Hepatocellular carcinoma specific TCR (T cell carcinoma) derived from convergence recombination and application thereof

Abstract

A T cell receptor (TCR) plays a crucial role in adaptive immune response by recognizing specific antigens. In the context of cancer immunotherapy, TCR sequences may be engineered to target specific tumor antigens. In this study, hepatocellular carcinoma (HCC) was simulated in mice by binding to genetic alterations repeatedly observed in human diseases, and HCC-specific TCR was identified using single cell TCR-seq. The TCR convergence is observed in a mouse carrying HCC, indicating that the TCR specific to the HCC antigen initiates the expansion of CD4 + T cells. In order to further clarify the function of such HCC-specific TCRs, TCR expression vectors are designed and constructed, which can be used in a variety of in vitro and in vivo applications, including identification of specific peptide sequences for TCR recognition, manufacture of TCR engineered T cells, study of T cell function, and screening for potential immunotherapy. These findings are of great significance to the development of T cell-based cancer immunotherapies.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 512,299, filed July 7, 2023. Application No. 63 / 512,299, filed July 7, 2023, is hereby incorporated herein by reference in its entirety.

[0003] References to sequence lists

[0004] According to 37 CFR § 1.834(c)(1), the sequence list XML was submitted as a file named “UHK_01396_PCT_ST26”, created on June 26, 2024, and has a size of 61,683 bytes, which is incorporated herein by reference. Invention Field

[0005] This invention relates generally to T-cell receptors (TCRs), and more specifically, to the discovery and application of hepatocellular carcinoma-specific TCRs derived from convergent recombination. Background of the Invention

[0007] T cell receptors (TCRs) are key components of the adaptive immune response, playing a crucial role in the recognition of specific antigens. A TCR consists of two distinct protein chains, α and β or γ and δ, linked together by disulfide bonds. The α and β chains contain variable (V), diverse (D), and joiner (J) gene segments that are rearranged during T cell development to generate unique TCR sequences that recognize specific antigens. This TCR rearrangement process, known as V(D)J recombination, is mediated by the RAG1 and RAG2 proteins, which recognize recombination signal sequences (RSS) flanking the V, D, and J gene segments. The RAG proteins cleave DNA at the RSS, producing double-strand breaks that are repaired via the non-homologous end joining (NHEJ) pathway. This process results in the formation of a unique TCR sequence specific to each T cell.

[0008] TCRs on T cells can recognize specific cancer antigens. Modified T cells targeting HCC antigens such as AFP, GPC3, and MAGE-A can be used in adoptive cell therapy (ACT) to destroy cancer cells. Viral antigens (HBsAg) are also targets of ACT. CD8 T cells have traditionally been considered the most effective immune cells for ACT, and infusion of CD8 CAR T cells alone has shown long-term B cell eradication. However, CD4 T cells also play a crucial role in the immune response, and they can enhance the cytolytic activity of CD8 T cells through cytokine production. Studies have shown that CD4 CAR T cells have demonstrated considerable effectiveness in directly killing target tumor cells in vitro and in vivo. Although CD4 CAR T cells may exhibit slower granzyme B secretion and tumor killing at onset, they are less prone to AICD and exhaustion compared to their CD8 counterparts, which confers relatively better persistence of the CD4 compartment after antigen exposure.

[0009] Ideally, the antigens selected for targets in ACT need to be highly expressed in tumor cells and not in the necessary healthy tissues. Several strategies can be employed to identify TCRs that specifically recognize tumor antigens. One such approach is single-cell TCR sequencing (TCR-seq), which allows for the identification and characterization of TCRs at the single-cell resolution disclosed herein.

[0010] Any discussion of documents, actions, materials, devices, articles of manufacture, etc., already included in this specification shall not be construed as an admission that any or all of these matters constitute part of the prior art or are common knowledge in the field relating to this disclosure prior to the priority date of each claim of this application.

[0011] Throughout this specification, the word “comprise” or variations thereof such as “comprises” or “comprising” shall be understood to imply inclusion of the said element, integer or step, or group of elements, integers or steps, but not to exclude any other element, integer or step, or group of elements, integers or steps. Summary of the Invention

[0012] Compounds, compositions, and methods relating to and usable for treating liver cancer are disclosed. Immunoproteins related to or derived from T-cell receptors (TCRs) found to be associated with liver cancer are disclosed. The disclosed immunoproteins typically contain and / or retain the functional portion of the identified TCR. For example, in some forms, the immunoproteins contain (a) a complementarity-determining region (CDR) having the sequences SGHSA (SEQ ID NO:53), FRNQAP (SEQ ID NO:54), and ASSLDRGQDTQY (SEQ ID NO:55), (b) a CDR having the sequences TISGNEY (SEQ ID NO:56), GLQQN (SEQ ID NO:57), and ILRGTGGNNKLT (SEQ ID NO:58), or (c) both.

[0013] In some forms, the immune protein includes (a) a TCRβ chain variable domain (TCR-β domain) containing a complementarity-determining region (CDR) having the sequences SGHSA (SEQ ID NO:53), FRNQAP (SEQ ID NO:54), and ASSLDRGQDTQY (SEQ ID NO:55), (b) a TCRα chain variable domain (TCR-α domain) containing a CDR having the sequences TISGNEY (SEQ ID NO:56), GLQQN (SEQ ID NO:57), and ILRGTGGNNKLT (SEQ ID NO:58), or (c) both. In some forms, the immune protein includes the T cell receptor (TCR) containing a TCR-β domain, a TCR-α domain, or both.

[0014] In some forms, the TCR-β domain shares at least 75% sequence identity with SEQ ID NO: 51 and the TCR-α domain shares at least 75% sequence identity with SEQ ID NO: 52. In some forms, the TCR-β domain shares at least 85% sequence identity with SEQ ID NO: 51 and the TCR-α domain shares at least 85% sequence identity with SEQ ID NO: 52. In some forms, the TCR-β domain shares at least 90% sequence identity with SEQ ID NO: 51 and the TCR-α domain shares at least 90% sequence identity with SEQ ID NO: 52. In some forms, the TCR-β domain shares at least 95% sequence identity with SEQ ID NO: 51 and the TCR-α domain shares at least 95% sequence identity with SEQ ID NO: 52. In some forms, the TCR-β domain has at least 75% sequence identity with SEQ ID NO: 51 and the TCR-α domain has at least 75% sequence identity with SEQ ID NO: 52. In some forms, the TCR-β domain contains the sequence SEQ ID NO: 51 and the TCR-α domain contains the sequence SEQ ID NO: 52.

[0015] In some forms, immune proteins target hepatocellular carcinoma (HCC) antigens.

[0016] Nucleic acids encoding one or more of the disclosed immune proteins are also disclosed. Vectors containing one or more of the disclosed nucleic acids are also disclosed. In some forms, the nucleic acid or vector contains an expression segment that includes coding regions in the following order: a signal sequence (such as fibL from silkworm moth), a TCR-β domain, a TCR β constant region, a furin cleavage site, a flexible linker, T2A, a TCR-α domain, and a TCR α constant region.

[0017] Cells expressing one or more of the disclosed immune proteins have also been disclosed. In some forms, the cells are genetically modified T cells.

[0018] Cell populations derived by amplifying one or more of the disclosed cells were also disclosed.

[0019] Pharmaceutical compositions are also disclosed. In some forms, the composition comprises one or more disclosed immune proteins and pharmaceutically acceptable buffers, carriers, diluents, or excipients. In some forms, the composition comprises one or more disclosed cell populations and pharmaceutically acceptable buffers, carriers, diluents, or excipients.

[0020] Methods for treating subjects with diseases, conditions, or illnesses are also disclosed. In some forms, the disease, condition, or illness is associated with targets of immune proteins. Typically, the method involves administering an effective amount of one or more of the disclosed pharmaceutical compositions to the subject.

[0021] In some forms, the subject has HCC or has been identified as having an increased risk of developing HCC. In some forms, the cell population is isolated from or derived from an amplification of cells obtained from a subject who had a disease, condition, or illness prior to the introduction of the cells. In some forms, the cell population is isolated from or derived from an amplification of cells obtained from a healthy donor.

[0022] The study also disclosed T-cell receptors (TCRs) that can specifically target liver cancer cells. These T-cell receptors (TCRs) contain the TCR β-chain variable domain of SEQ ID NO: 51 and the α-chain variable domain of SEQ ID NO: 52, wherein the three complementarity-determining regions (CDRs) of the β-chain variable domain include: CDR1: SGHSA (SEQ ID NO: 53), CDR2: FRNQAP (SEQ ID NO: 54), and CDR3: ASSLDRGQDTQY (SEQ ID NO: 55), and the three CDRs of the α-chain variable domain include: CDR1: TISGNEY (SEQ ID NO: 56), CDR2: GLQQN (SEQ ID NO: 57), and CDR3: ILRGTGGNNKLT (SEQ ID NO: 58).

[0023] T cells carrying specific TCRs are activated in the livers of patients with HCC, but not in normal livers. These TCRs also exhibit TCR convergence, indicating that T cells from multiple HCC-carrying subjects share the same TCR amino acid sequence, although encoded by different mRNA variants. TCR convergence across HCC-carrying mice indicates that this HCC antigen-specific TCR triggers CD4+. + T cell expansion.

[0024] In some forms, the disclosed TCR sequence can then be introduced into T cells, which can then be expanded and infused into a subject to target and treat cancer cells.

[0025] In some forms, the disclosed TCR can be used for adoptive T-cell immunotherapy to treat liver cancer.

[0026] In some forms, the disclosed TCRs can be engineered for the treatment of various other cancers, such as melanoma or leukemia.

[0027] In some forms, the disclosed TCRs can guide the identification of homologous tumor antigens, leading to the development of more therapeutic TCRs and other immunotherapy strategies for treating liver cancer. This can also aid in the design of vaccines or immunotherapies that target tumor antigens and stimulate an immune response against cancer cells.

[0028] In some forms, vectors expressing TCRs can be used to generate T cell clones that recognize specific tumor antigens. These T cell clones can then be used to screen peptide libraries derived from tumor antigens to identify specific peptide epitopes recognized by the TCRs. This could potentially facilitate the development of personalized immunotherapies that target specific tumor antigens in patients.

[0029] In some forms, the disclosed TCRs can be expressed in specific cells to study their function and activation in response to a variety of stimuli, which can help elucidate T cell biology.

[0030] Methods for identifying TCRs were also disclosed. These methods include generating animal models of liver cancer that mimic the multi-stage and multi-hit processes of human liver carcinogenesis. Liver cancer was induced by ablation of Trp53 and Pten using CRISPR and by overexpressing Myc using a transposon vector. Plasmids were delivered specifically to hepatocytes via fluid dynamics injection. After induction of liver cancer, TCR types were identified using single-cell TCR-seq. This technique was used to compare TCR libraries in healthy livers and livers with HCC to identify HCC-specific TCRs.

[0031] Additional advantages of the disclosed methods and compositions will be shown in part in the description below, and in part will be understood from the description, or may be learned by practice of the disclosed methods and compositions. The advantages of the disclosed methods and compositions will be realized and obtained by the elements and combinations particularly pointed out in the appended claims. It should be understood that the foregoing general description and the following detailed description are exemplary and illustrative only, and are not limiting of the claimed invention. Brief description of the attached diagram

[0033] Figure 1A-1H This study demonstrated the presence of immune surveillance in CRISPR- and transposon-induced hepatocellular carcinoma in mice. Figure 1A This diagram shows plasmids used to induce (top) CRISPR / Cas9-mediated Trp53 and Pten deletion and (bottom) transposon-mediated Myc overexpression in mouse liver. Arrows indicate tumor nodules. Figure 1B The image shows the injection (Figure 1). Figure 1A The image shows a representative macroscopic view of a mouse liver with the plasmid combination shown at a specified number of days post-injection. Controls received only the transposase vector. Figure 1CRepresentative histological sections of tumor-bearing livers are shown, obtained by immunostaining to detect MYC, P53, and PTEN. Figure 1D Showing from ( Figure 1C Distribution of hepatic tumor nodules in the liver as indicated by immunostaining. The pie chart summarizes two independent experiments, marking the number of tumor nodules with the indicated immunostaining patterns. Sections were excised from 3 mice / groups on day 25 of HCC induction. Figure 1E-1F This is a representative flow cytometry plot showing changes in the percentage of immune cell populations indicating the progression of liver cancer in mice. Figure 1E ) and quantitative ( Figure 1F ).exist Figure 1F In the diagram, each point represents a single mouse. Significance was assessed by an unpaired two-tailed t-test; *p<0.05 and **p<0.01 represent three independent experiments. The control group received only the transposase vector. Figure 1G The images show representative histological sections from livers carrying HCC, which were immunostained to detect MYC or CD3 in areas of (left) pretumor cells or (right) HCC. Figure 1H The survival of immunodeficient (NSG) and control WT mice (n=10 / group) after injection of Trp53 / Pten CRISPR and Myc overexpression plasmids to induce HCC development was shown. p=0.001 by log-rank test.

[0034] Figure 2A-2D CD4 in HCC is shown + scRNAseq of T cells. Figure 2A-2B CD4 in the livers of control and HCC mice is shown. + T cell transcriptional landscape. Total liver CD4 count based on scRNAseq analysis. + UMAP manifestations of T cells ( Figure 2A ), and partitioning based on HCC conditions ( Figure 1B CD4+ cells were isolated from 4 HCC mice and 4 control mice. + T cells. Cells from each mouse were stained with a unique barcode antibody. Figure 2C It is a comparison ( Figure 2A-2B Bubble graph of Chat and indicator marker gene expression in 11 clusters. Figure 2D It describes cluster C3 (Cxcr6) + Pdcd1 + ) and cluster C7 (Cxcr6 + Pdcd1 - A heatmap showing the relative expression of genes indicated in the diagram.

[0035] Figures 3A-3E CD4 in HCC is shown+ Clonal expansion of T cells. Figures 3A-3B A circos plot is shown, which displays data from the control group ( Figure 3A ) and carrying HCC ( Figure 3B CD4 in mice + Distribution of TCR types in T cells. T cells with the same TCR type are those that share the same TCR-α and TCR-β chains in their amino acid sequences. The first 30 TCRs are numbered and highlighted with different colors. Figure 3C The cloned CDR3 sequence encoding TCR #1 is shown.

[0036] The TCR-β CDR3 nucleotide sequence of clone 1's TCR #1 is SEQ ID NO:1; clone 3 is SEQ ID NO:2; clone 5 is SEQ ID NO:3; clone 10 is SEQ ID NO:4; clone 14 is SEQ ID NO:5; clone 21 is SEQ ID NO:6; clone 22 is SEQ ID NO:7; clone 25 is SEQ ID NO:8; clone 32 is SEQ ID NO:9; clone 42 is SEQ ID NO:10; clone 49 is SEQ ID NO:11; clone 54 is SEQ ID NO:12; clone 63 is SEQ ID NO:13; clone 64 is SEQ ID NO:14; clone 83 is SEQ ID NO:15; clone 124 is SEQ ID NO:16; clone 133 is SEQ ID NO:17; clone 134 is SEQ ID NO:18; clone 140 is SEQ ID NO:19; clone 209 is SEQ ID NO:20; clone 222 is SEQ ID NO:19. Clone NO:21; clone 248 is SEQ ID NO:22; clone 266 is SEQ ID NO:23; clone 461 is SEQ ID NO:24; and clone 862 is SEQ ID NO:25. The amino acid sequence of TCR-β CDR3 of TCR #1 is SEQ ID NO:55.

[0037] The nucleotide sequence of TCR-α CDR3 of TCR #1 of clone 1 is SEQ ID NO:25; clone 3 is SEQ ID NO:26; clone 5 is SEQ ID NO:27; clone 10 is SEQ ID NO:29; clone 14 is SEQ ID NO:30; clone 21 is SEQ ID NO:31; clone 22 is SEQ ID NO:32; clone 25 is SEQ ID NO:33; clone 32 is SEQ ID NO:34; clone 42 is SEQ ID NO:35; clone 49 is SEQ ID NO:36; clone 54 is SEQ ID NO:37; clone 63 is SEQ ID NO:38; clone 64 is SEQ ID NO:39; clone 83 is SEQ ID NO:40; clone 124 is SEQ ID NO:41; clone 133 is SEQ ID NO:42; clone 134 is SEQ ID NO:43; clone 140 is SEQ ID NO:44; clone 209 is SEQ ID NO:45; clone 222 is SEQ ID NO:25. Clone NO:46; clone 248 is SEQ ID NO:47; clone 266 is SEQ ID NO:48; clone 461 is SEQ ID NO:49; and clone 862 is SEQ ID NO:50. The amino acid sequence of TCR-α CDR3 of TCR #1 is SEQ ID NO:58.

