Modified t cell receptors for prevention and treatment of viral infections and cancer

Abstract

To provide a method for expressing a functional modified T cell receptor (TCR) in a T cell.SOLUTION: There is provided a method for expressing a functional modified T cell receptor (TCR) in a T cell, comprising transducing at least one nucleic acid encoding first and a second TCR peptide chains, wherein the first and second peptide chains of the modified TCR are combined with each other on a cell surface, but are not combined with endogenous TCR peptides expressed on the T cell surface. Also provided is a method for reducing or treating occurrence of cancer or viral infection, comprising administering to a patient a pharmaceutical composition comprising, in a therapeutically effective amount, T cells produced by the method.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 227,195, filed July 29, 2021, the entire disclosure of which is incorporated herein by reference.

[0002] Sequence Listing Reference This disclosure contains references to amino acid and nucleic acid sequences that have been submitted contemporaneously herewith as a sequence listing xml file entitled "17768IB-01-WO-POA_seq_listing.xml," file size 107 kilobytes (KB), created on July 25, 2022. The sequence listing is incorporated herein by reference in its entirety pursuant to 37 CFR § 1.52(e)(5).

[0003] The present disclosure relates to modified T cell receptors (TCRs) that can be administered to a subject for the prevention and / or treatment of viral infection and / or cancer. [Background technology]

[0004] The background discussion includes information that may be useful in understanding the compositions and methods described herein. It is not an admission that any of the information provided herein is prior art or relevant to the compositions and methods, nor is it an admission that any publication specifically or implicitly referenced is prior art.

[0005] The TCR is a transmembrane protein located on the surface of T cells that recognizes antigens presented by major histocompatibility complex (MHC) I or II molecules from antigen-presenting cells (APCs). Signaling through the T cell receptor with appropriate costimulation leads to the activation of T cells (e.g., helper CD4 + Release of pro-inflammatory cytokines by T cells or cytotoxic CD8 + Initiates signaling pathways that activate T cells to respond to antigen (through initiation of T cell cytolysis).

[0006] Cancers and viruses can evade T cell-mediated immune responses by attenuating TCR signaling, thereby downregulating T cell responses. Modifying TCRs to promote T cell responses can improve the host's immune response to cancer or viral infection.

[0007] T cell receptor (TCR) molecules function as dimers in a cellular context. Transgenic modification of T cell TCRs requires the addition of genes for each monomer unit in the dimer. However, because T cells already contain native TCRs, it is conceivable that transgenic TCR monomers heterodimerize with native TCRs. These hybrid TCRs can cause off-target effects in transgenic T cells, and the effects of transgenic and / or endogenous TCRs are reduced or eliminated by cross-linking their respective peptide chains (Govers & al. (2010) Trends Mol. Med. 16(2):77-87). Thus, there remains a need to provide modified T cell receptors to improve immune responses to specific antigens (e.g., antigens from cancer cells or viruses) for the treatment of cancer and / or viral infections. Summary of the Invention [Means for solving the problem]

[0008] Disclosed herein are modified TCRs that can be used to treat and / or prevent viral infections and / or cancer. The modified TCRs are heterodimers comprising two different peptide chains. Each of the individual peptide chains of the modified TCR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain comprises a variable region, a constant region, and a connecting peptide, and the variable region and the constant region are connected via a linker. The connecting peptide is located between the constant region and the transmembrane domain. In some embodiments, the intracellular domain comprises a CD28 region and a CD3ζ ITAM region.

[0009] Also disclosed are methods for expressing functional modified TCRs in T cells, wherein peptide fragments of the modified TCRs self-assemble with each other on the cell surface and do not self-assemble with endogenous TCR peptides also expressed on the T cell surface. The modified TCRs can be engineered to express variable regions comprising α and β chains with specificity for HLA-presented peptides. The modified TCRs can be engineered to express variable regions comprising Ig variable domains with specificity for tumor-specific antigens.

[0010] Also disclosed herein are cells comprising the modified TCRs.

[0011] Nucleic acids encoding the modified TCRs and vectors comprising nucleic acids encoding the modified TCRs are also disclosed herein.

[0012] Also disclosed herein are methods for preventing and / or treating cancer or viral infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising a modified TCR, a nucleic acid encoding a modified TCR, or a cell comprising a modified TCR.

