAP projections are calculated using the first 30 principal components. To identify clusters based on gene expression profiles, we performed Shared Nearest Neighbor (SNN) modular optimization clustering using a k-parameter of 20 and a resolution of 0.5.

TCR similarity analysis

For each of three replicates of the batch 17TIL TCR sequencing experiments we compared the tetramer+ library to the batch library. We calculated the ratio between the frequency in the tetramer+ library and the frequency in the tetramer library for each amino acid clonotype. If the sequence is missing from the batch library, we estimate its frequency to be 3×10 ^-5 Slightly below the minimum frequency in the library. We selected sequences with this ratio higher than 1 (i.e., with higher frequencies in the tetramer + population). We focused on the sequences that appear in this population for three replicates of all experiments. This procedure produced 10 α and 11 β chains. Within this population we find a low hamming distance sequence of N135.1. We calculated the sharing level of each beta sequence in the Emerson dataset ²⁷ And according to OLGA ²⁸ The provided standard human model calculates eachProbability of generation of the alpha or beta sequences, OLGA ²⁸ Is a model for estimating the probability of generation of a given CDR3 (α or β). Study of prevalence of HLA-A 01:01 in Emerson dataset was based on DeWitt et al ²⁹ Is a HLA comment of (2).

TCR overexpression in PBMC

This well-established technique is employed with minor modifications, as described below ³⁰ . CDR3 nucleotide sequences use IMGT references ³¹ The appropriate V and J sequences present in (a) are reconstructed and ligated to the mouse constant region as described previously. An optimized mouse constant region sequence was obtained. Each TCR chain is embedded between XbaI/NotI restriction sites. Double stranded DNA sequences were ordered from Twist bioscience and then sequence optimized for human expression using a vendor's platform. TCR chains were restriction cloned into pGEM-4Z-EGFP-a 64. The sequence verified cloning plasmid was linearized by SpeI digestion, transcribed in vitro using AmpliCap-Max T7 High Yield Message Maker kit (#c-ACM 04037, cellScript) and purified using rnaclearup kit (# 23600,Norgen Biotek). The mRNA formed was stored in 5. Mu.g aliquots at-80 ℃. PBMC were electroporated with mixed alpha/beta mRNA at 400V/500. Mu.s using Electrosquare Porator ECM (BTX) every 1X 10 ⁶ Each cell was 10. Mu.g. Electroporation cells were at least two hours of rest before use in downstream experiments. TCR expression was verified by flow cytometry using a mouse constant tcrp staining.

Peptide titration assay

As described above, HLA-A 01:01+B-LCL is used in a concentration range of 10 ^-11 M to 10 ^-5 M was pulsed with the synthetic peptide followed by TIL or electroporated lymphocytes in a 1:1 ratio at 37℃with 5% CO ₂ Is incubated in a 96-well U-shaped plate for 16-20 hours. After co-incubation, the plates were centrifuged at 300 g. To obtain 4-1BB readings, cell pellets were stained for CD19, CD3, CD8 and 4-1BB and analyzed by flow cytometry. The percentage of 4-1BB+ cells in the CD8+ population under different conditions was compared. For secreted ifnγ readings, supernatants were collected and analyzed using human ifnγ ELISA MAX deltaxe (Biolegend, # 430106), as described above.