[0038] Figure 3D This displays the composition of cell clusters within the T-cell clonoid indicated by TCR #1, coded by cell cluster color. Each bar represents a single clonoid and is labeled with a clone ID, such as ( Figure 3C As shown in the diagram. The horizontal axis labels indicate the cell numbers.

[0039] Figure 3E CD4 cells carrying TCR #1 induced in HCC + UMAP representation of T cells, encoded by cell cluster color and by mouse ID shape.

[0040] Figures 4A-4D The nucleotide and amino acid sequences of clone 5 of HCC TCR #1 are shown. Figures 4A-4B The TCR clone 5's Trbv (shown) Figure 4A (SEQ ID NO: 59) and Trav ( Figure 4B The nucleotide sequence of (SEQ ID NO: 60). Gene segments are represented by different colors. Figure 4C-4D It is TCR clone 5's TRBV ( Figure 4C (SEQ ID NO: 51) and Trav ( Figure 4DThe amino acid sequence of (SEQ ID NO: 52). Gene segments are represented by different colors.

[0041] Figures 5A-5B The development of an engineered expression cassette for HCC-specific TCRs is shown. Figure 5A This is a schematic diagram of an engineered construct used to express HCC-specific TCRs. Figure 5B Partial construct sequences are shown. The complete construct sequence is SEQ ID NO: 61. The various parts of the construct are: FiBL signal (amino acids 1 to 17 of SEQ ID NO: 61), TCRb variable region (amino acids 18 to 129 of SEQ ID NO: 61), TCRb constant region (amino acids 130 to 302 of SEQ ID NO: 51), Furin cleavage site (amino acids 303 to 306 of SEQ ID NO: 61), flexible linker (amino acids 307 to 310 of SEQ ID NO: 61), T2A (amino acids 311 to 328 of SEQ ID NO: 61), FiBL signal (amino acids 329 to 345 of SEQ ID NO: 61), TRa variable region (amino acids 346 to 457 of SEQ ID NO: 61), and TRa constant region (amino acids 458 to 593 of SEQ ID NO: 61). Invention Details

[0043] The disclosed methods and compositions can be more readily understood by referring to the following detailed description of specific embodiments and examples included therein, as well as the accompanying drawings and descriptions preceding and following them.

[0044] A. Definition

[0045] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0046] As used in this article, the singular forms “a,” “one,” and “the” include both singular and plural referents, unless the context clearly indicates otherwise.

[0047] When referring to the nucleotide sequence of a nucleic acid encoding a gene product of interest (e.g., a protein), "encoding" means including cases where the nucleic acid contains a nucleotide sequence identical to or a portion thereof to an endogenous sequence of a nucleic acid found in a cell or genome, which produces a gene product upon transcription and / or translation into a polypeptide. In some cases, the nucleotide sequence or nucleic acid encoding the gene product does not contain intron sequences. In specific cases, the nucleotide sequence or nucleic acid encoding a T-cell receptor contains a nucleotide sequence that can be translated in a computer into an amino acid sequence corresponding to the variable and constant domains of the T-cell receptor, without inserted intron sequences.

[0048] As used herein, the term “nucleic acid” in its broadest sense refers to any compound and / or substance incorporated into or potentially incorporated into an oligonucleotide chain. In some forms, nucleic acid is a compound and / or substance incorporated into or potentially incorporated into an oligonucleotide chain via a phosphodiester bond. In some forms, “nucleic acid” refers to a single nucleic acid residue (e.g., a nucleotide and / or nucleoside). In some forms, “nucleic acid” refers to an oligonucleotide chain containing a single nucleic acid residue. As used herein, the terms “oligonucleotide” and “polynucleotide” are used interchangeably. In some forms, “nucleic acid” encompasses any natural or synthetic linear and sequential array of RNA, as well as single-stranded and / or double-stranded DNA and / or cDNA, nucleotides, and nucleosides, such as replication RNA (repRNA), messenger RNA (mRNA), small interfering RNA (siRNA), transfer RNA (tRNA), microRNA (miRNA), guide strand RNA (sgRNA), polynucleotides, oligonucleotides, oligonucleosides, and their derivatives. Such nucleic acids may be collectively referred to as “constructs” or “plasmids.” Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and / or similar terms include nucleic acid analogs, i.e., analogs having a backbone different from that of a phosphodiester. For example, the so-called “peptide nucleic acid,” which is known in the art and has peptide bonds instead of phosphodiester bonds in its backbone, is considered to be within the scope of this invention. The term “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate in form of each other and / or encode the same amino acid sequence. Nucleotide sequences encoding proteins and / or RNA may include introns. Nucleic acids can be purified from natural sources, produced using recombinant expression systems, and optionally purified, chemically synthesized, etc. Where appropriate, for example in the case of chemically synthesized molecules, nucleic acids may include nucleoside analogs, such as analogs having chemically modified bases or sugars, backbone modifications, etc. Unless otherwise stated, nucleic acid sequences are presented in the 5' to 3' orientation. The term “nucleic acid segment” is used herein to refer to a nucleic acid sequence that is part of a longer nucleic acid sequence. In many forms, a nucleic acid segment contains at least 3, 4, 5, 6, 7, 8, 9, 10, or more residues.In some forms, nucleic acids are or include natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5-propynylcytidine, C-5-propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deadenine, 7-deadenine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanosine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases. The modified sugar (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or the modified phosphate group (e.g., thiophosphate and 5'-N phosphoramide bond).

[0049] The term "antigen" refers to any substance (e.g., peptide, protein, nucleic acid, lipid, small molecule, such as a part expressed by a pathogen or cancer cell or precancerous cell, or otherwise associated with a pathogen or cancer cell or precancerous cell) that serves as a target for a receptor in an adaptive immune response. Antigens can be structural components of pathogens, cancer cells, or precancerous cells.

[0050] As used herein, the term "T-cell antigen" refers to any antigen recognized by T cells and that triggers an immune response on T cells (e.g., an antigen specifically recognized by T-cell receptors on T cells via presentation of an antigen or a portion thereof that binds to the major histocompatibility complex molecule (MHC). In some forms, the antigen that is a T-cell antigen is also a B-cell antigen. In other forms, the T-cell antigen is not a B-cell antigen. T-cell antigens are typically proteins or peptides. T-cell antigens can be antigens that stimulate CD8+ T-cell responses, CD4+ T-cell responses, or both. Therefore, in some forms, nanocarriers can effectively stimulate both types of responses.

[0051] As used herein, the term “target” or “marker” refers to any entity capable of specifically binding to a particular target moiety. In some forms, a target is associated with one or more specific tissue types. In some forms, a target is associated with one or more specific cell types. For example, the expression level of a cell type-specific marker in that cell type is typically at least 2-fold higher than in a reference cell population. In some forms, a cell type-specific marker is present at a level at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 50-fold, at least 100-fold, or at least 1000-fold higher than its mean expression in a reference population. Detection or measurement of a cell type-specific marker can make it possible to distinguish one or more cell types of interest from many, most, or all other cell types. In some forms, a target may include proteins, carbohydrates, lipids, and / or nucleic acids, as described herein. If a substance specifically binds to a target, it is considered to be “targeted” for the purposes described herein. In some forms, the target moiety specifically binds to the target under stringent conditions. If the targeting portion specifically binds to the target, thereby delivering the entire nanocarrier to a specific organ, tissue, cell, and / or subcellular location, then an inventive nanocarrier containing the targeting portion, such as a vaccine nanocarrier, is considered "targeted".

[0052] As used herein, the term "therapeutic agent" means any agent that, when administered to a subject, has a therapeutic, preventive and / or diagnostic effect and / or causes the desired biological and / or pharmacological effect.

[0053] The terms “high,” “higher,” “increase,” “elevates,” or “elevation” refer to an increase, for example, above baseline levels compared to a control. The terms “low,” “lower,” “reduces,” or “reduction” refer to a decrease, for example, below baseline levels compared to a control.

[0054] As used herein, the term "modulation" refers to the ability of a compound to alter its activity in some measurable manner compared to a suitable control. The activity may increase or decrease due to the presence of the compound in the assay, compared to a control in the absence of the compound. Preferably, the activity increases by at least 25%, more preferably at least 50%, and most preferably at least 100% compared to the activity level in the absence of the compound. Similarly, the activity decreases by at least 25%, more preferably at least 50%, and most preferably at least 100% compared to the activity level in the absence of the compound. Compounds that increase known activity are "agonists." Compounds that decrease or inhibit known activity are "antagonists."

[0055] The term "inhibition" refers to a reduction or decrease in activity or expression. Inhibition can be a complete or partial inhibition of activity or expression. Inhibition can be compared to a control or standard level. Inhibition can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%. %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0056] As used herein, the term “monitoring” refers to any method in the art that can be used to measure activity.

[0057] As used herein, the term "providing" refers to any manner in which a compound or molecule is added to something known in the art. Examples of providing may include the use of pipettes, syringes, needles, tubes, guns, etc. It may be manual or automated. It may include any or any other method of transfection by providing nucleic acids to a culture dish, cells, tissue, cell-free system, and may be in vitro or in vivo.

[0058] As used in this article, the term "prevention" refers to the administration of a compound prior to the onset of clinical symptoms of a disease or condition in order to prevent abnormal bodily manifestations associated with the disease or condition.

[0059] As used herein, the term “requirement of treatment” refers to a judgment made by a caregiver (e.g., a physician, nurse, registered nurse, or individual in the case of a human; a veterinarian in the case of an animal (including non-human mammals)) that a subject needs or will benefit from treatment. This judgment is based on a number of factors within the caregiver’s scope of expertise, but includes knowledge that the subject is ill or will be ill due to a condition treatable with the disclosed compound.

[0060] As used herein, “subject” includes, but is not limited to, animals, plants, bacteria, viruses, parasites, and any other living organism or entity. Subjects can be vertebrates, more particularly mammals (e.g., humans, horses, pigs, rabbits, dogs, sheep, goats, non-human primates, cattle, cats, guinea pigs, or rodents), fish, birds, or reptiles or amphibians. Subjects can be invertebrates, more particularly arthropods (e.g., insects and crustaceans). The term does not indicate a specific age or sex. Therefore, it is intended to encompass adult and newborn subjects, as well as fetuses, regardless of male or female. A patient is a subject suffering from a disease or ailment. The term “patient” includes subjects in both human and veterinary settings.

[0061] "Treatment" and "treating" refer to the medical management of a subject with the aim of curing, improving, stabilizing, or preventing a disease, pathological condition, or symptom. This term includes active treatment, which is treatment specifically aimed at improving a disease, pathological condition, or symptom, and also includes etiological treatment, which aims to eliminate the cause of the relevant disease, pathological condition, or symptom. Furthermore, this term includes palliative treatment, which aims to relieve symptoms rather than cure a disease, pathological condition, or symptom; preventive treatment, which aims to minimize or partially or completely suppress the development of a relevant disease, pathological condition, or symptom; and supportive treatment, which is treatment used to supplement another specific therapy aimed at improving a relevant disease, pathological condition, or symptom. It should be understood that while treatment aims to cure, improve, stabilize, or prevent a disease, pathological condition, or symptom, it does not need to actually result in a cure, improvement, stabilization, or prevention. The effectiveness of treatment can be measured or evaluated using methods suitable for the disease, pathological condition, or symptom involved, as described herein and known in the art. Such measurements and evaluations can be performed using qualitative and / or quantitative methods. Therefore, for example, the characteristics or features of a disease, pathological condition or symptom and / or the symptoms of a disease, pathological condition or symptom can be reduced to any effect or any amount.

[0062] The cells can be in vitro. Alternatively, the cells can be in vivo and may be present in the subject. “Cell” can be a cell from any organism, including but not limited to bacteria.

[0063] As used herein, the term "effective amount" for compounds refers to an amount of a compound that is non-toxic but sufficient to provide the desired results. As will be noted below, the exact amount required will vary from subject to subject, depending on the subject's species, age and general condition, the severity of the disease being treated, the specific compound used, the method of administration, etc. Therefore, it is impossible to specify a precise "effective amount." However, an appropriate effective amount can be determined by a person skilled in the art using only routine experiments.

[0064] The dosage or amount of the compounds described herein is sufficient to produce the desired effect in the method of delivery. The dosage should not be so high as to cause adverse side effects, such as unwanted cross-reactions, allergic reactions, etc. Typically, the dosage will vary depending on the subject's age, condition, sex, and disease severity, and can be determined by someone skilled in the art. The dosage may be adjusted by an individual physician based on the clinical condition of the subject involved. The dosage, administration regimen, and route of administration may be modified.

[0065] "Pharmaceutical acceptable" means that the material is not biologically or otherwise undesirable, that is, the material can be administered to a subject together with the selected compound without causing any undesirable biological effects or interacting in a harmful manner with any other component of the pharmaceutical composition containing the material.

[0066] The term "vector" refers to a nucleic acid molecule or polynucleotide, such as replicating RNA, plasmid, bacteriophage, or granule, in which another nucleic acid sequence segment can be inserted to induce replication of the inserted segment. The vector may be an expression vector. A vector also refers to a molecule incorporating a nucleic acid sequence encoding a regulatory element for transcription, translation, transcript stability, replication, and other functions known in the art. A vector can be a nucleic acid such as a plasmid or other DNA vector. A vector may contain one or more genes in a linear or circular configuration. A vector may also have a "plasmid backbone" involved in the generation, manufacture, or analysis of a gene product. An "expression vector" is a vector that allows the production of a product encoded by the nucleic acid sequence contained within the vector. For example, the expression of a specific growth factor protein encoded by a specific gene. A "gene product" refers to a product encoded by the nucleic acid sequence of a vector.

[0067] The term "gene" or "multiple genes" refers to an isolated or modified nucleic acid sequence, including both RNA and DNA, that encodes genetic information for the synthesis of complete RNA, complete proteins, or any part of such complete RNA or complete proteins. Genes that are not naturally part of the genome of a particular organism are called "foreign genes," "heterologous genes," or "exogenous genes," while genes that are naturally part of the genome of a particular organism are called "endogenous genes." The term "gene" used with respect to genomic DNA includes inserted non-coding regions as well as regulatory regions, and may include both 5' and 3' ends.

[0068] The term "expressed" or "expression" refers to the transcription from DNA into an RNA nucleic acid molecule that is at least partially complementary to a region of one of the two nucleic acid strands of a gene. The term "expressed" or "expression" also refers to the translation from said RNA nucleic acid molecule to produce a protein or polypeptide or a portion thereof.

[0069] The term "coding region" refers to a portion of a nucleic acid containing a codon that can be translated into amino acids. Although "stop codons" (TAG, TGA, or TAA) are not translated into amino acids, they can also be considered part of a coding region. Unless otherwise stated, promoters, ribosome binding sites, transcription terminators, introns, etc., are not considered part of a coding region. The coding regions of this invention can be present in a single polynucleotide construct, such as on a separate vector, or in separate polynucleotide constructs, such as on separate (different) vectors. Furthermore, any vector can contain a single coding region, or can contain two or more coding regions. Additionally, vector, polynucleotide, or nucleic acid embodiments can encode heterologous coding regions fused to or not fused to nucleic acids encoding different heterologous polypeptides. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretory signal peptides or heterologous functional domains.

[0070] The term "regulatory element" refers to a DNA sequence that controls and regulates the transcription of another DNA sequence.

[0071] The term "promoter" refers to a nucleic acid sequence sufficient to direct gene transcription. This invention also includes promoter elements sufficient to make the expression of promoter-dependent genes controllable to cell type specificity, tissue specificity, or induced by external signals or reagents. Promoters that are induced to cause gene expression after exposure to or treatment of cells with promoter-inducing agents, biomolecules, chemicals, ligands, light, etc., are generally referred to as "inducible promoters" or "tunable" promoters.

[0072] As used herein, the terms “approximately” or “about” with respect to numbers are generally considered to include numbers that fall within the range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) unless otherwise stated or obvious from the context (unless such numbers would be less than 0% or more than 100% of the possible value).

[0073] The terms "immunologic," "immunological," or "immune" response refer to a beneficial humoral (antibody-mediated) and / or cellular (mediated by antigen-specific T cells or their secretory products) response against an immunogen in a recipient patient. Such responses can be active responses induced by immunogen administration or passive responses induced by antibody administration or triggered T cell responses. The relative contributions of humoral and cellular responses to the protective or therapeutic effects of an immunogen can be distinguished by isolating antibodies and T cells separately from an immunized syngeneic animal and measuring the protective or therapeutic effects in a second subject.