[0013] Various objects, features, aspects and advantages will become more apparent from the following detailed description of preferred embodiments, taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0014] [Figure 1] 1 shows one embodiment of a modified TCR of the present disclosure comprising a heterodimer of peptide chains P-NR-025 and P-NR-026. [Figure 2] 1 shows another embodiment of a modified TCR of the present disclosure comprising a heterodimer of peptide chains P-NR-027 and P-NR-028. [Figure 3A]

[0033] Figure 3 shows plasmid constructs encoding specific peptide chains of the modified TCRs of the present disclosure. Figure 3A shows a plasmid encoding peptide chain p-NR-025. Figure 3B shows a plasmid encoding peptide chain p-NR-026. Figure 3C shows a plasmid encoding peptide chain p-NR-027. Figure 3D shows a plasmid encoding peptide chain p-NR-028. [Figure 3B]

[0033] Figure 3 shows plasmid constructs encoding specific peptide chains of the modified TCRs of the present disclosure. Figure 3A shows a plasmid encoding peptide chain p-NR-025. Figure 3B shows a plasmid encoding peptide chain p-NR-026. Figure 3C shows a plasmid encoding peptide chain p-NR-027. Figure 3D shows a plasmid encoding peptide chain p-NR-028. [Figure 3C]

[0033] Figure 3 shows plasmid constructs encoding specific peptide chains of the modified TCRs of the present disclosure. Figure 3A shows a plasmid encoding peptide chain p-NR-025. Figure 3B shows a plasmid encoding peptide chain p-NR-026. Figure 3C shows a plasmid encoding peptide chain p-NR-027. Figure 3D shows a plasmid encoding peptide chain p-NR-028. [Figure 3D]

[0033] Figure 3 shows plasmid constructs encoding specific peptide chains of the modified TCRs of the present disclosure. Figure 3A shows a plasmid encoding peptide chain p-NR-025. Figure 3B shows a plasmid encoding peptide chain p-NR-026. Figure 3C shows a plasmid encoding peptide chain p-NR-027. Figure 3D shows a plasmid encoding peptide chain p-NR-028. [Figure 4-1]

[0049] Figures 4A and 4B show the results of a killing assay of activated natural killer (aNK) cells transfected with plasmid constructs encoding peptide chains of the modified TCRs of the present disclosure. Figures 4A and 4B show target cell lysis by aNK cells transfected with modified TCRs P-NR-025 + P-NR-026 and P-NR-027 + P-NR-028, respectively. Figures 4C-4E show target cell lysis by positive and negative control aNK cells. [Figure 4-2] This is a continuation of Figure 4-1. [Figure 4-3] This is a continuation of Figure 4-2. [Figure 5] 1 shows chimeric TCR expression in aNK (NK92) cells transfected with plasmid constructs encoding the peptide chains of chimeric TCRs P-NR-025+P-NR-026, P-NR-025+PWH295, PWH305+PWH308, and PWH303+PWH308. [Figure 6] 6 shows the results of a killing assay in aNK expressing wild-type and chimeric TCRs shown in FIG. 5. [Figure 7] 1 shows chimeric TCR expression in aNK transfected with plasmid constructs encoding the peptide chains of chimeric TCRs PWH305+PWH308 and PWH303+PWH308. [Figure 8] 8 shows the results of a killing assay in aNK expressing wild-type and chimeric TCRs shown in FIG. 7. DETAILED DESCRIPTION OF THE INVENTION

[0015] I. Definition The following definitions refer to various terms used above and throughout this disclosure.

[0016] "T cell receptor" or "TCR" refers to a dimeric polypeptide typically found on the surface of T cells. Each peptide chain of a TCR generally comprises an extracellular domain comprising variable and constant regions, a transmembrane domain, and an intracellular domain. The variable region is the portion of the TCR that interacts with antigens presented by MHC. The constant region is the region of each of the two peptide chains where the two peptide chains are covalently linked by disulfide bonds. The intracellular domain generally comprises CD3ζ, which contains one or more immunoreceptor tyrosine-based activation motifs (ITAMs). The ITAMs mediate the binding of the variable region to the appropriate intracellular signaling pathway.

[0017] "Encoding," when used in reference to a nucleic acid, refers to the fact that when transcription is initiated from a nucleic acid in a cell, the resulting transcript is translated into a given protein. That is, a nucleic acid "encodes" a peptide when a codon triplet of a tRNA produces a polypeptide from that nucleic acid in accordance with the normal operation of transcription and translation in a cell.