Caspase-3 cleavage assay

As previously described, intracellular staining of lysed caspase-3 was used to measure cytotoxic T cell-induced apoptosis of melanoma target cells. Briefly, melanoma cells were stained with CellTrace Far Red (Invitrogen, #c 34564) according to the manufacturer's protocol. Then 1X 10 ⁵ The individual labeled melanoma cells and lymphocytes were targeted at a 3:1 effector to target ratio of 5% CO at 37℃ ₂ Co-incubate in 96-well U-plates for 3 hours in a humidified incubator. Following incubation, cells were washed, fixed, permeabilized (permeized) and labeled with an anti-lytic caspase-3-PE antibody using the caspase-3 apoptosis PE kit (BD, # 550914) according to the manufacturer's protocol. The washed cells were analyzed by flow cytometry to determine the percentage of caspase-3+ cells from the celltrace+ target cells. Donor-derived lymphocytes electroporated with the TCR of interest were compared to negative controls of lymphocytes not electroporated with mRNA or melanoma cells alone. The experiments were all three biological replicates.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 9.0.2 version of MacOS (version 3.5.0) and Real Statistics Resource Pack software of Excel (version 7.7.1, www (.) real-statistics (.) com). Comparison of HLA allele frequencies between the pan-carcinoma, melanoma and ras.q61 mutated TCGA populations was performed using a Fisher exact test with FDR correction. The IFNg ELISA, 4-1BB based reactivity and cleaved caspase-3 assay was analyzed using one-way anova and Tukey multiple comparison assay. Two-way analysis of variance is used when multiple assays are performed simultaneously and thus can be reliably compared. Peptide titration assays were analyzed by two-way anova and Sidak multiplex comparison assays. Comparison of single cell phenotype ratios was performed using the chi-square test. Differences in activation scores between TCR clones were analyzed using Wilcoxon rank sum test and Bonferroni multiple hypothesis test correction. Corrected P values of 0.05 or less are considered significant.

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Example 1

Selection of neoantigen candidates

Data driven selection of neoantigen candidates

Nras.q61 is the second highly mutated protein site in melanoma, but not all neoantigens from which it derives relapse as well. To be a "hot spot of interest" neoantigen, the combined HLA allele/mutation frequency should be high in cancer patients. Here we have focused on high potential candidates by analyzing the genetic data of patients, using data driven methods to discover new antigens. Considering only the HLA alleles in the analysis dataset that occur simultaneously with the ras.q61 mutation, we used NetMHCpan to predict binding of 8-14 long ras.q61 derived neopeptides. Using these data, we assigned two scores for each HLA/mutation combination: (1) its frequency in the cluster; and (2) its optimal binding prediction score (i.e., the optimal% grade listed on predicted binding new peptide). We then apply stepwise filtering/sorting-setting the frequency threshold and then ordering the remaining candidates according to their estimated binding potential.

Since the three RAS isoforms (i.e., NRAS, KRAS, and HRAS) have the same N-terminal sequence, we incorporate all of them into our analysis, the four most common ras.q61 amino acid substitutions: the same is true of arginine, lysine, leucine and histidine.

Using the protocol described above, we analyzed three melanoma populations: TCGA, summarizing mutations and HLA-I annotations for 363 melanoma cases; the Hartwig database incubated 221 such patients; and the previously disclosed MELA-AU dataset for 69 patients.

In these groups, 95 (26%), 60 (27%) and 15 (22%) had mutations at ras.q61, respectively. As expected, we found that the most common amino acid substitutions at position 61 were arginine (46-60%) and lysine (35-47%); NRAS is the most abundant RAS isoform mutated at position 61 (96-100% ras.61 mutations).

Comparison of TCGA HLA allele frequencies between patients with ras.q61-mutant and β -wild type melanoma did not show significant prevalence bias (Fisher accurate test for FDR correction).

Plotting the scores of HLA/mutation combinations illustrates how the most promising candidate genes are prioritized. Of these three data sets, HLA-A 01:01 stands out by contributing to two best candidates (HLA-A 01:01/ras.q61r and HLA-A 01:01/ras.q61 k). We therefore selected HLA-A 01:01 for further analysis.

Consistent with its high abundance in the general population, 26-38% of patients in the analyzed cancer group carry HLA-A 01:01 ³ . Importantly, when limited to the ras.61-mutant population, the frequency of alleles was not reduced: 27-40% of patients with RAS.Q61 mutant melanoma possess this gene. HLA-A 01:01 appears in 7-8.7% of melanoma along with ras.q61 mutations. Specifically, in our analysis, 3.3-4.5% and 3-4.3% have HLA-A 01:01/ras.q61r combination and HLA-A 01:01/ras.q61k combination, respectively.

NetMHCpan 4.0 predicts 32 RAS.Q 61-derived neopeptides A.times.01:01, including 9 predicted strong binders. Importantly, the same "classical peptide" ildtagagxeey (SEQ ID NO: 40) is the best binding prediction for ras.q61r and ras.q61k (predicted binding affinities 202.4nM and 218.5nM, respectively), while its less common variants (ras.q61 l and ras.q61 h) are also predicted to bind the a.01:01 allele (58.2 nM and 101.8nM, respectively).