[0074] As used herein, the term "immunostimulant" refers to an agent that modulates an immune response to an antigen but is not an antigen or is not derived from an antigen. As used herein, "modulates" means to induce, enhance, inhibit, direct, or redirect an immune response. Such agents include immunostimulants that stimulate (or enhance) an immune response to an antigen, but are not an antigen or are not derived from an antigen as defined above. Therefore, immunostimulants include adjuvants. In some forms, the immunostimulant is on the surface of a nanocarrier and / or encapsulated within the nanocarrier. In some forms, the immunostimulant on the surface of the nanocarrier differs from the immunostimulant encapsulated within the nanocarrier. In some forms, the nanocarrier contains more than one type of immunostimulant. In some forms, more than one type of immunostimulant acts on different pathways. Examples of immunostimulants include those provided elsewhere herein.

[0075] As used herein, the term "single guide RNA" or "sgRNA" refers to a polynucleotide sequence containing a guide sequence, a tracr sequence, and a tracr pairing sequence. The "guide sequence" refers to a sequence of approximately 20 base pairs (bp) within the guide RNA that specifies a target site, and is used interchangeably with the terms "guide" or "spacer."

[0076] The terms "Cas9," "Cas9 protein," or "Cas9 nuclease" refer to RNA-directed endonucleases, specifically the Cas9 protein, which catalyzes site-specific cleavage of double-stranded DNA. They are also known as "Cas nucleases" or "CRISPR-associated nucleases."

[0077] The term "about" is used to describe values ​​that are about + / - 10% above or below the range of values; in other forms, the range of values ​​may be about + / - 5% above or below the range of values; in other forms, the range of values ​​may be about + / - 2% above or below the range of values; in other forms, the range of values ​​may be about + / - 1% above or below the range of values. The foregoing ranges are intended to be clear from the context and do not imply further limitations. All methods described herein may be performed in any suitable order unless otherwise stated herein or clearly contradicted by the context. Unless otherwise required, the use of any and all instances or exemplary language (e.g., "such as") provided herein is intended only to better illustrate this disclosure and does not constitute a limitation on the scope of this disclosure. No language in the specification should be construed as indicating that any unclaimed element is essential to the practice of this disclosure.

[0078] Each compound disclosed herein is intended and should be considered as specifically disclosed herein. Furthermore, each subset that can be identified in this disclosure is intended and should be considered as specifically disclosed herein. Therefore, any compound or subset of compounds that may be specifically included for use or excluded from use, or included in or excluded from the list of compounds, is specifically considered.

[0079] The components used to prepare the disclosed compositions and the compositions themselves used in the methods disclosed herein are disclosed. These and other materials are disclosed herein, and it should be understood that when combinations, subsets, interactions, groups, etc., of these materials are disclosed, while specific references to every various individual and collective combination and arrangement of these compounds may not be explicitly disclosed, each specific reference is specifically considered and described herein. For example, if a particular polypeptide is disclosed and discussed, and many modifications that can be made to many polypeptides are discussed, then every combination and arrangement of the polypeptide and possible modifications is specifically considered, unless specifically indicated to the contrary. Thus, if examples of classes of molecules A, B, and C and classes of molecules D, E, and F, and examples of the combination molecule AD are disclosed, then each is considered individually and collectively, even if each is not individually enumerated, meaning that combinations AE, AF, BD, BE, BF, CD, CE, and CF are considered disclosed. Similarly, any subsets or combinations of these are also disclosed. Thus, for example, a subset of AE, BF, and CE would be considered disclosed. This concept applies to all aspects of this application, including but not limited to the steps in methods for preparing and using the disclosed compositions. Therefore, if there are multiple additional steps that can be performed, it should be understood that each of these additional steps can be performed in any particular form or combination of forms of the disclosed method.

[0080] B. Composition

[0081] 1. Immune proteins

[0082] Immunoproteins related to or derived from T-cell receptors (TCRs) found to be associated with liver cancer are disclosed. The disclosed immunoproteins typically contain and / or retain the functional portion of the identified TCR. For example, in some forms, the immunoproteins contain (a) a complementarity-determining region (CDR) having the sequences SGHSA (SEQ ID NO:53), FRNQAP (SEQ ID NO:54), and ASSLDRGQDTQY (SEQ ID NO:55), (b) a CDR having the sequences TISGNEY (SEQ ID NO:56), GLQQN (SEQ ID NO:57), and ILRGTGGNNKLT (SEQ ID NO:58), or (c) both.

[0083] In some forms, the immune protein includes (a) a TCRβ chain variable domain (TCR-β domain) containing a complementarity-determining region (CDR) having the sequences SGHSA (SEQ ID NO:53), FRNQAP (SEQ ID NO:54), and ASSLDRGQDTQY (SEQ ID NO:55), (b) a TCRα chain variable domain (TCR-α domain) containing a CDR having the sequences TISGNEY (SEQ ID NO:56), GLQQN (SEQ ID NO:57), and ILRGTGGNNKLT (SEQ ID NO:58), or (c) both. In some forms, the immune protein includes the T cell receptor (TCR) containing a TCR-β domain, a TCR-α domain, or both.

[0084] In some forms, the TCR-β domain shares at least 75% sequence identity with SEQ ID NO: 51 and the TCR-α domain shares at least 75% sequence identity with SEQ ID NO: 52. In some forms, the TCR-β domain shares at least 85% sequence identity with SEQ ID NO: 51 and the TCR-α domain shares at least 85% sequence identity with SEQ ID NO: 52. In some forms, the TCR-β domain shares at least 90% sequence identity with SEQ ID NO: 51 and the TCR-α domain shares at least 90% sequence identity with SEQ ID NO: 52. In some forms, the TCR-β domain shares at least 95% sequence identity with SEQ ID NO: 51 and the TCR-α domain shares at least 95% sequence identity with SEQ ID NO: 52. In some forms, the TCR-β domain has at least 75% sequence identity with SEQ ID NO: 51 and the TCR-α domain has at least 75% sequence identity with SEQ ID NO: 52. In some forms, the TCR-β domain contains the sequence SEQ ID NO: 51 and the TCR-α domain contains the sequence SEQ ID NO: 52.

[0085] In some forms, immune proteins target hepatocellular carcinoma (HCC) antigens.

[0086] 2. T cell receptors

[0087] Convergent TCR sequences in T cells reactive to specific tumor antigens and their amplification in hepatocellular carcinoma (HCC) in individual animals were observed. These findings suggest that T cells carrying these specific TCR amino acid sequences are important for anti-tumor immune responses. Convergent TCRs are particularly important in cancer immunotherapy because they provide a more efficient method for identifying and isolating tumor-reactive T cells for adoptive cell therapy. By isolating T cells from liver tissue carrying HCC and performing single-cell RNA sequencing on both the transcriptome and TCR, the identified tumor-specific TCR sequences were more correlated with tumor-reactive T cells in vivo. Furthermore, TCR convergence and clonal amplification were observed in different mice, indicating the presence of specific and immunogenic tumor antigens corresponding to these TCRs. This method provides an important pathway for developing novel immunotherapies for liver cancer using identified HCC-specific TCR sequences.

[0088] A T-cell receptor (TCR) capable of specifically targeting liver cancer cells has been disclosed. The exemplary T-cell receptor (TCR) contains a TCR β-chain variable domain and an α-chain variable domain. The TCR β-chain variable domain has the following sequence:

[0089] NAGVIQTPRHKVTGKGQEATLWCEPISGHSAVFWYRQTIVQGLEFLTYFRNQAPIDDSGMPKERFSAQMPNQSHSTLKIQSTQPQDSAVYLCASSLDRGQDTQYFGPGTRLLVL (SEQ ID NO:51). TRB-CDR1 is bold; TRB-CDR2 is italic; TRB-CDR3 is bold italic. The α-chain variable structural domain has the following sequence:

[0090] DAKTTQPDSMESTEGETVHLPCSHATISGNEYIYWYRQVPLQGPEYVTHGLQQNTTNSMAFLAIASDRKSSTLILPHVSLRDAAVYHCILRGTGGNNKLTFGQGTVLSVIP (SEQ ID NO:52). TRA-CDR1 is in bold; TRA-CDR2 is in italics; TRA-CDR3 is in bold italics. The three complementarity-determining regions (CDRs) of the β-chain variable structural domain include: CDR1:SGHSA (SEQ ID NO:53), CDR2:FRNQAP (SEQ ID NO:54), and CDR3:ASSLDRGQDTQY (SEQ ID NO:55), and the three CDRs of the α-chain variable structural domain include: CDR1:TISGNEY (SEQ ID NO:56), CDR2:GLQQN (SEQ ID NO:57), and CDR3:ILRGTGGNNKLT (SEQ ID NO:58).

[0091] As used herein, “T-cell receptor” or “TCR” refers to a polypeptide expressed on the membrane surface of CD4+ and CD8+ T lymphocytes. TCRs are antigen receptors that function as components of the immune system, recognizing peptides that bind to major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells. A TCR can be a heterodimer of two disulfide-linked transmembrane polypeptide chains α and β, or γ and δ. Each of these four TCR polypeptide chains is encoded by a different genetic locus containing multiple discontinuous gene segments. These include variable (V) regions, linking (J) regions, and constant (C) regions. The β and δ chains contain additional elements called diversity (D) regions. The variable regions help the TCR identify specific antigens and MHC molecules to which it binds. As used herein, the term TCR includes each of the four individual polypeptide chains, as well as its biologically active fragment, including aqueously soluble fragments, single chains, or linked two chains. The biologically active fragment can maintain the ability to bind specifically to a particular antigen.

[0092] As used herein, a TCR "isotype" refers to a group of TCR polypeptide chains belonging to the α, β, γ, or δ chains. Therefore, in some cases, TCR polypeptides belonging to the same isotype may have different variable regions but may have the same constant regions.

[0093] In some forms, the TCRα and TCRβ variable structural domains disclosed herein can be fused with any TCR constant region.

[0094] In some forms, the CDRs of the TCRα and TCRβ disclosed herein can be fused with the variable regions of antibodies to form antigen-binding domains. The term antibody as used herein refers to a natural or synthetic polypeptide that binds to a target antigen. This term includes polyclonal and monoclonal antibodies, including intact antibodies and functional (e.g., antigen-binding) antibody fragments, including Fab fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments including single-chain variable fragments (scFv), and single-domain antibody fragments (e.g., sdAb, sdFv, nanobody). This term encompasses genetically engineered and / or other modified forms of immunoglobulins, such as intracellular antibodies, peptide antibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugate antibodies, multispecific (e.g., bispecific) antibodies, biantibodies, triantibodies and tetraantibodies, tandem di-scFv, and tandem tri-scFv. The term also encompasses complete or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD. Therefore, although usually discussed in the context of IgG, the target antibody for the variable domain of an Ig-Fc specific immunoglobulin can be another IgG subtype, or a homotype such as IgM, IgE, IgA, or IgD.

[0095] In some forms, the TCRα and TCRβ variable domains and CDR disclosed herein can form part of the antigen-binding domain of CAR-T cells.

[0096] 3. Human cells

[0097] In some forms, the cells are obtained from a human subject. For example, in some forms, the cells are autologous cells, i.e., cells obtained from the subject before the introduction of the TCR peptide and / or the nucleic acid or vector encoding the TCR peptide, and reintroduced into the same subject after modification. In other forms, the cells are heterologous cells, i.e., cells obtained from a subject different from the intended recipient. In some forms, the cells are frozen before or after the introduction of the TCR peptide and / or the nucleic acid or vector encoding the TCR peptide. Methods and compositions for freezing and thawing live eukaryotic cells are known in the art. In some forms, the cells are autologous immune cells, such as T cells or progenitor cells / stem cells.

[0098] In some forms, cells are obtained from healthy subjects. In other forms, cells are obtained from subjects identified as having a disease or condition (such as cancer and / or autoimmune diseases) or at risk of having a disease or condition.

[0099] In some forms, the introduction of TCR peptides into cells occurs through cellular genetic modification. In some forms, cellular genetic modification involves introducing nucleic acids or vectors encoding TCR peptides into cells to induce intracellular expression and presentation of the TCR peptide on the cell surface. In some forms, cellular genetic modification involves transduction using transposons encoding TCR peptides. In some forms, TCR peptides are introduced into cells in vitro by transducing cells with nucleic acids encoding transposons containing TCR.

[0100] i. T cells

[0101] In some forms, the cells are human immune cells, such as T cells. Therefore, in some forms, T cells are obtained from a diseased or healthy subject before amplification and genetic modification. In some forms, the T cells are autologous T cells obtained from the subject before in vitro genetic modification (i.e., to express the TCR of interest) and reintroduction in vivo into the same subject as genetically modified T cells expressing the TCR of interest (such as those identified herein).

[0102] T cells can be obtained from a variety of samples, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In some forms, T cells are obtained from blood units collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL™ isolation. In a preferred form, cells from an individual's circulating blood are obtained via apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. Cells collected via apheresis can be washed to remove plasma fractions and placed in appropriate buffers or culture media for subsequent processing steps. In some forms, cells are washed with phosphate-buffered saline (PBS). In some forms, the wash solution is calcium-deficient and may be magnesium-deficient or may be deficient in many (if not all) divalent cations. After washing, cells can be resuspended in various biocompatible buffers, such as Ca2+-free, Mg2+-free PBS, PLASMALITE A, or other buffered saline solutions. Alternatively, unwanted components of the apheresis sample can be removed, and the cells can be resuspended directly in culture medium.

[0103] In some formulations, T cells are isolated from peripheral blood lymphocytes by lysing erythrocytes and exhausted monocytes, for example by centrifugation through a PERCOLL™ gradient or by countercurrent centrifugation. In certain formulations, specific subsets of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, are further isolated using positive or negative selection techniques. For example, in some formulations, T cells are isolated by incubating with beads conjugated to anti-CD3 / anti-CD28 (i.e., 3 × 28) such as DYNABEADS® M450 CD3 / CD28 T for a duration sufficient to allow for positive selection of the desired T cells. Thus, T cells expressing the heterologous TCRs identified herein are provided on the surface of the T cells.

[0104] In some forms, T cells are genetically modified T cells. For example, in some forms, in addition to containing and expressing the same TCRs within the T cell, the T cells are genetically modified to reduce, prevent, or otherwise alter the expression of one or more genes within the “wild-type” T cells. In some forms, T cells are modified to reduce or prevent the expression of one or more surface receptors that may interfere with the function or structure of the TCRs disclosed herein.

[0105] ii. Delivery medium

[0106] Any disclosed composition, including but not limited to disclosed TCR proteins and / or nucleic acids, can be delivered to target cells using a delivery medium. Delivery media may be, for example, polymer particles, inorganic particles, silica particles, liposomes, micelles, multilayer vesicles, etc.

[0107] The delivery medium can be microparticles or nanoparticles. Nanoparticles are commonly used for intertissue applications, cell penetration, and certain routes of administration. Nanoparticles can have any desired size for the intended use. Nanoparticles can have any diameter from 10 nm to up to about 1,000 nm. Nanoparticles can have diameters of 10 nm to 900 nm, 10 nm to 800 nm, 10 nm to 700 nm, 10 nm to 600 nm, 10 nm to 500 nm, 20 nm to 500 nm, 30 nm to 500 nm, 40 nm to 500 nm, 50 nm to 500 nm, 50 nm to 400 nm, 50 nm to 350 nm, 50 nm to 300 nm, or 50 nm to 200 nm. In some forms, nanoparticles can have diameters less than 400 nm, less than 300 nm, or less than 200 nm. This range can be from 50 nm to 300 nm.

[0108] Therefore, in some forms, the delivery medium is a nanoscale composition, for example, from 10 nm to about 1 micrometer, but excluding about 1 micrometer. However, it should be understood that in some forms, and for some applications, the particles may be smaller or larger (e.g., microparticles, etc.). Although many compositions may be referred to as nanoparticle or nanocarrier compositions, it should be understood that in some forms and for some applications, the carrier may be slightly larger than the nanoparticles. Such compositions may be referred to as microparticle compositions. The size of the microparticles may have a diameter, for example, from 0.1 to 100 μm.

[0109] 4. Pharmaceutical Composition

[0110] In some forms, a pharmaceutical composition containing genetically modified cells or a population of genetically modified cells expressing the disclosed TCR protein may be administered to a subject.