[0018] An "effective amount" or "therapeutically effective amount" refers to the amount and / or dosage and / or dosing regimen of one or more agents necessary to produce a desired result, e.g., an amount sufficient to prevent a viral infection in a subject, an amount sufficient to reduce the occurrence of a viral infection in a subject, and / or an amount sufficient to treat a viral infection in a subject. Alternatively, an effective amount or therapeutically effective amount refers to an amount and / or dosage and / or dosing regimen sufficient to reduce the occurrence of cancer in a subject and / or an amount sufficient to treat cancer in a subject.

[0019] "Cancer" refers to one or more disease states including the development of a tumor, neoplasm, or otherwise unwanted, abnormal, and / or unregulated cell growth in a patient's body, tissue, or organ. In certain embodiments, cancer includes bladder cancer, bone cancer, brain cancer (including medulloblastoma, meningioma, and neuroblastoma), breast cancer, cancer of the central nervous system, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, gallbladder cancer, head and neck cancer, gastric cancer, and the like. cancer), HIV / AIDS-related cancer, kidney cancer, leukemia (including acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), B-cell leukemia (BCL), chronic lymphocytic carcinoma (CLL), chronic myeloid leukemia (CML), and chronic T-cell lymphocytic leukemia (CTLL)), liver cancer, lung cancer (including non-small cell and small cell), lymphoma (including non-Hodgkin's lymphoma and Hodgkin's lymphoma), melanoma, multiple myeloma, nasopharyngeal carcinoma, oral cancer (including cancer of the mouth, tongue, salivary glands, or gums), neuroendocrine cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and vaginal cancer. In certain embodiments, the cancer is bladder cancer, breast cancer, colon cancer, or pancreatic cancer.

[0020] Percent "identical" or "identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared over a comparison window and aligned for maximum correspondence. For purposes of this disclosure, the degree of identity of amino acid or nucleic acid sequences is determined using the BLAST algorithm described in Altschul et al. (199) J. Mol. Biol. 215:403-10, which is publicly available through software provided by the National Center for Biotechnology Information (web address www.ncbi.nlm.nih.gov). This algorithm identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in a query sequence that match or meet a positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). Initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score falls by an amount X from its maximum achieved value; when the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments; or when the end of either sequence is reached. To determine percent identity of amino acid or nucleic acid sequences, the default parameters of the BLAST program can be used. For analysis of amino acid sequences, the BLASTP defaults are a word length (W) of 3; an expectation (E) of 10; and the BLOSUM62 scoring matrix.For the analysis of nucleic acid sequences, the BLASTN program defaults are a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The TBLASTN program (which queries nucleotide sequence databases using protein sequences) uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

[0021] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) Proc. Nat'l. Acad. Sci. USA 90:5873-87). The smallest sum propability (P(N)) provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid with the reference nucleic acid is less than about 0.01.

[0022] A "viral infection" refers to a condition in which a virus invades a host, e.g., a patient, and replicates. A viral infection does not require that the host exhibit symptoms of the viral infection. The term "virus" is not particularly limited and refers to both DNA and RNA viruses. DNA viruses can be single-stranded or double-stranded and can belong to any family of DNA viruses, including, but not limited to, herpesviridae, adenoviridae, polyomavididae, and poxviridae. Specific embodiments of DNA viruses include human herpesviruses and varicella-zoster viruses. RNA viruses can also be single-stranded or double-stranded and can belong to any family of RNA viruses, including, but not limited to, reoviridae, coronaviruses, picornaviridae, flaviviridae, hepeviridae, togaviridae, filoviridae, paramyxoviridae, pneumoviridae, rhabdoviridae, hantaviridae, and orthomyxoviridae. Particular embodiments of RNA viruses include rotavirus, coronavirus, SARS virus, poliovirus, rhinovirus, hepatitis A virus, yellow fever virus, West Nile virus, hepatitis C virus, dengue virus, Zika virus, rubella virus, Sindbis virus, chikungunya virus, Ebola virus, Marburg virus, measles virus, mumps virus, respiratory syncytial virus, rabies virus, influenza A virus, influenza B virus, influenza C virus, and influenza D virus. In some embodiments, the virus is a human immunodeficiency virus.

[0023] "Subject," "individual," and "patient" refer interchangeably to a mammal, preferably a human or non-human primate, but also to domestic mammals (e.g., dogs or cats), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs), and agricultural mammals (e.g., horses, cows, pigs, sheep). In certain embodiments, a subject can be a human (e.g., an adult male, adult female, juvenile male, juvenile female, boy, girl) under the care of a physician or other healthcare professional. In certain embodiments, a subject can be not under the care of a physician or other healthcare professional.