Notably, the pan-carcinoma analysis summarises our melanoma-based candidate priorities. RAS.Q61 mutations are still very common, occurring in 3.3-5.9% of cases of ubiquity (individuals 226/6824 and 123/2091 from TCGA and Hartwig datasets, respectively). Thus, the correlation of HLA-A 01:01/ras.q61 neoantigen will not be limited to melanoma.

Direct identification of HLA-A 01:01/ras.q61 derived neoantigen using a single allele overexpression system

Unlike ras.q61r, no new antigen of highly recurrent HLA-A 01:01/ras.q61k combination has been previously reported. We therefore set out to query positively for HLA-A 01:01 binding neoantigenic landscape with the focus of ras.q61k. To this end, we established a 721.221 HLA-A 01:01 monoallelic cell line which co-overexpressed the 25-mer ras.q61k minigene and performed HLa-peptide histology analyses thereon. This over-expression setting has the advantages of usability and neoantigen amplification while still maintaining processing and presentation of the native antigen. The results of Mass Spectrometry (MS) were analyzed using MaxQuant and queried from the human proteomic dataset (Uniprot), we manually added the minigene sequence. For 721.221 ^{A*01:01；mRAS.Q61K} A total of 2403 peptides were detected. Although NetMHCpan predicted seven different HLA-A-01:01 binding neopeptides of ras.q61k, a single decapeptide ILDTAG was identifiedKEEY (SEQ ID NO: 1). This finding was further verified by comparing the identification spectrum with the spectrum of the synthetic peptide. Interestingly, ILDTAG in the context of HLA-A 01:01KEEY (SEQ ID NO: 1) and closely related ILDTAG REEY (SEQ ID NO: 34) is the best scoring neopeptide prediction for RAS.Q61K and RAS.Q61R, respectively. We quantified the presentation of new peptides in the over-expression system using high sensitivity absolute targeting MS and validated ras.q61 r-derived ILDTAGREEY (SEQ ID NO: 34). For 721.221 ^{A*01:01；mRAS.Q61K} And 721.221 ^{A *01:01；mRAS.Q61R} ,ILDTAGKEEY (SEQ ID NO: 1) and ILDTAGRThe EEY (SEQ ID NO: 34) was detected at concentrations of 3.2850 and 38.2375. Mu. Mol/1X 10, respectively ⁶ Individual cells.

ILDTAGKEEY (SEQ ID NO: 1) is robustly presented on tumors expressing HLA-A 01:01/RAS.Q61K

To further confirm our findings in a malignant background and endogenous mutation/HLA expression, we performed HLA-peptide histology analysis on 17T tumor derived cell lines. Since the previous whole exome study identified 17T melanoma as nras.q61k mutant, HLA typing conferred its HLA-a 01:01+, we used MS in discovery mode to positively identify new antigens, regardless of prediction or previous identification. The analysis was performed as described above, and 17T mutated protein sequences (including nras.q61k variants) were added manually to the human proteome as a database. In fact, of the 2356 peptides, a single new peptide, decapeptide ILDTAG, was detectedKEEY (SEQ ID NO: 1) (FIG. 4).

To investigate the robustness of presentation, we planned a panel of four snap frozen tumors and five additional tumor-derived cell lines, all of which endogenously expressed HLA-A 01:01/nras.q61k combinations. We included Hs940T, an HLA-A 01:01/nras.q61r melanoma cell line in the assay to test ILDTAGREndogenous presentation of EEY (SEQ ID NO: 34). All cell lines were confirmed to express mutated NRAS transcripts.

ILDTAG in all nras.q61 k-mutant samples was identified using HLA-peptide histology with high sensitivity absolute targeting MSKEEY (SEQ ID NO: 1) novel peptides in an amount ranging from 0.1250 to 2.9138. Mu. Mol/1X 10 ⁶ Each cell, 1X 10 per 1mg of tumor tissue was assumed ⁸ Individual cells. ILDTAGREEY (SEQ ID NO: 34) was identified as meeting the expectations of Hs940T ⁴¹ . Importantly, our panel of cell lines HuT78 was derived from T cell lymphomas, indicating cross-cancerous relevance of the identified neoantigens.