[0111] In some forms, the pharmaceutical composition comprises one or more of a pharmaceutically acceptable buffer, carrier, diluent, or excipient. In some forms, the pharmaceutical composition comprises a specific number or population of cells, such as a population of cells expanded by culturing and expanding genetically modified cells isolated (e.g., T cells expressing the disclosed TCR), such as a homogeneous population. Thus, in some forms, the pharmaceutical composition comprises a homogeneous population of modified cells including and / or expressing the disclosed TCR peptide. In other forms, the pharmaceutical composition comprises a population of cells containing variable or different genetic modifications, such as a heterogeneous population. In some forms, the pharmaceutical composition comprises bispecific or multispecific cells. Any composition may comprise one or more human monoclonal antibodies, such as specific antigens targeted by T cells expressing the disclosed TCR.

[0112] In some forms, cells have been isolated from diseased or healthy subjects before the genes are modified to express the disclosed TCR peptide.

[0113] The term "pharmaceutically acceptable carrier" describes a pharmaceutically acceptable material, composition, or medium involved in carrying or transporting a compound of interest from one tissue, organ, or part of the body to another tissue, organ, or part of the body. For example, in some forms, a carrier is a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of a carrier must be "pharmaceutically acceptable" because it must be compatible with the other components of the formulation. It must also be suitable for use in contact with any tissue or organ it may come into contact with, meaning it must not carry the risk of toxicity, irritation, allergic reactions, immunogenicity, or any other complications that excessively outweigh its therapeutic benefits.

[0114] In some forms, the pharmaceutical composition comprises buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The pharmaceutical composition may be formulated for delivery via any route of administration. The term "route of administration" may refer to any route of administration known in the art, including but not limited to aerosol, nasal, oral, intravenous, intramuscular, intraperitoneal, inhalation, transmucosal, transdermal, parenteral, implantable pump, continuous infusion, topical application, capsule, and / or injection. The pharmaceutical composition is preferably formulated for intravenous administration.

[0115] Typically, the disclosed pharmaceutical compositions are administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration are usually determined by factors such as the patient's condition and the type and severity of the patient's disease, but the appropriate dosage can be determined through clinical trials.

[0116] The disclosed pharmaceutical compositions may be delivered in a therapeutically effective amount. The precise therapeutically effective amount is the amount of composition that will produce the most effective result in terms of therapeutic efficacy in a given subject. This amount will vary depending on a variety of factors, including, but not limited to, the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dose, and type of drug), the nature of one or more pharmaceutically acceptable carriers in the formulation, and the route of administration. Those skilled in the art in the clinical and pharmacological fields will be able to determine the therapeutically effective amount through routine experiments, for example, by monitoring the subject's response to the administered compound and adjusting the dose accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

[0117] C. Usage Method

[0118] A method is provided for using a composition comprising the disclosed TCR protein and / or T cells expressing the disclosed TCR.

[0119] In a particular form, this method provides enhanced antitumor activity by administering T cells expressing the disclosed TCR. Methods for treating a subject's disease include administering cells expressing the disclosed TCR to the subject.

[0120] 1. Treatment methods

[0121] A treatment method is described, comprising cells and other therapeutic agents containing the disclosed TCR peptide. In a preferred form, the method includes adoptive cell therapy (ACT) using T cells expressing the disclosed TCR protein.

[0122] Exemplary methods involve treating a subject (e.g., a human) with a disease, condition, or symptom associated with the presence or proliferation of unwanted cells by administering to the subject an effective amount of a pharmaceutical composition comprising genetically modified cells containing a TCR polypeptide targeting the unwanted cells (i.e., target cells). In some forms, the method administers to a subject (e.g., a human) genetically engineered T cells expressing a recombinant TCR protein in an amount effective for treating the disease, condition, or symptom. For example, in some forms, the method treats a disease or symptom associated with elevated or specific expression of an antigen by administering to the subject an effective amount of a pharmaceutical composition comprising cells modified to express the TCR protein disclosed herein.

[0123] In some forms, the method treats a subject suffering from a disease, condition, or illness associated with elevated or specific expression of an antigen by administering to the subject an effective amount of a pharmaceutical composition comprising T cells modified to contain a disclosed TCR targeting the antigen. In some forms, the disease, condition, or illness associated with elevated or specific expression of the antigen is cancer, and the antigen is a cancer antigen.

[0124] Typically, this method includes ACT, for example, by providing disclosed T cells carrying a TCR for in vivo therapeutic efficacy. In some forms, ACT methods that include administering disclosed T cells carrying a TCR have enhanced in vivo efficacy compared to ACTs using conventional CAR-T cells.

[0125] A method for treating a subject suffering from a disease, condition, or illness is provided, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising live, viable cells engineered to express a disclosed TCR protein. In some forms, when the method treats a subject suffering from a disease, condition, or illness associated with elevated or specific expression of an antigen, the method comprises administering to the subject an effective amount of T cells modified to express the disclosed TCR protein targeting the antigen. For example, in some forms, the method treats a subject suffering from a disease, condition, or illness associated with elevated or specific expression of an antigen by administering to the subject an effective amount of a pharmaceutical composition comprising genetically modified cells, wherein the cells are modified by introducing the following substance into the cells:

[0126] (i) Nucleic acid, such as DNA or RNA, such as viral RNA or mRNA, optionally but preferably a vector, optionally containing a transposon encoding a TCR protein; and

[0127] (ii) Enable TCR protein to be expressed by cells.

[0128] Prior to gene modification, the cells may have been isolated from a subject suffering from a disease, symptom, or condition, or from a healthy donor. In some forms, the method also includes administering an effective amount of an antibody targeting an antigen, such as a human monoclonal antibody, to the subject. hmAbs can target antigens selected from the group consisting of: cancer antigens, inflammatory disease antigens, neuronal disorder antigens, HIV / AIDS antigens, diabetes antigens, cardiovascular disease antigens, infectious disease antigens (including viral antigens, protozoan antigens, bacterial antigens, and allergens), autoimmune disease antigens, and combinations thereof.

[0129] 2. Disease to be treated

[0130] This provides a method for treating diseases and / or conditions in subjects who require treatment. Subjects to be treated may have diseases, conditions, or illnesses such as, but not limited to, cancer, immune system disorders (such as autoimmune diseases), inflammatory diseases, neuronal disorders, HIV / AIDS, diabetes, cardiovascular diseases, infectious diseases, or combinations thereof. The disease, condition, or illness may be associated with elevated or specific expression of an antigen.

[0131] i. Cancer

[0132] In some forms, the method treats or prevents cancer. In some forms, the method treats or prevents cancer or other proliferative diseases or conditions in subjects identified as having cancer or other proliferative diseases or conditions or at risk of having cancer or other proliferative diseases or conditions. Cancer is a genetically unstable disease that allows cancer cells to acquire features proposed by Hanahan and Weinberg, including (i) self-sufficiency in growth signals; (ii) insensitivity to growth signals; (iii) evasion of apoptosis; (iv) persistent angiogenesis; (v) tissue invasion and metastasis; (vi) unlimited replication potential; (vii) reprogramming of energy metabolism; and (viii) evasion of immune destruction (Cell., 144:646–674, (2011)).

[0133] Tumors that can be treated according to the disclosed methods are classified based on their embryonic origin from the tissue from which they are derived. Carcinomas are tumors that arise in the epithelial lining of endoderm or ectoderm tissues such as the skin or internal organs and glands. Sarcomas occur less frequently and are derived from mesodermal connective tissues such as bone, fat, and cartilage. Leukemia and lymphoma are malignant tumors of hematopoietic cells in the bone marrow. Leukemia occurs as a single-celled proliferation, while lymphoma tends to grow as a tumor mass. Malignant tumors can occur in many organs or tissues of the body to develop into cancer.

[0134] Table 1: Targeting Cancer Antigens of the Disclosed Methods and Compositions. The disclosed TCRs can target exemplary cancers with specific or related antigens.

[0135]

[0136]

[0137] The disclosed compositions and methods can be used to treat one or more cancers provided in Table 1.

[0138] The disclosed compositions and methods of treatment are generally applicable to the treatment of cancer, sarcoma, lymphoma, and leukemia. The described compositions and methods can be used to treat or alleviate the symptoms of benign or malignant tumors in a subject by delaying or inhibiting the growth / proliferation or viability of tumor cells, reducing the number, growth, or size of tumors, inhibiting or reducing tumor metastasis, and / or inhibiting or reducing symptoms associated with tumor development or growth.

[0139] The types of cancers that can be treated with the provided compositions and methods include, but are not limited to, cancers such as angiomyocarcinoma, multiple myeloma, adenocarcinomas and sarcomas of the bone, bladder, brain, breast, cervix, colorectal, esophagus, kidney, liver, lung, nasopharynx, pancreas, prostate, skin, stomach, and uterus. The following experiments support the conclusion that the methods of this disclosure are effective in treating solid tumors. Therefore, in some forms, the target cancer is a solid tumor. In some forms, the composition is used to treat multiple cancer types simultaneously. The composition can also be used to treat metastatic tumors or tumors in multiple sites.

[0140] Exemplary tumor cells include, but are not limited to, cancerous tumor cells, including leukemia, including but not limited to acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia such as myeloblasts, promyelocytic leukemia, myelomonocytes, monocytic leukemia, erythroleukemia, and myelodysplastic syndromes; chronic leukemia such as, but not limited to, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, and hairy cell leukemia; polycythemia vera; lymphoma, such as, but not limited to, Hodgkin's disease and non-Hodgkin's disease; multiple myeloma, such as, but not limited to, smoldering multiple myeloma, non-secreting myeloma, osteosclerosing myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma; Waldenström macroglobulin. Blood disorders; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas, such as but not limited to osteosarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, osteofibrosarcoma, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain tumors, including but not limited to glioma, astrocytoma, brainstem glioma, ependymoma, oligodendroglioma, nonglioma. Tumors, including but not limited to: acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineal cell carcinoma, pinealoblastoma, and primary brain lymphoma; breast cancer, including but not limited to adenocarcinoma, lobular (small cell) carcinoma, ductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease, and inflammatory breast cancer; adrenal cancer, including but not limited to pheochromocytoma and adrenocortical carcinoma; and thyroid cancer, such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer. Pancreatic cancer, including but not limited to insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumors, and carcinoid or islet cell tumors.Pituitary cancer, including but not limited to Cushing's disease, prolactin-secreting tumors, acromegaly, and diabetes insipidus; eye cancer, including but not limited to ocular melanoma such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancer, including but not limited to squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, including but not limited to squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancer, including but not limited to squamous cell carcinoma and adenocarcinoma; uterine cancer, including but not limited to endometrial cancer and uterine sarcoma; ovarian cancer, including but not limited to ovarian epithelial cancer, borderline tumors, germ cell tumors, and stromal tumors; esophageal cancer, including but not limited to squamous cell carcinoma, adenocarcinoma, and adenoid cystic carcinoma. Carcinoma, including but not limited to mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; gastric cancer, including but not limited to adenocarcinoma, fungal (polypoid), ulcerative, superficial, diffuse, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancer; rectal cancer; liver cancer, including but not limited to hepatocellular carcinoma and hepatoblastic carcinoma; gallbladder cancer, including but not limited to adenocarcinoma; cholangiocarcinomas, including but not limited to papillary, nodular, and diffuse; lung cancer, including but not limited to non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung cancer; testicular cancer, including but not limited to germinal tumor, seminoma, anaplastic, classic (typical), spermatogenic, non-seminomatous, embryonal carcinoma, teratoma, choriocarcinoma (yolk sac tumor); prostate cancer, including but not limited to adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancer. Cancer); oral cancer, including but not limited to squamous cell carcinoma; basal carcinoma; salivary gland cancer, including but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; pharyngeal cancer, including but not limited to squamous cell carcinoma and verrucous carcinoma; skin cancer, including but not limited to basal cell carcinoma, squamous cell carcinoma, melanoma, superficial spreading melanoma, nodular melanoma, lentigines-like malignant melanoma, acral lentigines-like nevus melanoma; renal cancer, including but not limited to renal cell carcinoma, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell carcinoma (renal pelvis and / or uterus); Wilms' tumor;Bladder cancer, including but not limited to transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and carcinosarcoma. A review of this disease can be found in Fishman et al., 1985, Medicine, 2d Ed.; JBLippincott Co.; Philadelphia and Murphy et al., 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery (Viking Penguin, Penguin Books USA, Inc., United States of America).

[0141] ii. Immune system disorders

[0142] In some forms, this method administers modified T cells containing TCR-protein to treat or prevent one or more immune system disorders, including autoimmune diseases.

[0143] In some cases, the immune system's ability to distinguish between self-antigens and foreign antigens can be misdirected towards healthy tissues, leading to unintended attacks and destruction of normal host cells (i.e., autoimmune diseases). Autoimmune diseases encompass more than 100 types of illnesses, and their etiology and prognosis can vary across different populations based on factors such as the affected region, age of onset, response to treatments, and clinical presentation (Muhammad et al., Chimeric Antigen Receptor Based Therapy as a Potential Approach in Autoimmune Diseases: How Close Are We to the Treatment, Frontiers in Immunology, 11 (2020)).

[0144] B lymphocytes that secrete autoantibodies and autoreactive T lymphocytes play a crucial role in the development of autoimmune diseases. Autoimmunity is divided into two main categories based on the extent of tissue damage: organ-specific and systemic autoimmunity. The former involves specific areas of the body, such as type 1 diabetes (T1D), multiple sclerosis (MS), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), and myasthenia gravis (MG), while the latter affects multiple areas of the body, leading to systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS). Therefore, in some forms, this method treats or prevents one or more organ-specific autoimmune diseases in a subject. In other forms, this method treats or prevents one or more systemic autoimmune diseases in a subject.

[0145] In some forms, the method reduces or prevents one or more physiological processes associated with the development or progression of an autoimmune disease in a subject. For example, in some forms, the method reduces or prevents the spread of one or more epitopes, such as where infection alters a primary epitope to a secondary epitope or forms several new epitopes on antigen-presenting cells; bystander activation or pre-initiated autoreactive T cell activation in a T cell receptor (TCR)-independent manner; persistent viral infection or the persistence of viral antigens that induce an immune response; or immune cross-reactivity between the host and the pathogen, for example, due to shared immune epitopes or sequence similarity.