[0024] "Treating" and "treatment" each refer to a method of reducing, inhibiting, or otherwise ameliorating an infection by administering a therapeutic agent to a subject in need of treatment. In some embodiments, a subject in need of treatment may include a subject who has, is diagnosed with, or is suspected of having an infection, e.g., a viral infection. In certain embodiments, treating or treatment involves administering a therapeutic agent to a subject who has, is diagnosed with, or is suspected of having a disease, disorder, or condition (e.g., cancer or a viral infection). In some embodiments, the subject may be asymptomatic. Treatment involves administration of a modified TCR, a cell comprising the modified TCR, a nucleic acid encoding the modified TCR, and / or a vector comprising a nucleic acid encoding the modified TCR.

[0025] "In combination" or "in combination" includes administering an agent (e.g., a modified TCR, a cell comprising a modified TCR, and / or a nucleic acid encoding a modified TCR) in the presence of an additional agent. Concomitant administration in a therapeutic treatment method includes methods in which a first, second, third, or additional agent is administered simultaneously. Concomitant administration also includes methods in which a first or additional agent is administered in the presence of a second or additional agent, which may, for example, have been previously administered. A combination therapeutic treatment method may be performed stepwise by different actors. For example, one actor may administer a first agent to a subject and a second actor may administer a second agent to the subject, and the administering steps may be performed simultaneously or near simultaneously. The actor and subject may be the same entity (e.g., a human). Thus, the term encompasses both simultaneous administration and substantially simultaneous administration, i.e., near simultaneous administration.

[0026] II. Engineered T Cell Receptors The modified TCR of the present invention relates to a dimeric peptide based on the TCR structure. In particular, the modified TCR comprises two peptide chains, each of which comprises an extracellular domain (including a variable region, a constant region, and a connecting peptide), a transmembrane domain, and an intracellular domain. In a specific embodiment, the variable region and the constant region are connected via a linker. In another specific embodiment, the connecting peptide is located between the constant region and the transmembrane domain. In a further specific embodiment, the two peptide chains are linked to each other by a disulfide bond between the connecting peptides of each peptide chain.

[0027] The extracellular domain comprises a variable region, a constant region, and a connecting peptide. The exact sequence of the variable region is not particularly limited, except that it can recognize an antigen presented on an MHC molecule. By convention, the variable region on one of the modified TCR peptide chains may be referred to as "Vα" and the variable region on the other peptide chain may be referred to as "Vβ." In some embodiments, the variable regions on both peptide chains are the same. In alternative embodiments, the variable regions on each peptide chain are different. In certain embodiments, Vα comprises the sequence of SEQ ID NO: 15 (Vα-1) or SEQ ID NO: 30 (Vα-2). In another embodiment, Vβ comprises the sequence of SEQ ID NO: 16 (Vβ-1) or SEQ ID NO: 31 (Vβ-2). Alternatively, the variable region comprises a sequence having at least 70% sequence identity (i.e., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity) to SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:30, or SEQ ID NO:31. In one embodiment, the variable region comprises a sequence having at least 70% sequence identity (i.e., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity) to SEQ ID NO: 15 or SEQ ID NO: 30, but 100% identity to any or all three complementarity determining regions (CDRs) of SEQ ID NO: 15 or SEQ ID NO: 30.In one embodiment, the variable region comprises a sequence having at least 70% sequence identity (i.e., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity) to SEQ ID NO: 16 or SEQ ID NO: 31, but 100% identity to any or all three complementarity determining regions (CDRs) of SEQ ID NO: 16 or SEQ ID NO: 31.

[0028] The constant region represents the peptide sequence between the variable region and the connecting peptide. In specific embodiments, the constant region comprises an immunoglobulin (Ig) domain or a coiled-coil domain. In some embodiments, the constant regions of both peptide chains are the same. In alternative embodiments, the constant regions of each of the peptide chains are different.