We conclude that ILDTAGKEEY (SEQ ID NO: 1) is a robust, naturally processed, novel peptide derived from RAS.Q61K, present in the context of HLA allele A.times.01:01And (3) downwards.

Example 2

Identification of novel TCRs that bind to neoantigens

Results

As shown above, mutant ILDTAGKEEY (SEQ ID NO: 1) was predicted to bind strongly to HLA-A 01:01 and this has been previously verified (WO 2020/234875).

Targeting HLA-A 01:01/ILDTAGKThe TCR of the novel antigen of EEY (SEQ ID NO: 1) has a translation value; ACT using their engineered T cells is expected to be applicable to 3% of melanoma cases and 2.9:1000 pan-cancerous individuals. Tetramer-sorted TILs were subjected to RNA-based TCR sequencing to identify related alpha and beta chains. For each 17 and 135TIL, three cell populations were sequenced separately: batch CD 4-and A.times.01:01/ILDTAGKEEY (SEQ ID NO: 1) -tetramer+ and tetramer-sub-populations. Tetramer+ and tetramer-libraries were compared to extract chains enriched in tetramers (i.e., tetramer-specific), while sequencing a bulk population to place these chains in the background. Sorting and sequencing experiments were performed in three biological replicates and were highly consistent. Although each sequencing experiment revealed hundreds to thousands of different, efficient TCR chains, both 17TIL and 135TIL clearly showed an oligoclonal distribution. Limited to 1% or more of the amino acid sequence in the transcript, 11 beta (12 alpha) chains predominate in the CD4 pool of 17TIL, with 75.4% cumulative coverage of the transcript. Similarly, for 135TIL, six β (7 α) chains account for 92.2% of the CD 4-pool. Tetramer+ and tetramer-subsets also feature an oligoclonal TCR profile. Also of interest are TCR chains with a frequency of transcription enrichment of at least 100 fold (tetramer+)/(tetramer-) and four β and five α chains were found to have neoantigen specificity for 17 TIL. The cumulative frequency of these four beta strands in the tetramer+, batch CD 4-and tetramer-populations was 68.5%, 3% and 0.005%, respectively. Similarly, the cumulative frequency of five alpha chains in the tetramer+, batch CD 4-and tetramer-populations was 68.9%, 3.2% and 0%, respectively. For 135TIL, the single αβ pair met the above criteria (strand NA135.1 and NB 135.1), with frequencies within tetramer+135 TIL of 89.6% and 85%, respectively.

TCR-sequencing of the sorted 4-1bb+til after one night of co-incubation with homologous melanoma confirmed that both chains of interest did participate in the melanoma reactivity pool, with both a and one β chain significantly enriched in the 4-1bb+ subgroup (binomial unilateral assay, corrected with Benjamini Hochberg).

Single cell characterization of TCR library

To further study these TCR libraries and reveal tetramer binding αβ pairing, single cell TCR sequencing was performed. After one night of co-incubation of TIL and syngeneic melanoma (i.e., 17TIL with 17T, 135TIL with 135T), the samples were sorted into the population of interest; double RNA-and TCR-single cell sequencing was then performed using a 10X platform. Similar to batch sequencing experiments, the inventors focused on three TIL populations: batch CD8+ and HLA-A 01:01/ILDTAGKEEY (SEQ ID NO: 1) -tetramer+ and tetramer-sub-populations. Tetramer-specific clones were studied using (tetramer+)/(tetramer-) frequency enrichment.

As expected, the batch and single cell sequencing data are highly consistent. Confirmation of single cell data the candidate strand pairs deduced from bulk TCR sequencing were confirmed by single cell data.

The inventors focused on clones represented by at least 2 cells in a single cell tetramer+sub-population. The major tetramer-specific clones in 17TIL were designated N17.1-5 in descending order of frequency of appearance. The single dominant tetramer-specific clone of 135TIL is designated N135.1. N17.2, N17.4, N17.5 and N135.1 are all alpha beta cells, and N17.3 is an alpha beta cell. Conventional analytical lines consider N17.1 to be an αβ clone. However, as will be further elucidated below, this clone actually has a second alpha chain rearranged with the delta gene TRDV 1. The corresponding TCR chain of the above clone will be named as follows: n { chain type: A/B { TIL:17/135} { clone number } { chain number } ¹ }. For example, clone N17.2 was written as NB17.2, while clone N17.3 was written as NA17.3.2 as the second alpha chain.