[0146] In some forms, the method applies T cells expressing the disclosed TCR, which are involved in initiating or otherwise developing or maintaining immune system diseases or conditions. Non-limiting examples of immune system disorders that can be treated or prevented by the methods described include 22q11.2 deletion syndrome, achondroplasia and severe combined immunodeficiency, adenosine deaminase 2 deficiency, adenosine deaminase deficiency, adult-onset immunodeficiency with anti-interferon-γ autoantibodies, agammaglobulinemia, non-Bruton type, Aicardi-Goutieres syndrome, Aicardi-Goutieres syndrome type 5, allergic bronchopulmonary aspergillosis, alopecia, total alopecia, generalized alopecia, amyloidosis (AA), familial amyloidosis, ataxia-telangiectasia, autoimmune lymphoproliferative syndrome, and autoimmune lymphoproliferative syndrome due to CTLA4 haploinsuffiency.(haploinsuffiency), type 1 autoimmune polyglandular syndrome, autosomal dominant high IgE syndrome, autosomal recessive early-onset inflammatory bowel disease syndrome, autosomal recessive high IgE syndrome, naked lymphocyte syndrome 2, Barth syndrome, Blau syndrome, Bloom syndrome, obliterative bronchiolitis, C1q deficiency, familial chronic mucocutaneous candidiasis, autosomal recessive, chondrodysplasia, CHARGE syndrome, Chediak-Higashi syndrome, cherubism, chronic atypical neutrophilic dermatitis with lipoatrophy and fever syndrome, chronic graft-versus-host disease, chronic granulomatous disease, chronic neurocutaneous joint syndrome of infants, chronic mucocutaneous candidiasis (CMC), Cohen syndrome, combined immunodeficiency with cutaneous granuloma, common variable immunodeficiency, complement component 2 deficiency, type 1 complement deficiency. Complement 8 deficiency, type 2 complement 8 deficiency, congenital alveolar proteinosis, cryoglobulinemia, cutaneous mast cell tumor, cyclic neutropenia, interleukin-1 receptor antagonist deficiency, dendritic cell, monocyte, B lymphocyte and natural killer lymphocyte deficiency, congenital dyskeratosis, autosomal dominant congenital dyskeratosis, autosomal recessive congenital dyskeratosis, X-linked congenital dyskeratosis, verrucous epidermal dysplasia, familial amyloidosis, Finnish type, familial cold autoinflammatory syndrome, familial Mediterranean fever, familial mixed cryoglobulinemia, Felty syndrome, type 1B glycogen storage, type 2 Griscelli syndrome, Hashimoto's encephalopathy, Hashimoto's syndrome, hemophagocytic lymphohistiocytosis, Hennekam syndrome, hepatic vein occlusive disease with immunodeficiency, hereditary folate malabsorption. folatema labsorption), Hermansky-Pudlak syndrome 2, herpes simplex encephalitis, Hoyera alHreidarsson syndrome, hyper-IgE syndrome, hyper-IgD syndrome, ICF syndrome, idiopathic acute eosinophilic pneumonia, idiopathic CD4-positive T-lymphopenia, IL12RB1 deficiency, immunodeficiency due to thymus agenesis, immunodeficiency due to calcium entry defect 1 with T-cell inactivation, immunodeficiency due to calcium entry defect 2 with T-cell inactivation, type 1 hyper-IgM immunodeficiency, type 2 hyper-IgM immunodeficiency, type 3 hyper-IgM immunodeficiency, type 4 hyper-IgM immunodeficiency, type 5 hyper-IgM immunodeficiency, thymoma immunodeficiency, immunodeficiency due to anhidrosis of ectodermal dysplasia, immune dysregulation, X-linked polyendocrinopathy, and Enteropathy, Immunoglobulin A deficiency 2, Multiple intestinal atresia, IRAK-4 deficiency, Type 3 solitary growth hormone deficiency, Kawasaki disease, Large granular lymphocytic leukemia, Type 1 leukocyte adhesion deficiency, LRBA deficiency, Lupus, Lymphocytic hypophysitis, Majeed syndrome, Melkersson-Rosenthal syndrome, Type 1 MHC deficiency, Muckle-Wells syndrome, Multifocal fibrosis, Multiple sclerosis, MYD88 deficiency, Neonatal systemic lupus erythematosus, Netherton syndrome, Neutrophil-specific granuloma agenesis, Niemhen rupture syndrome, Omenn syndrome, Osteosclerosis autosomal recessive 7, Relapsing rheumatism, Papillon Lefevre syndrome, partial androgen insensitivity syndrome, PASLI, Pearson syndrome, multiple sclerosis in children, periodic fever, aphthous stomatitis, pharyngitis and adenitis, PGM3-CDG, porcine dyschromia with neutropenia, pruritic urticaria of pregnancy, purine nucleoside phosphorylase deficiency, pyogenic arthritis, pyoderma gangrenosa and acne, relapsing polychondritis, reticular dysplasia, sarcoidosis, Say Barber Miller syndrome, Schimke immune bone dysplasia, Schnitzler syndrome, selective IgA deficiency, selective IgM deficiency, severe combined immunodeficiency, severe combined immunodeficiency due to complete RAG1 / 2 deficiency, severe combined immunodeficiency with ionizing radiation sensitivity, severe combined immunodeficiency, severe congenital neutropenia (autosomal recessive inheritance), X-linked severe congenital neutropenia, Shwachman-Diamond syndrome, Singleton-Merten syndrome, SLC35C1-CDG(CDG-IIc), specific antibody deficiency, spondyloenchondrodysplasia, Stevens-Johnson syndrome, T-cell immunodeficiency, congenital alopecia and nail dystrophy, TARP syndrome, Trichohepatoenteric syndrome, tumor necrosis factor receptor-related periodic syndrome, twin-to-twin transfusion syndrome, Vici syndrome, WHIM syndrome, Wiskott-Aldrich syndrome, Woods-Black-Norbury syndrome, X-linked agammaglobulinemia, X-linked lymphoproliferative syndrome, X-linked lymphoproliferative syndrome 1, X-linked lymphoproliferative syndrome 2, X-linked magnesium deficiency with Epstein-Barr virus infection and tumors, X-linked severe combined immunodeficiency and ZAP-70 deficiency.

[0147] The disclosed compositions and methods can also be used to treat autoimmune diseases or conditions. Exemplary autoimmune diseases or conditions that are not mutually exclusive with the above-mentioned immune system conditions include achalasia, Addison's disease, adult-onset Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune autonomic dysfunction, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, and autoimmune orchitis. Autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neuronal neuropathy (AMAN), Baló disease, Behcet's disease, benign mucosal pemphigoid, bullous pemphigoid, Castleman's disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis (EGPA), cicatricial pemphigoid, Cogan syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, herpetic dermatitis, dermatomyositis, Devic disease (neuromyelitis optica), discoid lupus, Dressler syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, primary mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrotic alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture syndrome, granulomatous disease with polyangiitis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, hemolytic anemia, allergic purpura (HSP), herpes gestationis or pemphigoid of pregnancy (PG), hidradenitis suppurativa (HS) (acne paradox) Inversa, hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosis, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytic clotting vasculitis, lichen planus, lichen sclerosus (Lichen)sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus, chronic Lyme disease, Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatism (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, parsplanitis (peripheral uveitis) Pemphigoid, peripheral neuropathy, peripheral encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyadenomas type I, II, and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrene, Raynaud's phenomenon, reactive arthritis, recurrent polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff person syndrome. Susac syndrome (SPS), subacute bacterial endocarditis (SBE), Susac syndrome, sympathetic ophthalmitis (SO), Takayasu arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes mellitus, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, and Wegener's granulomatosis (or granulomatosis with polyangiitis (GPA)).

[0148] iii. Other diseases or conditions

[0149] In some forms, the method administers modified T cells containing the disclosed TCR protein to a subject in need. For example, in some forms, the method treats a subject with one or more genetic diseases or conditions, such as hereditary genetic diseases or conditions, or somatic genetic diseases or conditions in the subject.

[0150] Any method may include treatment for a subject with an underlying disease or condition. For example, in some forms, the method treats a patient with another disease or condition (such as diabetes, a bacterial infection (e.g., tuberculosis), a viral infection (e.g., hepatitis, HIV, HPV infection, etc.), or a drug-related disease or condition (such as cancer or an autoimmune disease). In some forms, the method treats a subject with an immunocompromised. In some forms, the method treats a subject with a disease of the kidney, liver, heart, lungs, brain, bladder, reproductive system, intestines / guts, stomach, bone, or skin.

[0151] D. Application

[0152] In some forms, the method involves administering modified T cells containing the disclosed TCR protein in an amount sufficient to effectively treat or prevent one or more diseases or conditions in a subject.

[0153] An effective or therapeutically effective amount of a pharmaceutical composition comprises a dose of modified cells, such as therapeutic T cells expressing the disclosed TCR, which may be sufficient to treat, suppress or alleviate one or more symptoms of a disease or condition (such as cancer or an autoimmune disease), or otherwise provide a desired pharmacological and / or physiological effect (e.g., reducing, suppressing or reversing one or more underlying pathophysiological mechanisms of a disease or condition (such as cancer or an autoimmune disease)).

[0154] In some forms, when administration of a pharmaceutical composition comprising modified cells (such as therapeutic T cells, including T cells expressing the disclosed TCR) elicits an anticancer response, the amount administered can be expressed as the amount that effectively achieves the desired anticancer effect in the recipient. For example, in some forms, the amount of a pharmaceutical composition comprising modified cells (such as therapeutic T cells expressing the disclosed TCR) effectively inhibits the viability or proliferation of cancer cells in the recipient. In some forms, the amount of a pharmaceutical composition comprising modified cells (such as therapeutic T cells expressing the disclosed TCR) effectively reduces the tumor burden or the total number of cancer cells in the recipient, and combinations thereof.

[0155] In other forms, the amount of a pharmaceutical composition containing modified cells (such as T cells expressing the disclosed TCR) is effective in reducing one or more symptoms or signs of cancer in cancer patients, or in effectively reducing signs of autoimmune diseases or conditions in patients. Signs of cancer may include cancer markers, such as PSMA levels in the patient's blood.

[0156] The effective amount of a pharmaceutical composition containing modified cells (such as T cells expressing the disclosed TCR) required will vary from subject to subject, depending on the subject's species, age, weight and general condition, the severity of the condition being treated, and the mode of administration. Therefore, it is not possible to specify an exact amount for each pharmaceutical composition. However, an appropriate amount can be determined by those skilled in the art using only the routine experiments given in the teachings herein. For example, the effective dose and schedule for administering a pharmaceutical composition containing T cells expressing the disclosed TCR can each be determined empirically, and such determination is within the scope of the art. In some forms, the dose range for administering a composition containing T cells expressing the disclosed TCR is sufficiently large to achieve a reduction in cancer cell proliferation or viability, or, for example, a reduction in tumor burden.

[0157] The dosage should not be so high as to cause adverse side effects, such as unwanted cross-reactions or allergic reactions. Typically, the dosage will vary depending on the patient's age, condition, and sex, route of administration, whether other medications are included in the regimen, and the type, stage, and location of the disease being treated. If any contraindications occur, the dosage may be adjusted by an individual physician. It should also be understood that the effective dose of the composition used for treatment, containing T cells expressing the disclosed TCR, may be increased or decreased during a specific treatment course. Dosage variations can arise from and become apparent from diagnostic assay results.

[0158] Dosage can vary and can be administered once or multiple times daily for one or more days. Guidelines for appropriate dosages for a given class of drug products can be found in the literature. Optimal dosing schedules can be calculated based on measurements of drug accumulation in a subject or patient. Optimal dosage, administration method, and repetition rate can be easily determined by a competent technician. Optimal dosages can vary depending on the relative potency of individual drug compositions and can often be estimated based on the EC50 found to be effective in in vitro and in vivo animal models.

[0159] Generally speaking, a pharmaceutical composition containing T cells expressing the disclosed TCR can be administered at a dose of 10⁴ to 10⁹ cells / kg body weight, preferably 10⁵ to 10⁷ cells / kg body weight (inclusive of all integer values ​​within those ranges). In some forms, a patient can be treated by infusion of the disclosed pharmaceutical composition containing T cells (e.g., T cells) expressing the disclosed TCR at a rate of about 10⁴ to 10¹² cells or more per square meter of body surface area (cells / m²).

[0160] Infusions can be repeated at a frequency and number of times that the patient can tolerate until the desired response is achieved. T cells expressing the disclosed TCR composition can also be administered once or multiple times at these doses. Cells can be administered using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a particular patient can be readily determined by a person skilled in the medical field by monitoring the patient for signs of disease and adjusting treatment accordingly. In some forms, the unit dose of T cells expressing the disclosed TCR is a unit dosage form for intravenous injection. In some forms, the unit dose is a unit dosage form for oral administration. In some forms, the unit dose is a unit dosage form for inhalation. In some forms, the unit dose is a unit dosage form for intratumoral injection.

[0161] Treatment can last a sufficient amount of time to achieve one or more desired treatment goals, such as a reduction in the amount of cancer cells relative to the start of treatment or the complete absence of cancer cells in the recipient. Treatment can continue for the desired period of time, and progress can be monitored using any known means for monitoring the progression of anticancer treatment in a patient. In some forms, it is administered every day, or weekly, or for each part of the week. In some forms, the treatment regimen is administered over a period of up to two, three, four, or five days, several weeks, or several months, or up to six months, or longer than six months, such as up to one, two, three, or five years.

[0162] The efficacy of a pharmaceutical composition containing modified cells (such as therapeutic T cells) administered at a specific dose according to the methods described herein can be determined by assessing aspects of a subject’s medical history, signs, symptoms, and objective laboratory tests known to be useful for assessing the condition of a subject in need of treatment for cancer or other diseases and / or conditions. These signs, symptoms, and objective laboratory tests will vary depending on the specific disease or condition being treated or prevented, as is known to any clinician treating such patients or researchers conducting experiments in the field. For example, a particular treatment regimen would be considered effective if, based on comparison with an appropriate control group and / or knowledge of the normal progression of disease in the general population or a particular individual: (1) the subject’s physical condition shows improvement (e.g., the tumor has partially or completely regressed), (2) the progression of the disease or condition shows stabilization, slowing, or reversal, or (3) the need for other medications used to treat the disease or condition is reduced or eliminated. In some forms, efficacy is assessed as a measure of the reduction in tumor volume and / or tumor mass at a specific time point after treatment (e.g., 1–5 days, weeks, or months).

[0163] In some forms, the method administers a combination of modified T cells expressing the disclosed TCR protein and a pharmaceutically acceptable carrier. The compositions described herein can be readily formulated into pharmaceutical compositions consisting of one or more compounds combined with a pharmaceutically acceptable carrier. See, for example, Remington's PharmaceuticalSciences, latest edition, EW Martin Mack Pub. Co., Easton, PA, which discloses classic carriers and conventional methods for preparing pharmaceutical compositions that can be used in conjunction with the preparation of formulations of the therapies described herein, and which are incorporated herein by reference. These are most typically standard carriers for administering the compositions to humans. In one aspect, for both humans and non-humans, these include solutions such as sterile water, saline, and buffered solutions of physiological pH. Other therapies may be administered according to standard procedures used by those skilled in the art.

[0164] In addition to the selected therapeutic agent, the pharmaceutical compositions comprising modified cells (such as therapeutic T cells) described herein may also include, but are not limited to, carriers, thickeners, diluents, buffers, preservatives, surfactants, etc.

[0165] Pharmaceutical compositions comprising one or more modified cells (such as therapeutic T cells expressing the disclosed TCR) and optionally one or more additional therapeutic agents can be administered to a subject in a variety of ways, depending on whether local or systemic treatment is required and the area to be treated. Thus, for example, a pharmaceutical composition comprising modified cells (such as therapeutic T cells expressing the disclosed TCR) can be administered as an intravenous infusion or injected directly into a specific site, such as into or around a tumor. Furthermore, the pharmaceutical composition can be applied to the surface of the subject's eye, vagina, rectum, nose, orally, by inhalation, or parenterally, such as via intradermal, subcutaneous, intramuscular, intraperitoneal, intrarectal, intraarterial, intralymphatic, intravenous, intrathecal, and intratracheal routes. In some forms, the composition is applied directly to a tumor or tissue, for example, directed.

[0166] Parenteral administration (if used) is typically characterized by injection. Injectable formulations can be prepared in conventional forms, as liquid solutions or suspensions, as solid forms suitable for dissolution or suspension in a liquid prior to injection, or as emulsions. Recently revised parenteral administration methods involve the use of sustained-release or sustained-release systems to maintain a constant dose. See, for example, U.S. Patent No. 3,610,795, which is incorporated herein by reference. Suitable routes of parenteral administration include intravascular administration (e.g., intravenous bolus, intravenous infusion, intra-arterial bolus, intra-arterial infusion, and catheter infusion into the vascular system); peritectonic and intratissue injections (e.g., intraocular, intraretinal, or subretinal injections); subcutaneous injections or depositions, including subcutaneous infusions (such as via an osmotic pump); and direct application via catheters or other placement devices (e.g., implants comprising porous, non-porous, or gel-like materials).

[0167] Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, and may also contain buffers, diluents, and other suitable additives. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions, including saline and buffer media. Parenteral media include sodium chloride solutions, Ringer's dextran, dextran and sodium chloride, lactated Ringer's, or non-volatile oils. Intravenous media include fluids and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextran), etc. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases.

[0168] The administration of a pharmaceutical composition containing cells with one or more gene modifications (e.g., T cells expressing the disclosed TCR) can be local (i.e., a specific region, physiological system, tissue, organ, or cell type) or systemic.

[0169] It should be understood that, unless otherwise stated, the disclosed methods and compositions are not limited to specific synthetic methods, specific analytical techniques, or specific reagents, and therefore can vary. It should also be understood that the terminology used herein is for descriptive purposes only and is not intended to be limiting.

[0170] E. Combination therapy

[0171] In some forms, the method involves administering modified T cells expressing the disclosed TCR protein in combination with other therapeutic agents or forms of treatment. Any disclosed pharmaceutical composition containing modified cells (such as therapeutic T cells, e.g., a population of T cells expressing the disclosed TCR) can be used alone or in combination with other therapeutic agents or forms of treatment (e.g., chemotherapy or stem cell transplantation). As used herein, “combination” or “in combination” means the administration of therapeutic agents concurrently with, simultaneously with, or sequentially.

[0172] In some forms, the pharmaceutical composition and other therapeutic agents are administered separately via the same route of administration. In other forms, the pharmaceutical composition and other therapeutic agents are administered separately via different routes of administration. The combination may be administered concurrently (e.g., as a mixture), separately but simultaneously (e.g., via a separate intravenous line to the same subject; one agent is administered orally while another agent is administered by infusion or injection, etc.), or sequentially (e.g., one agent is administered first, followed by the second agent).

[0173] Examples of preferred alternative therapeutic agents include other conventional therapies known in the art for treating the desired disease, condition, or illness. In some forms, the therapeutic agent is one or more other targeted therapies (e.g., targeted cancer therapies) and / or immune checkpoint blockers (e.g., anti-CTLA 4, anti-PD1, and / or anti-PDL1 agents, such as antibodies).