[0029] In certain embodiments, the constant region is an Ig domain. Ig domains are not particularly limited and may include IgA, IgD, IgE, IgG, and IgM. The constant region may comprise Ig-Cκ, IgG-CH-1, or IgM-CH-1. In a specific embodiment, Ig-Cκ comprises the sequence of SEQ ID NO: 17. In another embodiment, IgG-CH-1 comprises the sequence of SEQ ID NO: 18 (IgG-CH-1a) or SEQ ID NO: 32 (IgG-CH-1b). In yet another embodiment, IgM-CH-1 comprises the sequence of SEQ ID NO: 33. In certain embodiments, the constant region of one peptide chain of the modified TCR comprises the sequence of Ig-Cκ, and the constant region of the other peptide chain of the modified TCR comprises the sequence of Ig-CH-1, e.g., IgG-CH-1a, IgG-CH-1b, and IgM-CH-1. Alternatively, the constant region comprises a sequence having at least 70% sequence identity (i.e., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity) to SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:32, or SEQ ID NO:33.

[0030] In certain alternative embodiments, the constant region is a coiled-coil domain, e.g., a WinZip domain. WinZip domains have been described. See, e.g., U.S. Patent No. 6,897,017, incorporated herein by reference in its entirety. In a specific embodiment, the WinZip domain is selected from the group consisting of WinZip-A2 (corresponding to SEQ ID NO: 20) and WinZip-B1 (corresponding to SEQ ID NO: 19). In certain embodiments, the constant region of one peptide of the modified TCR comprises WinZip-A2, and the constant region of the other peptide of the modified TCR comprises WinZip-B1.

[0031] The linker connecting the variable and constant regions can be a flexible linker. In certain embodiments, the linker comprises the amino acid sequence GGSGG (SEQ ID NO: 2).

[0032] A connecting peptide links the constant region to the transmembrane domain. In some embodiments, the connecting peptide comprises an amino acid sequence selected from the group consisting of GSG or GGCGG (SEQ ID NO: 1).

[0033] The extracellular domain of each peptide chain (including the variable region, constant region, and connecting peptide) is covalently engaged to a transmembrane domain. In some embodiments, the sequence of the transmembrane domain is selected from human leukocyte antigens (HLA). In some embodiments, the transmembrane domains of both peptide chains are the same. In other embodiments, the transmembrane domains of both peptide chains are different. In certain embodiments, the transmembrane domain comprises HLA-DRA, HLA-DRB1, or HLA-DRB2. In specific embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 21 (HLA-DRA), SEQ ID NO: 22 (HLA-DRB1), or SEQ ID NO: 34 (HLA-DRB2). Alternatively, the transmembrane domain comprises a sequence having at least 70% sequence identity (i.e., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity) to SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:34. In one embodiment, the transmembrane domain for one peptide chain comprises HLA-DRA and the transmembrane domain for the other peptide chain of the modified TCR comprises HLA-DRB, e.g., HLA-DRB1 or HLA-DRB2. In another specific embodiment, the transmembrane domain for one peptide chain of the modified TCR comprises the amino acid sequence of SEQ ID NO:21 and the transmembrane domain for the other peptide chain of the modified TCR comprises the amino acid sequence of SEQ ID NO:22 or SEQ ID NO:34.

[0034] The peptide chains of the modified TCR further comprise an intracellular domain, each comprising a CD28 region and a CD3ζ ITAM region. In a specific embodiment, the CD28 region comprises the amino acid sequence of SEQ ID NO: 23. In another embodiment, the CD3ζ ITAM region comprises the amino acid sequence of SEQ ID NO: 24. Alternatively, the constant region comprises a sequence having at least 70% sequence identity (i.e., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity) to SEQ ID NO: 23 or SEQ ID NO: 24. In an even further embodiment, the intracellular domain of each peptide chain of the modified TCR comprises the amino acid sequence of SEQ ID NO: 23 and the amino acid sequence of SEQ ID NO: 24.

[0035] In one embodiment, each of the peptide chains of the modified TCR are the same as each other, hi another embodiment, each of the two peptide chains in the modified TCR are different from each other.

[0036] Table 1 describes specific combinations of peptide chains that dimerize to form modified TCRs.

[0037] [Table 1]

[0038] Table 2 describes certain peptide chains that can homodimerize with each other or heterodimerize with other peptide chains, including extracellular domains (including variable regions, constant regions, and connecting peptides), transmembrane domains, and intracellular domains.

[0039] [Table 2]

[0040] The peptide chains in Table 1 or Table 2 may form homodimers or heterodimers to generate modified TCRs. For example, P-NR-025 (SEQ ID NO: 5) and P-NR-026 (SEQ ID NO: 6) may dimerize to form a modified TCR (FIG. 1). Alternatively, P-NR-027 (SEQ ID NO: 3) and P-NR-028 (SEQ ID NO: 4) may dimerize to form another modified TCR (FIG. 2). Additional peptide chain combinations to form chimeric TCRs are shown in Table 3 below.