The sequences of the receptors are summarized in table 1 below.

TABLE 1

Verification and characterization of the novel antigen binding TCR repertoire

Having identified a number of tetramer-specific T cell clones, the inventors aimed to verify their activity against HLA-A 01:01/ILDTAGKThe individual sensitivity and specificity of the novel antigen of EEY (SEQ ID NO: 1).

To this end, they overexpress the α/β combination of interest in peripheral T cells of healthy donors. As shown in fig. 1A, tetramer binding experiments demonstrated neoantigen binding of N17.2, N17.5 and N135.1 and identified the relevant αβ combination of αβ clone N17.3 (NA17.3.2/NB 17.3). Surprisingly, the αβ pair of the most prominent N17.1 in tetramer-specific clones belonging to 17TIL was not verified, although expression was effective (fig. 1A). As observed in single cell RNA sequencing, the clonal expression of delta gene TRDV1 in this clone allowed us to review TCR sequencing data and found the second alpha chain of N17.1, NA17.1.2 due to the rare event of TRDV1/TRAJ27 recombination ⁴⁶ . NA17.1.2 has been identified in bulk TCR sequencing as the same frequency as NA17.1.1. No other TRDV genes are involved in alpha-chain rearrangement. As expected, the NA17.1.2/NB17.1 strand combination was verified on tetramer staining (FIG. 1A).

The inventors continued to engineer T cells with different TCR and with different concentrations of ILDTAGKEEY (SEQ ID NO: 1) neopeptide or its wild-type counterpart pulsed HLA-A 01:01+B-LCL co-incubation. All five tested TCRs demonstrated neoantigen specificity, as demonstrated by the different reactivity to mutant peptides at the supraphysiological pulse peptide concentration (fig. 1B). Peptide titration experiments further showed that these TCRs were able to efficiently recognize ILDTAG at concentrations ranging from 0.01nM to 10nMKEEY (SEQ ID NO: 1) novel peptides (FIG. 1C). Interestingly, a trend was observed in 17TIL in which there was an inverse relationship between TCR frequency and sensitivity. To further determine the reactivity and cytotoxic ability of TCR to endogenously expressed neoantigens, the inventors co-incubated TCR-engineered T cells with HLA-A 01:01+nras.q61k+ tumor cell lines. Reactivity assays against a panel of tumor cell linesTwo of the most effective TCRs, N17.1.2 and N17.2, summarised the immunopeptidomic results (fig. 2A). In addition, in the case of HLA-A 01:01+KSignificant upregulation of 4-1BB was observed upon co-incubation of the RAS.Q61K+ lung adenocarcinoma cell line calu6, demonstrating HLA-A 01:01/ILDTAGKCross isotype, cross cancer type correlation of the new antigen of EEY (SEQ ID NO: 1) as predicted. All five TCRs tested had cytotoxic ability to express melanoma of the neoantigen as shown by the cleaved caspase 3 assay (fig. 2B).

Since most tetramer bound 17TIL also binds HLA-A 01:01/ILDTAGREEY (SEQ ID NO: 34) -tetramer cross-binds, and N17.1.2 is therefore presumed to possess both of these reactivities. Notably, in fact N17.1.2 proved to be able to recognize ILDTAG at concentrations as low as 1nMREEY (SEQ ID NO: 34) and induces 4-1BB upregulation upon co-incubation with HLA-A 01:01+/NRAS.Q61R+ cell line (FIGS. 1C and 2A). The remaining four TCRs were confirmed not to recognize ILDTAGREEY (SEQ ID NO: 34) novel peptides.

IntrA-And inter-patient sequence fusion of neoantigen-specific T cell receptors

The inventors investigated whether some of the undiscovered alpha/beta chains (as shown in Table 1) are interchangeable. As shown in FIGS. 3A-F, NH1 (NA 135.1/NB 17.5) is a hypothetical clone that causes P for both alpha and beta within a similarity cluster _gen Maximization. NH1 was tested for neoantigen reactivity and validated by tetramer staining and neoantigen-specific ifnγ release (fig. 3A-F). The opposite combination NA17.5/NB135.1 was also verified.