[0174] The compositions and methods described herein can be used as a first, second, third, or combination therapy with other types of therapies known in the art, such as chemotherapy, surgery, radiotherapy, gene therapy, immunotherapy, bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radiofrequency ablation, etc., in an adjuvant or neoadjuvant setting.

[0175] The disclosed pharmaceutical compositions and / or other therapeutic agents, procedures, or forms may be administered during active disease or during remission or less active disease. The pharmaceutical compositions may be administered before, concurrently with, after, or during disease or symptom remission. When administered in combination, the disclosed pharmaceutical compositions and additional therapeutic agents (e.g., a second or third agent) or all of them may be administered at a higher, lower, or the same amount or dose than each agent used alone (e.g., as a monotherapy). In some forms, the disclosed pharmaceutical compositions, additional therapeutic agents (e.g., a second or third agent), or all of them are administered at a lower amount or dose than each agent used alone (e.g., as a monotherapy) (e.g., as required to achieve the same therapeutic effect) (e.g., at least 20%, at least 30%, at least 40%, or at least 50%).

[0176] F. Methods for identifying TCR

[0177] 1. Generate tumor models

[0178] This paper discloses a method for introducing vectors into hepatocytes that can induce tumor formation in mice using a hydrodynamic injection technique. These vectors consist of a Sleeping Beauty transposon vector expressing an oncogene and a CRISPR vector carrying a guide RNA containing a tumor suppressor gene. The combined effect of proto-oncogene expression and tumor suppressor gene deletion leads to the development of hepatocellular carcinoma (HCC) in mice. This method provides a convenient, efficient, liver-specific, and non-viral approach that can be applied to both wild-type and genetically modified mice without the need for transgenic mice or complex crossbreeding.

[0179] In some forms, transposon vectors can be used to overexpress proto-oncogenes such as c-myc.

[0180] In some forms, CRISPR vectors can be used for deletions or mutations in the second exon of Trp53 and the first exon of Pten.

[0181] Hepatocellular carcinoma (HCC) develops due to the combined effects of Myc expression and Trp53 and Pten ablation. In mice, Trp53 and Pten ablation, along with Myc overexpression, to develop HCC mimics the multi-stage and multi-hit process of human liver carcinogenesis.

[0182] Non-human animal models are suitable for research purposes, such as studying the molecular and genetic mechanisms associated with the development of recurrent HCC tumors, identifying potential therapeutic targets, and testing potential compounds for the treatment of recurrent HCC.

[0183] Therefore, a non-human animal model is a genetically modified non-human animal. As used herein, the term "genetically modified non-human animal" refers to a non-human animal that possesses foreign DNA in at least one chromosome of its genome. In some forms, at least one or more cells of the genetically modified non-human animal, for example, at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, or 50% of its cells, possess foreign DNA in their genome. Cells possessing foreign DNA (i.e., target cells) can be various types of cells, such as endogenous cells, somatic cells, immune cells, T cells, B cells, or endogenous tumor cells. In some forms, the genetically modified non-human animal contains a modified endogenous locus containing a foreign sequence (e.g., a human sequence), for example, replacing one or more non-human sequences with one or more human sequences. Non-human animals typically cannot pass on the modification to their offspring, i.e., through germline transmission.

[0184] In some embodiments, the target cells are liver cells. Exemplary target liver cells include, but are not limited to, hepatocytes, Kupffer cells, stellate cells, sinusoidal endothelial cells (SEC), bile duct cells, bile duct epithelial cells (BEC), hepatic progenitor cells (LPC), pit cells, or liver-associated natural killer (NK) cells, dendritic cells, and fenestrated sinusoidal endothelial cells (LSEC). In a preferred embodiment, the target liver cells are hepatocytes.

[0185] Non-human animal models develop focal HCC tumors some time after integration with the model generation system. In some forms, the timeframe for focal HCC tumor development is approximately 3 to 5 weeks, more preferably approximately 4 weeks, after integration with the model generation system. Typically, non-human animal models develop one or more recurrent HCC liver tumors some time after surgical resection of the focal tumor. In some forms, the timeframe for recurrent HCC tumor development is approximately 6 to 11 weeks after surgical resection of the focal tumor. In some forms, the timeframe for recurrent HCC tumor development is approximately 6 weeks to 7 months after surgical resection of the focal tumor. In some forms, the timeframe for recurrent HCC tumor development is approximately 6 weeks, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, and approximately 7 months after surgical resection of the focal tumor.

[0186] Recurrent HCC tumor cells and tissues are defined structurally and functionally as described herein, using methods and assays similar to those described below. Because recurrent HCC tumor cells and tissues are known to evolve phenotypedly and functionally over time with the occurrence of additional gene mutations, they can change phenotypedly and functionally over time in non-human animal models. However, recurrent HCC tumor cells and tissues can be consistently isolated and / or identified using the methods described herein and employing biomarkers disclosed herein. In some forms, HCC tumor cells and tissues express cell surface biomarkers, including but not limited to alpha-fetoprotein and phosphatidylinositol proteoglycan 3.

[0187] Typically, non-human animal models are immunocompetent animals. As used herein, an "immunocompetent" animal is one in which its innate or naturally occurring innate and adaptive immune system has been preserved (i.e., not artificially altered), allowing the animal to retain its normal ability to generate an immune response against foreign antigens. One advantage is that the animal model already possesses specific tolerance to certain xenogeneic cells and / or tissues (e.g., cells and / or tissues generated by the model-generating system), preventing it from recognizing such xenogeneic cells and / or tissues as foreign. Therefore, cells and / or tissues generated by the model-generating system can be obtained without resorting to germline genetic modification of the recipient animal's innate immune system or the use of immunosuppressants to prevent the animal from rejecting xenogeneic cells and / or tissues. Another advantage is that the animal retains its ability to generate a normal immune response against cells and / or tissues generated by the model-generating system. Therefore, such immunocompetent animals will typically have normal and / or unaltered T, B, and / or NK cells (provided they are not artificially altered or engineered; it should be understood that natural mutations may occur, but otherwise they will not significantly impair the animal's normal immune response). Non-human animal models exhibit immune tolerance to HCC tumor cells while maintaining a capable immune system. Animal models are "tolerable" to HCC tumor cells, meaning they are immune-tolerant, allowing them to maintain a tolerant state towards HCC tumor cells rather than initiating an immune response or rejection of them. As described herein, the term "tolerable" generally refers to animals that tolerate HCC tumors rather than being immunocompromised or immunodeficient through the use of germline genetic modifications or immunosuppressants. This contrasts with immunodeficient or immunocompromised animals, where the natural or innate immune response is attenuated, weakened, or reduced, altering the animal's ability to fight foreign antigens. Such animals typically possess atypical T, B, and / or NK cells. In a preferred form, the tumor is derived from an immune-active mouse; therefore, the tumor is not rejected by the mouse's immune system.

[0188] The target animal used to accommodate the model generation system is preferably a mammal. In some forms, the mammal is a rodent, such as a mouse, rat, squirrel, prairie dog, porcupine, beaver, guinea pig, and hamster. In some forms, the target animal is a rabbit. In some forms, the target animal is a zebrafish. In a preferred form, the target animal is a rodent, preferably a mouse or rat. The selected target animal will be used for various purposes, including modeling and research of human diseases and evaluation of treatments. In one or more forms, the target animal can be used as a model of recurrent HCC and / or a method for modeling recurrent HCC using the aforementioned model generation system. For example, HCC tumors can be established in recipient animals via the model generation system for testing drugs, cell therapies, photodynamic therapy, magnetothermal therapy, gene therapy, etc. The model generation system can be used to promote the development of HCC tumor cells and / or tissues, particularly in the liver. Therefore, it is desirable to restrict the movement of tumor cells to promote tumor formation in the liver.

[0189] In some forms, the non-human animal is selected from the following C57BL strain mice: C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / Ola. In some forms, the mice are 129 strain mice selected from the following groups of strains: 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, and 129T2. These mice are described, for example, in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10:836 (1999); and Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines (2000), both of which are incorporated herein by reference in their entirety. In some forms, the mice are a hybrid of the 129 strain and the C57BL / 6 strain. In some forms, the mice are a hybrid of the 129 strain or the BL / 6 strain. In some forms, the mice are a BALB strain, such as the BALB / c strain. In some forms, the mice are a hybrid of the BALB strain and another strain. In some forms, the mice are derived from hybrid lines (e.g., 50% BALB / c-50% 12954 / Sv; or 50% C57BL / 6-50% 129).

[0190] In some forms, the non-human animal is a rat. The rat can be selected from Wistar rats, the LEA strain, the Sprague Dawley strain, the Fischer strain, F344, F6, and Dark Agouti. In some forms, the rat strain is a mixture of two or more strains selected from the following group: Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

[0191] In some forms, CRISPR-mediated somatic knockout of tumor suppressor genes has been used to induce hepatocellular carcinoma (HCC) in mice.

[0192] In some forms, CRISPR-mediated gene knockout involves the deletion of one or both of the tumor suppressor genes Trp53 and Pten.

[0193] The term “CRISPR” (clustered regularly spaced short palindromic repeats) is an acronym for a DNA locus containing multiple short, orthogonally repeated base sequences. The prokaryotic CRISPR / Cas system has been adapted for gene editing (silencing, enhancing, or altering specific genes) in eukaryotes (see, for example, Cong, Science, 15:339(6121):819–823(2013) and Jinek et al., Science, 337(6096):816-21(2012)). Methods for preparing compositions for genome editing using the CRISPR / Cas system are described in detail in WO 2013 / 176772 and WO2014 / 018423 (which are specifically incorporated herein by reference in their entirety).

[0194] Generally, the term "CRISPR system" collectively refers to transcripts and other elements involved in the expression of CRISPR-related ("Cas") genes or directing their activity, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active tracrRNA), tracr-mate sequences (in the context of endogenous CRISPR systems, encompassing "positive repeats" and partially positive repeats processed by tracrRNA), guide sequences (also referred to as "spacer regions" in the context of endogenous CRISPR systems), or other sequences and transcripts derived from CRISPR loci. One or more tracr-mate sequences operatively linked to the guide sequence (e.g., positive repeat-spacer-positive repeat) may also be referred to as pre-crRNA (pre-CRISPR RNA) or crRNA processed by nucleases. Typically, the CRISPR-Cas9 system comprises guide RNA (gRNA) and Cas9 nuclease, together forming a ribonucleoprotein (RNP) complex. The presence of a specific pre-spacer adjacent motif (PAM) in the genomic DNA is essential for gRNA binding to target sequences. The Cas9 nuclease then creates a double-strand break in the DNA. If a DNA template is present, the endogenous repair mechanism triggered by the double-strand break can lead to gene knockout via frameshift mutation or knock-in of the desired sequence.

[0195] In some forms, tracrRNA and crRNA are linked to form a chimeric crRNA-tracrRNA hybrid, in which the mature crRNA is fused with a portion of the tracrRNA via a synthetic stem-loop to mimic the natural crRNA:tracrRNA duplex, as described in Cong, Science, 15:339(6121):819–823 (2013) and Jinek et al., Science, 337(6096):816-21 (2012). A single fused crRNA-tracrRNA construct can also be referred to as a guide RNA or gRNA (or a single guide RNA (sgRNA)). Within the sgRNA, the crRNA portion can be identified as the “target sequence,” and the tracrRNA is often referred to as the “scaffold.”

[0196] CRSIPR systems with enhanced editing activity and high genome-wide targeting specificity typically consist of two components: (1) a single guide RNA configured for enhanced editing activity; and (2) a Cas enzyme.

[0197] In some forms, TALEN-mediated tumor suppressor gene knockout can be used to induce hepatocellular carcinoma (HCC) in mice.

[0198] In some forms, the element that induces single- or double-strand breaks in the genome of a target cell is one or more nucleic acid constructs encoding transcription activator-like effector nucleases (TALENs). TALENs have an overall architecture similar to ZFNs, the main difference being that their DNA-binding domains are derived from TAL effector proteins, i.e., transcription factors from plant pathogens. The DNA-binding domain of a TALEN is a tandem array of amino acid repeat sequences, each approximately 34 residues long. The repeat sequences are very similar to each other; typically, they differ primarily at two positions (amino acids 12 and 13, called repeat variable double residues, or RVDs). Each RVD specifies the preferential binding to one of four possible nucleotides, meaning that each TALEN repeat binds to a single base pair, although NN RVDs are known to bind adenine in addition to guanine. The mechanism of DNA binding of TAL effectors is less well understood than that of zinc finger proteins, but their seemingly simpler encoding may prove highly beneficial for the design of engineered nucleases. TALENs also function as dimer cleavages, have relatively long target sequences (the shortest target sequence reported to date binds 13 nucleotides per monomer), and appear to have less stringent requirements regarding the length of the spacer region between binding sites than ZFNs. Monomeric and dimer TALENs can contain more than 10, 14, 20, or 24 repeating sequences.

[0199] Engineered TALs are described in Cermak et al., Nucl. Acids Res. 1-11 (2011) for their binding to specific nucleic acids. U.S. Publication No. 2011 / 0145940 discloses TAL effectors and methods for modifying DNA using them. Miller et al., Nature Biotechnol 29:143 (2011) reported the preparation of TALENs for site-specific nuclease architectures by linking truncated TAL variants to the catalytic domain of the Fok1 nuclease. The resulting TALENs were shown to induce gene modification in immortalized human cells. The general design principles of TAL binding domains can be found, for example, in WO 2011 / 072246.

[0200] In some forms, ZFN-mediated tumor suppressor gene knockout can be used to induce hepatocellular carcinoma (HCC) in mice.

[0201] In some forms, the element that induces single-strand or double-strand breaks in the genome of the target cell is one or more nucleic acid constructs encoding zinc finger nucleases (ZFNs). ZFNs are typically fusion proteins that contain a DNA-binding domain derived from a zinc finger protein linked to a cleavage domain.

[0202] The most common cleavage domain is the IIS-type enzyme Fok1. Fok1 catalyzes double-strand cleavage of DNA, at a distance of 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand. See, for example, U.S. Patent Nos. 5,356,802; 5,436,150 and 5,487,994; and Li et al., Proc., Natl.Acad.Sci.USA 89 (1992):4275-4279; Li et al., Proc.Natl.Acad.Sci.USA, 90:2764-2768 (1993); Kim et al., Proc.Natl.Acad.Sci.USA. 91:883-887 (1994a); Kim et al., J. Biol.Chem.269:31,978-31,982 (1994b). One or more of these enzymes (or fragments of their enzyme function) can be used as a source of cleavage domains.

[0203] In principle, DNA-binding domains that can be designed to target any genomic location of interest can be tandem arrays of Cys2His2 zinc fingers, each typically recognizing three to four nucleotides in the target DNA sequence. The Cys2His2 domain has a general structure: Phe (sometimes Tyr)-Cys-(2 to 4 amino acids)-Cys-(3 amino acids)-Phe (sometimes Tyr)-(5 amino acids)-Leu-(2 amino acids)-His-(3 amino acids)-His. By linking multiple fingers together (the number varies: in published studies, three to six fingers are used per monomer), ZFN pairs can be designed to bind genomic sequences 18–36 nucleotides long.

[0204] Engineering methods include, but are not limited to, rational design and various types of empirical selection methods. Rational design includes, for example, using a database containing triplet (or tetrad) nucleotide sequences and individual zinc finger amino acid sequences, wherein each triplet or tetrad nucleotide sequence is associated with one or more amino acid sequences of a zinc finger that binds to a specific triplet or tetrad sequence. See, for example, U.S. Patent Nos. 6,140,081; 6,453,242; 6,534,261; 6,610,512; 6,746,838; 6,866,997; 7,067,617; U.S. Publication Nos. 2002 / 0165356; 2004 / 0197892; 2007 / 0154989; 2007 / 0213269; and International Patent Application Publication Nos. WO 98 / 53059 and WO 2003 / 016496.

[0205] In some forms, genome editing compositions optionally contain donor polynucleotides. Modifications of target DNA resulting from NHEJ and / or homology-directed repair can be used to induce gene correction, gene replacement, gene markers, transgene insertion, nucleotide deletion, gene disruption, gene mutation, etc.

[0206] Therefore, DNA cutting using genome editing compositions can be used to remove nucleic acid material from a target DNA sequence by cutting the target DNA sequence and allowing the cell to repair the sequence in the absence of exogenously provided donor polynucleotides. Thus, thematic approaches can be used to knock out genes (resulting in the complete absence or alteration of transcription) or knock genetic material into selected loci in the target DNA.