While the present disclosure has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

It is intended that all publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. Furthermore, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. As for the chapter titles used, they should not be construed as necessarily limiting. Furthermore, the entire contents of any one or more priority files of the present invention are incorporated herein by reference in their entirety.

Sequence listing

<110> Yeda research and development Co., ltd (Yeda Research And Development Co. Ltd.)

Adena-Samuir (SAMUELS, yardena)

Neille Friedman (Nir)

Avia-petri (PERI, aviyah)

Angstrom Lei Ci grid Lin Sitan (GREENSTEIN Erez)

Mi Haer A long (ALON, michal)

<120> T CELL receptor for RAS-derived recurrent neoantigen and method for identifying same (T CELL recitos DIRECTED AGAINST RAS-DERIVED RECURRENT)

NEOANTIGENS AND METHODS OF IDENTIFYING SAME）

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<151> 2021-04-29

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<151> 2021-07-19

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Claims (36)

1. A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a population of T cells, wherein at least 10% of the T cells of the population have a CDR3 amino acid sequence as set forth in SEQ ID No. 6 and the β chain of the CDR has a CDR3 amino acid sequence as set forth in SEQ ID No. 7, thereby treating the cancer in the subject.

2. A method of treating cancer in a subject, comprising:

(a) Ascertaining an HLA profile of the subject;

(b) Determining whether the object expresses nras.q61k or nras.q61r; and

(c) When the subject is identified as HLA-A 01/nras.q61k or HLA-A 01/nras.q61r, treating the subject with a therapeutically effective amount of a population of T cells expressing a TCR, wherein the alpha chain of the TCR has a CDR3 amino acid sequence as set forth in SEQ ID NO:6 and the beta chain of the TCR has a CDR3 amino acid sequence as set forth in SEQ ID NO:7, thereby treating the cancer.

3. A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a population of T cells, wherein the α chain of the TCR of the T cells of the population has a CDR3 amino acid sequence as set forth in SEQ ID No. 2, and the β chain of the CDR has a CDR3 amino acid sequence as set forth in SEQ ID No. 17, thereby treating the cancer in the subject.

4. A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a population of T cells, wherein the α chain of the TCR of the T cells of the population has a CDR3 amino acid sequence as set forth in SEQ ID No. 16, and the β chain of the CDR has a CDR3 amino acid sequence as set forth in SEQ ID No. 3, thereby treating the cancer in the subject.

5. The method of claim 3, wherein at least 10% of the T cells in the population have a CDR3 amino acid sequence as set forth in SEQ ID No. 2 and the β chain of the CDR has a CDR3 amino acid sequence as set forth in SEQ ID No. 17.

6. The method of claim 4, wherein at least 10% of the T cells in the population have a CDR3 amino acid sequence as set forth in SEQ ID No. 16 and the β chain of the CDR has a CDR3 amino acid sequence as set forth in SEQ ID No. 3.

7. The method of any one of claims 1 to 6, wherein the TCR binds to a peptide of the sequence shown in SEQ ID No. 1 or SEQ ID No. 34 in a complex having an HLA-A 01:01 allele in the subject.

8. The method of any one of claims 1 to 7, wherein the T cells are autologous to the subject.

9. The method of any one of claims 1 to 7, wherein the T cells are non-autologous to the subject.

10. The method of any one of claims 1-7, wherein the T cell is genetically modified to express the T cell receptor.

11. The method of any one of claims 1 to 10, wherein the T cells comprise cd8+ T cells.

12. The method of any one of claims 1 to 11, wherein the cancer is selected from the group consisting of melanoma, colon cancer, breast cancer, thyroid cancer, gastric cancer, colorectal cancer, leukemia cancer, bladder cancer, lung cancer, ovarian cancer, breast cancer, and prostate cancer.

13. The method of any one of claims 1 to 12, wherein the cancer is melanoma.

14. The method of any one of claims 1 to 13, further comprising treating the subject with a checkpoint inhibitor.

15. An isolated population of T cells genetically modified to express a T Cell Receptor (TCR), wherein the alpha chain of the TCR has a CDR3 amino acid sequence as set forth in SEQ ID No. 6, and the beta chain of the CDR has a CDR3 amino acid sequence as set forth in SEQ ID No. 7.

16. An isolated population of T cells, wherein at least 10% of the α chains of the TCRs of the T cells in the population have a CDR3 amino acid sequence as set forth in SEQ ID No. 6, and the β chains of the CDRs have a CDR3 amino acid sequence as set forth in SEQ ID No. 7.

17. An isolated population of T cells, wherein the α chain of the TCR of the T cells of the population has a CDR3 amino acid sequence as set forth in SEQ ID No. 2, and the β chain of the TCR has a CDR3 amino acid sequence as set forth in SEQ ID No. 17.

18. An isolated population of T cells, wherein the α chain of the TCR of the T cells of the population has a CDR3 amino acid sequence as set forth in SEQ ID No. 16, and the β chain of the TCR has a CDR3 amino acid sequence as set forth in SEQ ID No. 3.

19. The isolated population of T cells of claim 17 or 18, genetically modified to express the TCR.

20. The isolated population of T cells of claim 15 or 16, which are cd8+ T cells.

21. Use of the isolated population of T cells of any one of claims 15 to 20 for the treatment of cancer.

22. A method of selecting a neoantigen that can be presented by a targeted recurrent HLA in the treatment of cancer immunotherapy, the method comprising:

(a) Analyzing the frequency of occurrence of cancer-associated muteins in the context of a single HLA allele in tumor cells of a plurality of cancer patients; and

(b) Determining the binding affinity of a peptide of 8-14 amino acids in length derived from the cancer-associated mutein to the single HLA allele, wherein the peptide comprises a mutation compared to the wild-type protein,

Wherein a candidate peptide that binds with an affinity above a first predetermined level to an HLA allele that occurs more frequently than a second predetermined level is selected as a neoantigen that can be presented by a targeted candidate HLA in the treatment of cancer immunotherapy;

(c) Confirming presentation of the candidate peptide by the HLA allele in an in vitro expression system of antigen presenting cells; and

(d) Confirming that the validated peptide is presented by the HLA allele in tumor cells.

23. The method of claim 22, wherein the determining comprises predicting the binding affinity using a prediction algorithm.

24. The method of claim 23, wherein the predictive algorithm comprises NetMHCpan.

25. The method of claim 22, wherein the confirming is performed using targeted mass spectrometry.

26. The method of any one of claims 22 to 25, wherein the HLA comprises an HLAI class.

27. The method of claim 22, wherein the plurality of patients comprises patients resistant to at least one cancer therapy.

28. The method of claim 22, further comprising analyzing the proportion of tumor cells containing the cancer-associated mutein prior to step (c), wherein a candidate peptide that binds to HLA alleles with an affinity above a first predetermined level and with a frequency above a second predetermined level is selected as a candidate HLA-presented neoantigen, the candidate peptide being comprised in the proportion of tumor cells above the predetermined level, the candidate peptide being targetable in the cancer immunotherapy treatment.

29. The method of claim 22, wherein the in vitro expression system comprises an extended peptide that expresses 25-27 amino acids or 45-47 amino acids, wherein the extended peptide comprises the amino acid sequence of the cancer-associated mutein.

30. The method of claim 22, wherein the antigen presenting cells comprise B cells.

31. The method of any one of claims 22 to 30, wherein the cancer-associated mutein is a member of the RAS family.

32. The method of claim 31, wherein the member is selected from the group consisting of NRAS, KRAS, and HRAS.

33. The method of claim 31, wherein the member is an NRAS.

34. The method of any one of claims 22-30, wherein the cancer-associated mutein is RAF kinase.

35. The method of claim 34, wherein the RAF kinase is B-RAF.

36. The method of any one of claims 22-35, wherein the cancer patient comprises a melanoma patient, a thyroid cancer patient, a pheochromocytoma patient, a seminoma patient, a gastric adenocarcinoma patient, a cholangiocarcinoma patient, a pancreatic cancer patient, a colorectal adenocarcinoma, a leukemia patient, a bladder urothelial cancer patient, an endometrial cancer patient, a thymus epithelial tumor patient, a non-small cell lung cancer patient, a sarcoma patient, an ovarian cancer patient, and a prostate cancer patient.