[0207] Alternatively, if the genome editing composition contains a donor polynucleotide sequence that includes at least a segment homologous to the target DNA sequence, the method can be used to add (i.e., insert or replace) nucleic acid material (e.g., "knock-in" nucleic acids encoding proteins, siRNAs, miRNAs, etc.), add tags (e.g., 6xHis, fluorescent proteins (e.g., green fluorescent protein; yellow fluorescent protein, etc.), hemagglutinin (HA), FLAG, etc.), add regulatory sequences to genes (e.g., promoters, polyadenylation signals, internal ribosome entry sequences (IRES), 2A peptides, start codons, stop codons, splicing signals, localization signals, etc.), modify nucleic acid sequences (e.g., introduce mutations), etc. Therefore, the composition can be used to modify DNA in a site-specific (i.e., "targeted") manner, such as gene knockout, gene knock-in, gene editing, gene tagging, etc., as in gene therapy, for example.

[0208] In applications where the goal is to insert a polynucleotide sequence into a target DNA sequence, the cell is also provided with a polynucleotide containing the donor sequence to be inserted. A “donor sequence,” “donor polynucleotide,” or “donor oligonucleotide” refers to the nucleic acid sequence to be inserted at the cleavage site. The donor polynucleotide typically contains sufficient homology to the genomic sequence at the cleavage site, such as 70%, 80%, 85%, 90%, 95%, or 100% homology to nucleotide sequences flanking the cleavage site (e.g., within about 50 bases, 15 bases, 10 bases, or 5 bases, or immediately adjacent to the cleavage site), to support homology-directed repair between the donor polynucleotide and the homologous genomic sequence. The donor sequence is typically different from the genomic sequence it replaces. Conversely, the donor sequence may contain at least one or more single-base alterations, insertions, deletions, inversions, or rearrangements relative to the genomic sequence, provided there is sufficient homology to support homology-guided repair. In some forms, the donor sequence includes non-homologous sequences flanking two homologous regions, such that homology-directed repair between the target DNA region and the two flanking sequences results in the insertion of the non-homologous sequence at the target region.

[0209] The donor sequence may also contain a vector backbone containing sequences that are dissimilar to the DNA region of interest and are not intended to be inserted into that region. Typically, the homologous regions of the donor sequence will have at least 50% sequence identity with the genome sequence to be recombined. In some forms, sequence identity is 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9%. Depending on the length of the donor polynucleotide, any value from 1% to 100% sequence identity can exist.

[0210] Donor sequences may contain certain sequence differences compared to the genomic sequence, such as restriction sites, nucleotide polymorphisms, and optional markers (e.g., drug resistance genes, fluorescent proteins, enzymes, etc.), which can be used to assess successful insertion of the donor sequence at the cleavage site or, in some cases, for other purposes (e.g., indicating expression at a target genomic locus). In some cases, if located in a coding region, such nucleotide sequence differences will not alter the amino acid sequence or will produce silent amino acid changes (i.e., changes that do not affect the protein's structure or function). Alternatively, these sequence differences may include flanking recombination sequences, such as FLP, loxP sequences, etc., which can be activated at a later time to remove the marker sequence.

[0211] The donor sequence can be single-stranded DNA, single-stranded RNA, double-stranded DNA, or double-stranded RNA. The donor sequence can be introduced into the cell in a linear or circular form. If introduced in a linear form, the ends of the donor sequence can be protected (e.g., from exonuclease degradation) by methods known to those skilled in the art. For example, one or more dideoxynucleotide residues can be added to the 3' end of a linear molecule and / or a self-complementary oligonucleotide can be attached to one or both ends. See, for example, Chang et al., Proc. Natl. Acad. Sci. USA, 84:4959-4963 (1987); Nehls et al., Science 272:886-889 (1996). Other methods for protecting exogenous polynucleotides from degradation include, but are not limited to, adding terminal amino groups and using modified internucleotide linkages, such as, for example, phosphate thioesters, aminophosphate esters, and O-methylribose or deoxyribose residues.

[0212] As an alternative to protecting the ends of the linear donor sequence, an additional length of sequence can be included outside the homologous region that can be degraded without affecting recombination. The donor sequence can be introduced into the cell as part of a vector molecule containing additional sequences, such as, for example, origin of replication, promoter, and genes encoding antibiotic resistance.

[0213] 2. Single-cell sequencing to identify TCRs

[0214] As disclosed herein, sequencing methods, such as single-cell TCR-seq, can be used to identify TCRs that respond to activation of tumor antigens in livers carrying HCC, rather than in normal livers.

[0215] In some forms, individual T cells are analyzed through high-throughput multiplex amplification and sequencing of nucleic acids encoding the T cell receptor (TCR) and various other T cell phenotypic markers. This method typically involves sorting individual T cells to individual locations (e.g., individual wells of a multi-well titration plate) and then performing nested polymerase chain reaction (PCR) amplification of the nucleic acids encoding the TCR and T cell phenotypic markers. The amplicon is barcoded to identify its source cell, pooled, and analyzed via deep sequencing. This method also involves reconstructing the TCR from individual T cells for functional studies, ligand discovery, or therapeutic screening.

[0216] In some forms, different sequencing methods are better suited for sequencing certain samples (e.g., rare samples can be sequenced more optimally using plate-based methods or single-nucleus sequencing). In some forms, plate-based single-cell RNA sequencing can be used (see, for example, Picelli, S et al., 2014, “Full-length RNA-seq from singlecells using Smart-seq2” Nature protocols 9, 171-181, doi:10.1038 / nprot.2014.006).

[0217] In some formats, high-throughput single-cell RNA-seq and / or targeted nucleic acid profiling (e.g., sequencing, quantitative reverse transcription polymerase chain reaction, etc.) can be used, where RNA from different cells is individually labeled, allowing the creation of a single library while preserving the cell identity of each read. In this regard, see the following: Macosko et al., 2015, “Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets” Cell 161, 1202-1214; International Patent Application No. PCT / US2015 / 049178, published March 17, 2016 as WO2016 / 040476; Klein et al., 2015, “Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets” Cell 161, 1202-1214; International Patent Application No. PCT / US2016 / 027734, published October 20, 2016 as WO2016168584A1; Zheng et al., 2016, “Haplotyping germline and cancer genomes with high-throughput linked-read sequencing” Nature Biotechnology 34. 303-311; Zheng et al., 2017, “Massively parallel digital transcriptional profiling of single cells” Nat. Commun. 8, 14049 doi:10.1038 / ncomms14049; International Patent Publication No. WO 2014210353 A2; Zilionis et al., 2017, “Single-cell barcoding and sequencing using droplet microfluidics” Nat Protoc.January; 12(1):44-73; Cao et al., 2017, “Comprehensivesingle cell transcriptional profiling of a multicellular organism bycombinatorial indexing” bioRxiv preprint first posted online Feb. 2, 2017, doi:dx.doi.org / 10.1101 / 104844; Rosenberg et al., 2017, “Scaling single cell transcriptomics through split pool barcoding" bioRxiv preprint first posted online Feb. 2, 2017, doi:dx.doi.org / 10.1101 / 105163; Vitak et al., "Sequencingthousands of single-cell genomes with combinatorial indexing" Nature Methods, 14(3):302-308, 2017; Cao et al., Comprehensive single-cell transcriptional profiling of a multicellular organism.Science, 357(6352):661-667, 2017; and Gierahn et al., “Seq-Well:portable, low-cost RNA sequencing of single cells at high throughput”, Nature Methods 14, 395-398 (2017), the entire contents and publications of which are incorporated herein by reference in their entirety.

[0218] In some forms, assessing the cell types (subtypes) and states present in an in vivo system may include analyzing expression matrices from scRNA-seq data, performing dimensionality reduction, graph-based clustering, and deriving a list of cluster-specific genes to identify the cell types and / or states present in the in vivo system. These biomarker genes can then be used to correlate the cell types (subtypes) and states of the ex vivo system with those of the in vivo system. The same analysis can then be applied to material sources based on ex vivo cell systems. An initial distribution of gene expression data is obtained from two sets of scRNA-seq analyses. In some forms, the distribution may be a count-based measure of the number of transcripts of each gene present in the cells. Further, clustering and gene expression matrix analysis allow for the identification of differences in the expression of key genes, such as key transcription factors, between the initial ex vivo system and the target in vivo system. In some instances, this can be accomplished by performing differential expression analysis. Example

[0219] method

[0220] Mice. Mice in the C57BL / 6J background were purchased from Jackson Laboratory and bred in the animal facility at Princess Margaret Cancer Centre. All animal experiments were approved by the University Health Network Animal Care Committee.

[0221] CRISPR and transposon vectors. sgRNAs targeting Trp53 and Pten were as described previously. The following guide oligonucleotides were designed to express (1) mouse Pten sgRNA: forward primer 5'- CACCGCTAACGATCTCTTTGATGA -3' (SEQ ID NO:62) and reverse primer 5'- AAACTCATCAAAGAGATCGTTAGC -3' (SEQ ID NO:63); and (2) mouse p53 sgRNA: forward primer 5'- CACCGCCTCGAGCTCCCTCTGAGCC -3' (SEQ ID NO:64) and reverse primer 5'- AAACGGCTCAGAGGGAGCTCGAGGC -3' (SEQ ID NO:65). The annealed double-stranded guide oligonucleotides were cloned into the BbsI cleavage site of the pX330 vector. The p53 sgRNA cassette in pX330-p53 was amplified by PCR using primers 5'-GCTTCTAGACATGTGAGGGCCTATTTC-3' (SEQ ID NO:66) and 5'-TACAGCTAGCGCCATTTGTCTGCAGAATTGG-3' (SEQ ID NO:67). This additional sgRNA cassette was then cleaved with NheI and XbaI and subcloned into the NheI site of pX330-Pten to obtain the duplex CRISPR vector pX330-p53-Pten.

[0222] A transposon system with SB100X and pT2 / BH was previously described. Mouse c-Myc CDS was cloned into pT2 / BH using EcoR1 and NotI restriction endonucleases to obtain the pT2-Myc plasmid.

[0223] HCC was induced by hydrodynamic injection. For the delivery of transposons and CRISPR vectors, mice (8–20 weeks old) were injected with a volume of 100 mL / kg body weight containing 25 μg pX330-p53-Pten, 0.66 μg SB100X, and 5 μg pT2-Myc. The molar ratio of SB100X to pT2-Myc was 1:5. Hydrodynamic injection into the lateral tail vein took 5–7 seconds. Blinding was achieved during injection by placing littermates of different genotypes into new cages lacking mouse information.

[0224] To assess and quantify the development of HCC, the appearance of the perceptible tumor in mice was monitored daily, defined as a identifiable enlargement of the abdomen. The humanitarian endpoint was defined as death or a liver weight exceeding 5 grams. The exact date on which mice reached the humanitarian endpoint was determined after euthanasia of mice carrying significant HCC. Liver weights collected at this stage ranged from 3 to 7 g, with the precise endpoint adjusted for one day per gram of live body weight. Early death (<5 days) was considered injection-related and excluded from the analysis. The number of liver tumors was quantified by counting tumor nodules on the liver surface or by normalizing the number of tumor clones relative to the area of ​​liver sections. Blinding was performed when quantifying liver sections rather than surface tumor nodules.

[0225] Hepatic mononuclear cell isolation. Mice were euthanized by CO2 asphyxiation and immediately perfused systemically with ice-cold PBS containing 10 mM EDTA. Liver tissue was collected, lysed, and passed through a 70 μm sieve to obtain a single-cell suspension. Mononuclear cells (MNCs) were enriched by centrifugation at 2000 rpm for 20 min using a 40 / 80% Percoll gradient.

[0226] Flow cytometry. Antibodies and tetramers used for staining liver MNCs included anti-mouse CD4 BUV737, anti-mouse CD8 PerCP-Cy5.5, anti-mouse CD45 Alexa Fluor 700, anti-mouse CD19 BUV395, anti-mouse NK1.1 BV605, and anti-mouse CD11b BV510 (all from BioLegend); and a tetramer labeled with mouse CD1dPBS-57 BV421 from the NIH Tetramer Facility.

[0227] Flow cytometry analysis was performed using a BD LSR Fortessa cell analyzer.

[0228] Single-cell RNA-seq and data analysis. Hepatocellular carcinoma (HCC) was induced in Chat-GFP mice using pX330-p53-Pten plus SB100X and pT2-Myc. On day 26 of HCC induction, HCC livers (from two male and two female mice) and control livers (from sex-matched littermates) were collected for liver MNC isolation. Liver MNCs were stained with antibodies against CD4, CD8, CD19, TCR-β, NK1.1, CD45, and CD1d tetramers, as well as barcode antibodies (TotalSeq-C0304, C0305, C0306, or C0307) against tagged cells from individual mice from HCC or control groups. CD45 cells were sorted using a FACSAria™ Fusion Cell Sorter (BD). + DAPI - NK1.1 -CD1dTetramer - CD19 - TCR-β + CD4 + CD8 - GFP + (Chat-GFP + CD4 + T cells and CD45 + DAPI - NK1.1 - CD1dTetramer - CD19 - TCR-β + CD4 + CD8 - GFP - (Chat-GFP - CD4 + A T-cell population, from which two CD4 cells were individually selected from each mouse. + T cell compartment. The same CD4 cells from mice that received the same treatment. + The T cell compartments were merged into 4 samples: control Chat-GFP. + Control Chat-GFP - HCC Chat-GFP + and HCCChat-GFP - After sorting and merging, samples were immediately submitted to the Princess Margaret Genomics Centre for downstream processing. Four samples were loaded into a 10x Chromium Controller, and libraries were prepared using the Chromium Next GEMSingle Cell 5' HT Reagent Kits v2 (Dual Index) (10x Genomics). The libraries were sequenced on an Illumina NovaSeq 6000 instrument. Sequencing depths were: GEX ~50,000 reads / cell, TCR ~5,000 reads / cell, and TotalSeq C ~2,000 reads / cell.

[0229] For single-cell RNA-seq data analysis, sequencing data were processed using Cell Ranger (version 7.0.0) and aligned with annotated mouse genomes (mm10). The Cell Ranger VDJ pipeline was used to call TCR sequences. Clonogenetic analysis was performed on the fusion contig annotations of the four samples. Connections of the V, D, and J regions were determined using the IMGT database. Filtered feature barcode matrices from the filtered contig annotations in Hierarchical Data Format (.h5 files) and .csv files from the four samples were analyzed using PartekFlow software (version 10.0.23.0214, licensed under CPOS Bioinformatics Core), and analyzed together. Single-cell counts were split into two data nodes: gene expression and antibody capture using a feature-type splitting tool. Gene expression was normalized using the recommended CPM (counts per million) method after excluding low-quality cells (counts <30,000; % mitochondrial counts <30). Antibody capture was normalized using the recommended method (add 1.0, divide by the geometric mean, add 1.0, and take the logarithm of 2.0), and then multiplets and cells with fuzzy hashing were filtered out. The two datasets were then fused using a merge matrix tool to obtain filtered, uniquely labeled, and normalized counts. This count data node was re-segmented to generate new gene expression and antibody capture nodes. Dimensionality reduction and visualization were performed on the new gene expression data nodes using PCA (principal components: 100, feature contribution: by variance, and by sample segmentation: no), graph-based clustering (using default parameters but with resolution set to 1.0), and UMAP (using default parameters). Biomarker calculations were performed on the graph-based clustering results to identify marker genes for each cell cluster. Differential analysis was performed using GSA, and visualization was done using heatmaps and volcano plots.

[0230] Immunohistochemical analysis. Sections excised from formalin-fixed paraffin-embedded (FFPE) mouse liver blocks were used for immunohistochemistry (IHC). After dewaxing and rehydration, endogenous peroxidase was inactivated in 3% H2O2 (20 ml 30% H2O2 + 180 ml PBS) at room temperature for 15 min. Antigen retrieval was performed with 10 mM sodium citrate buffer (pH 6.0) prior to immunostaining. Primary antibodies used for IHC included rabbit anti-CD3 (Abcam, ab5690) and rabbit anti-c-Myc (CellSignaling, #5605). The polymer-conjugated secondary antibody was HRP horse anti-rabbit IgG (MP-7405) from Vector Laboratories. ImmPACT Vector DAB peroxidase substrate (Vector Laboratories, SK-4100) was used for colorimetric detection. Immunostained histological sections were scanned using a NanoZoomer 2.0-HT slide scanner from Hamamatsu.

[0231] Statistical analysis. Paired comparisons were assessed using a two-tailed unpaired Student's t-test unless otherwise specified in the legend. A p-value < 0.05 was considered statistically significant. Unless otherwise specified, data are presented as mean ± SEM.

[0232] Data availability. The single-cell RNA sequencing data of mouse HCC reported in this article are stored in the Gene Expression Omnibus (GEO) database, accession code: GSE231322.

[0233] result

[0234] HCC was induced using CRISPR and transposon technology.

[0235] A mouse model of hepatocellular carcinoma (HCC) was established to identify a tumor suppressor gene (TCR) that is specific to HCC but not to normal tissue. This was achieved by incorporating genetic alterations repeatedly observed in human disease. These alterations include mutations in the TP53 and PTEN tumor suppressor genes and overexpression of the MYC proto-oncogene. TP53 is commonly altered in human hepatocellular carcinoma, while PTEN protein is reduced or absent in approximately 40% of HCC patients. Hepatocyte-specific loss of PTEN in mice similarly leads to the development of HCC. Chromosomal amplification involving MYC is one of the most common DNA copy number alterations in human HCC, and activation of MYC transcription is a central feature associated with the transformation of pretumor lesions into HCC.

[0236] Regarding the engineering of the aforementioned mutations, CRISPR-mediated somatic knockout of tumor suppressor genes and transposon-based expression of proto-oncogenes have been shown to induce HCC in mice. To mimic the multi-stage and multi-hit process of human liver carcinogenesis, a combination of two approaches was used: CRISPR ablation of Trp53 and Pten, and overexpression of Myc using a transposon vector. For this purpose, a double-stranded CRISPR vector with guide RNA targeting the second exon of Trp53 and the first exon of Pten was generated, along with a Sleeping Beauty transposon vector in which the Myc coding sequence is driven by the CAG promoter. Figure 1A These vectors were delivered to mice via hydrodynamic injection, allowing for specific plasmid delivery to hepatocytes. Rapid development of HCC was observed in injected mice due to the combined effect of Myc expression and the combined Trp53 and Pten ablation. Vesicles were visible on the liver surface at 15 days post-injection, and significant tumor nodules were present at day 25. Figure 1B Immunostaining confirmed that most of these tumor clones were negative for p53 and PTEN, and positive for MYC. Figure 1C-1D ).

[0237] Inducing immune surveillance in HCC models

[0238] The immune microenvironment exerts selective pressure on the clonal expansion of tumor cells. To investigate whether an immune response is induced during tumorigenesis in a model, tumor-infiltrating immune cells during HCC development were studied. It was found that liver CD4+ cells increased with the progression of HCC. + T cells and CD8 + T cell expansion, while the percentage of NKT cells decreased. Figure 1E-1F Immunohistochemical analysis showed that infiltrating immune cells were located in MYC. + Around pretumor cells and in established HCC ( Figure 1G Therefore, immune responses, particularly T-cell-mediated responses, are activated in HCC models. To determine whether the immune system actively shapes tumorigenesis in the model, HCC was induced in severely immunodeficient NSG mice. Compared to immunocompetent animals, NSG mice developed more severe disease and had shorter survival times. Figure 1H These results indicate that, under these circumstances, immune cells are involved in protecting against the development of liver cancer.

[0239] CD4 in HCC + T cell transcriptional landscape

[0240] To describe CD4 +To explore the heterogeneity and complexity of T cells and elucidate their induction in HCC, sorted CD44 cells were analyzed from four control and four HCC-carrying livers. + T cells underwent single-cell RNA sequencing (scRNA-seq). Cells from individual mice were barcoded with different antibodies to facilitate sample deconvolution, merging, and processing for CITE-seq coupled with TCR-seq. A total of 11 CD4 groups were identified. + T cell clusters: Clusters C1 and C8 are two naive T cell clusters; C2 contains Th17 cells and cells expressing the IL18 receptor gene; C3 cells co-express Cxcr6 and Pdcd1; C4 contains follicular helper T cells (Tfh) and follicular regulatory T cells (Tfr); C5 cells are actively circulating; C6 cells express Ccl5 and Nkg7, but rarely express other markers; C7 cells express Cxcr6, but are negative for Pdcd1; C9 cells are classic Treg cells; C10 cells show strong expression of cytotoxic genes such as Eomes, Prf1, Gzmk, Fasl, and Gzmb; and C11 is a minor cluster showing high expression of interferon-stimulated genes. Figure 2A-2C When these clusters were compared between control and HCC-carrying livers, a significant transition from naive T cells to effector T cells was observed in the presence of HCC, particularly the induction of the C3 cluster.

[0241] Cxcr6 is a marker of resident T cells in the liver, primarily enriched in the C3 and C7 clusters. In HCC livers, CD4... + T cells contain a large amount of C3 (Cxcr6) + Pdcd1 + Cells, but lacking C7 (Cxcr6) + Pdcd1 - )cell( Figure 2D These C3 cells showed high expression of suppressive immune receptors and exhaustion marker genes such as Pdcd1, Havcr2, Ptpn11, Lag3, Tox, and Tigit. In contrast, C7 cells strongly expressed differentiation, cytotoxicity-related, and other functional genes such as Il2ra, Il4, Il2, Fasl, Gzmb, and Csf2. These results suggest that Chat expression is associated with dysfunctional Pdcd1. + It is related to the appearance of T cells.

[0242] CD4 expression of Chat in liver cancer driven by tumor antigens + T cell clonal expansion

[0243] The proliferative characteristics of Chat-expressing T cells in HCC livers allowed us to investigate the driving forces behind their expansion. Single-cell TCR-seq revealed repetitive TCR types in all animals (e.g., defined by the shared amino acid sequence of both TCR-α and TCR-β chains). Figures 3A-3B Of the top 30 most common TCR types, only 5 were present in control mice, while the remaining 25 were observed in their littermates carrying HCC. Figures 3A-3B These results demonstrate the presence of CD4 in liver cancer. + TCR-specific amplification in T cells.

[0244] The most prevalent TCR type in HCC is TCR #1, encoded by 25 clonal variants (defined by shared mRNA sequences of both TCR-α and TCR-β chains). These clonal variants consist of synonymous mRNA variants using the same combination of the V, D, and J genes. These variations originate from different connections of the V, D, and J segments appearing in different mice carrying HCC, but due to codon redundancy, the resulting amino acid sequences are identical. Figure 3C This TCR convergence indication across mice carrying HCC indicates that HCC antigen-specific TCRs induce CD4+. + T cell proliferation, especially in Chat-GFP + CD4 + The T cell compartment was then examined. The properties of cells carrying this specific TCR type were then observed. T cells carrying TCR #1 primarily originated from those carrying Pdcd1. + C3 cluster of Tconv ( Figure 3D-3E These results support the use of PD-1 expressing Chat in mice. + Clonal expansion of Tconv.

[0245] Development of HCC-specific TCR engineered expression cassettes

[0246] Clone 5 was detected in all analyzed HCC mice out of 25 clones. VDJ of clone 5 β The region contains the Trbv16, Trbd1, and Trbj2-5 gene segments, while VJ α The region contains the Trav21-dv12 and Traj56 gene segments, with an N nucleotide addition ( Figures 4A-4D ).

[0247] To further elucidate the function of this HCC-specific TCR, a vector expressing the TCR was designed and constructed. Figures 5A-5B The design and optimization include:

[0248] 1. Replace the endogenous signal sequence with the FiBL signal sequence (from the silkworm moth's filamentin).

[0249] 2. Co-expression of α and β TCRs using the T2A self-cleaving ribosomal jumping peptide system.

[0250] 3. Inserting a furin recognition site upstream of 2A allows for the removal of the 2A residue (which would otherwise attach to TCRβ).

[0251] 4. Codon optimization was performed on all TCR sequences of mice using the GenSmart algorithm (expression increase >8-fold).

[0252] 5. The TCR co-expression construct was cloned into different gene delivery and expression systems, including transposons, lentiviruses, and retroviruses.

[0253] discuss

[0254] TCRs exist on the surface of T cells and recognize specific antigens presented by MHC. In cancer immunotherapy, TCRs can be modified to recognize specific cancer antigens, and these modified T cells are then infused into the patient's bloodstream to target and destroy cancer cells. Several HCC tumor antigens have been identified, including alpha-fetoprotein (AFP), phosphatidylinositol proteoglycan-3 (GPC3), and melanoma-associated antigen-A (MAGE-A). AFP is a marker of fetal liver development and is also expressed in some HCC cells. GPC3 is a glycoprotein overexpressed in HCC cells but not in normal hepatocytes. MAGE-A is a cancer-testis antigen expressed in various tumor types, including HCC. Furthermore, antigens expressed by cancer cells derived from oncogenic viruses (VHB, VHC) are also targets for HCC immunotherapy. In 2015, TCR-engineered T cells were used to combat viral antigens (HBsAg), producing modified T cells that survived and reduced HBsAg levels without toxicity, but without efficacy in end-stage metastatic disease. In LBO12, Sangro et al. provided data on AFPc332T cells in HCC patients (NCT03132792). One complete response and one stable disease were observed in four patients who received approximately 5 billion or more transduced cells, while five patients in the lower-dose cohort experienced stable disease. Cholestasis occurred, but no T-cell-related hepatotoxicity or DLT was observed. The first patient in cohort 3 had a confirmed partial response with a rapid and sustained decrease in serum AFP levels.

[0255] TCR convergence is the phenomenon where T cells from different individuals, despite having different TCR nucleotide sequences, recognize the same antigens with the same TCR amino acid sequence. This occurs due to the limited number of V, D, and J gene segments available for TCR gene rearrangement, resulting in the random generation of TCRs with the same amino acid sequence but different nucleotide sequences. Although the nucleotide sequences of the TCRs differ, the amino acid sequences remain identical, allowing T cells to recognize the same antigens. The TCR convergence observed in this study is significant for the development of T cell-based cancer immunotherapies because it facilitates the identification and isolation of T cells with specific antigen recognition capabilities.

[0256] In this study, a TCR expression vector was designed and constructed based on the amino acid sequence of an HCC-specific TCR, allowing for the expression of this specific TCR in cells. This TCR expression vector can be used for various in vitro and in vivo applications, such as:

[0257] 1. They can be used to identify specific peptide sequences recognized by TCRs. These TCR-expressing vectors can be used to generate T cell clones that recognize specific tumor antigens. These T cell clones can then be used to screen peptide libraries derived from tumor antigens to identify specific peptide epitopes recognized by TCRs. This can help design vaccines or immunotherapies that target tumor antigens and stimulate an immune response against cancer cells.

[0258] 2. Manufacturing TCR-engineered T cells: Vectors expressing TCR can be used to engineer T cells with specific TCR for use in adoptive cell therapy.

[0259] 3. Studying T cell function: By expressing this specific TCR in cells, we can study its function and activation in response to various stimuli. This can help us understand T cell biology and develop new immunotherapies for diseases.

[0260] 4. Screening for potential immunotherapies: Vectors expressing TCRs can be used to screen for the ability of potential immunotherapies to activate or suppress T-cell responses. This can help identify new treatments for diseases such as cancer, autoimmune disorders, and infectious diseases.

[0261] It should be understood that the disclosed methods and compositions are not limited to the specific methods, schemes, and reagents described, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which will be limited only by the appended claims.

[0262] Throughout the description and claims of this specification, the word “comprise” and variations thereof, such as “comprising” and “comprises”, mean “including but not limited to” and are not intended to exclude, for example, other additives, components, integers or steps.

[0263] "Optional" or "optionally" means that the event, situation, or material described below may or may not occur or may or may not exist, and the description includes instances in which the event, situation, or material occurs or exists as well as instances in which the event, situation, or material does not occur or does not exist.

[0264] Unless the context clearly indicates otherwise, the use of the word "can" indicates a choice or capability of the object or condition mentioned. Generally, using "can" in this way implies an explicit statement of the choice or capability, while also leaving open the possibility that the choice or capability may not exist in other forms or embodiments of the mentioned object or condition. Unless the context clearly indicates otherwise, the use of the word "may" indicates a choice or capability of the object or condition mentioned. Generally, using "may" in this way implies an explicit statement of the choice or capability, while also leaving open the possibility that the choice or capability may not exist in other forms or embodiments of the mentioned object or condition. Unless the context clearly indicates otherwise, "may" as used herein does not refer to unknown or questionable characteristics of the object or condition.

[0265] A range herein may be expressed as from “about” one particular value and / or to “about” another particular value. When expressing such a range, unless the context specifically indicates otherwise, a range from one particular value and / or to another particular value is also specifically anticipated and considered to be disclosed. Similarly, unless the context specifically indicates otherwise, when a value is expressed as an approximation using the antecedent “about,” it should be understood that the particular value forms another specifically anticipated implementation that should be considered disclosed. It will be further understood that, unless the context specifically indicates otherwise, each endpoint of a range is meaningful relative to and independent of the other endpoint. It should be understood that, unless the context specifically indicates otherwise, all individual values ​​and subranges of values ​​contained within an explicitly disclosed range are also specifically anticipated and should be considered disclosed. Finally, it should be understood that all ranges refer both to the range as a range and to the set of individual numbers from (and including) a first endpoint to (and including) a second endpoint. In the latter case, it should be understood that any individual number may be chosen as a form of the quantity, value, or characteristic referred to by the range. In this way, a range describes a set of numbers or values ​​from a first endpoint (and including the first endpoint) to a second endpoint (and including the second endpoint), from which a single member of the set (i.e., a single number) can be selected as the quantity, value, or characteristic referred to by the range. The foregoing applies regardless of whether some or all of these embodiments are explicitly disclosed in a particular case.

[0266] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed methods and compositions pertain. While any methods and materials similar to or equivalent to those described and used herein may be used to practice or test the methods and compositions of the invention, those methods, apparatus, and materials are particularly useful as described. Nothing herein should be construed as an admission that the invention is not entitled to rely on prior invention prior to such disclosure. No references are acknowledged as prior art. The discussion of references states the claims of their authors, and the applicant reserves the right to question the accuracy and relevance of the cited documents. It should be clearly understood that although numerous publications are cited herein, such citations do not constitute an admission that any of these documents constitutes part of common general knowledge in the art.

[0267] Those skilled in the art will recognize or be able to determine many equivalents of specific embodiments of the methods and compositions described herein using only conventional experiments. These equivalents are intended to be covered by the appended claims.

Claims

1. An immunoprotein comprising (a) a TCRβ chain variable domain (TCR-β domain) containing a complementarity-determining region (CDR) having the sequences SGHSA (SEQ ID NO:53), FRNQAP (SEQ ID NO:54), and ASSLDRGQDTQY (SEQ ID NO:55), (b) a TCRα chain variable domain (TCR-α domain) containing a CDR having the sequences TISGNEY (SEQ ID NO:56), GLQQN (SEQ ID NO:57), and ILRGTGGNNKLT (SEQ ID NO:58), or (c) both.

2. The immune protein of claim 1, wherein the immune protein comprises a T cell receptor (TCR), wherein the TCR comprises the TCR-β domain, the TCR-α domain, or both.

3. The immune protein according to claim 1 or 2, wherein the TCR-β domain has at least 75% sequence identity with SEQ ID NO: 51, and wherein the TCR-α domain has at least 75% sequence identity with SEQ ID NO:

52.

4. The immune protein according to claim 3, wherein the TCR-β domain comprises the sequence SEQ ID NO: 51, and wherein the TCR-α domain comprises the sequence SEQ ID NO:

52.

5. The immune protein according to any one of claims 1-4, wherein the immune protein targets hepatocellular carcinoma (HCC) antigen.

6. A pharmaceutical composition comprising the immune protein of any one of claims 1-5 and a pharmaceutically acceptable buffer, carrier, diluent or excipient.

7. A nucleic acid that encodes an immune protein according to any one of claims 1-5.

8. A vector comprising the nucleic acid of claim 7.

9. The vector according to claim 8, wherein the nucleic acid comprises an expression segment comprising coding regions in the following order: a signal sequence (e.g., fibL from silkworm moth), the TCR-β domain, the TCRβ constant region, a furin cleavage site, a flexible linker, T2A, the TCR-α domain, and the TCRα constant region.

10. A cell expressing the immune protein according to any one of claims 1-5.

11. The cell of claim 10, wherein the cell is a genetically modified T cell.

12. A cell population derived by amplifying the cells of claim 11.

13. A pharmaceutical composition comprising the cell population of claim 12 and a pharmaceutically acceptable buffer, carrier, diluent, or excipient.

14. A method for treating a subject suffering from a disease, symptom, or condition, the method comprising administering to the subject an effective amount of the pharmaceutical composition of claim 6 or 13.

15. The method of claim 14, wherein the subject has HCC or has been identified as having an increased risk of developing HCC.

16. The method of claim 14 or 15, wherein the cell population is isolated from or derived from a cell amplification, and the cells are obtained from a subject who had the disease, condition, or illness prior to the introduction of the cells.

17. The method of claim 14 or 15, wherein the cell population is isolated from or derived from cell amplifications, and the cells are obtained from a healthy donor.

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