[0041] [Table 3]

[0042] SEQ ID NOs: 15-24, 31-34, and 39-46 are provided merely as examples of suitable portions of the peptide chains that make up the modified TCRs (i.e., the defined variable region, constant region, connecting peptide, transmembrane domain, CD28 region, and CD3ζ ITAM region sequences), although many variations of these sequences are also useful for antiviral or anticancer therapeutic purposes. For example, polypeptides having at least 70% sequence identity (i.e., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity) to any one of SEQ ID NOs: 15-24, 31-34, and 39-46 are also useful for therapeutic purposes, provided that the molecule broadly retains the overall binding site, structure, and / or orientation of the individual molecule of SEQ ID NOs: 15-24, 31-34, and 39-46.

[0043] III. Polynucleotides and Vectors Molecular biologists now understand how to generate nucleic acids that express the peptide chains described herein and how to express such nucleic acids in cells to obtain the associated proteins. Further embodiments provided herein include nucleic acids or polynucleotides that encode the peptide chains that make up the modified TCRs. For example, nucleic acids encoding the modified TCRs described herein are provided herein as SEQ ID NOS: 7-14 and 35-38. A molecular biologist of ordinary skill in the art will understand how to modify the nucleotide sequences of SEQ ID NOs: 8, 10, 12, 14, and 35-38 to encode peptide chains of SEQ ID NOs: 5, 6, 3, 4, and 26-29, respectively, and suitable variants thereof (e.g., variants having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to any one of SEQ ID NOs: 5, 6, 3, and 4. Non-limiting examples of nucleic acids encoding peptide chains of SEQ ID NOs: 5, 6, 3, 4, and 26-29 are provided herein as SEQ ID NOs: 8, 10, 12, 14, and 35-38, respectively.

[0044] In certain embodiments, the nucleic acids described above can be expressed in supporter cell lines. Mammalian cell lines, such as Chinese hamster ovary (CHO) cells or 293T cells, are particularly suitable for these purposes. The proteins described herein are generally soluble and therefore excreted from the producing cells unless they are modified for intracellular retention. The proteins thus produced can be purified from the culture medium. Optionally, the proteins can be tagged with (for example) a poly-histidine tag or other such commercially available tags to facilitate purification. The proteins thus produced and purified can then be administered to a subject in need thereof as described below.

[0045] Alternatively, the nucleic acid can be expressed in primary T cells, e.g., T cells obtained from peripheral blood, tumors, and / or lymph nodes. Primary T cells can be harvested and manipulated as is routine in the art. The primary T cells can be from a subject with a condition treatable with the modified TCRs described herein. Alternatively, the primary T cells can be from another subject with primary T cells that are immunocompatible with the subject to be treated.

[0046] Additionally or alternatively, the nucleic acids described herein can be incorporated into a vector (e.g., a transfection vector or a viral transduction vector). Such a vector can then be transfected or transduced into the subject's own cells. In this manner, the subject's own cells produce the modified TCR. Non-limiting examples of vectors comprising the above nucleic acids are provided herein as SEQ ID NOS: 7-14. The interrelationship between the vector and the peptide chain of the modified TCR is shown in Table 2 above. Figures 3A-3D show embodiments of SEQ ID NOS: 8, 10, 12, and 14, respectively.

[0047] In some embodiments, the nucleic acid encoding the modified TCR is incorporated into cells. Such cells can translate the nucleic acid encoding the modified TCR to express the TCR. The cells can be the subject's own cells (e.g., autologous cells) or cells from a suitable donor (e.g., xenogeneic cells).

[0048] IV. Methods of Prevention or Treatment The above-described proteins, peptides, cells, nucleic acids, and vectors can be used to treat and / or prevent viral infections and / or cancers and / or reduce their occurrence. To treat and / or prevent viral infections and / or cancers, the modified TCRs, cells comprising the modified TCRs, nucleic acids encoding the modified TCRs, and vectors comprising nucleic acids encoding the modified TCRs described herein can be administered in therapeutically effective amounts to a subject in need thereof. The subject can be symptomatic or asymptomatic. Therapeutically effective amounts of these modified TCRs include, but are not limited to, 1 μg of the modified TCR per kg of subject body weight, 5 μg / kg, 10 μg / kg, 50 μg / kg, 100 μg / kg, 500 μg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 50 mg / kg, 100 mg / kg, 500 mg / kg, and 1 mg / kg or more.

[0049] When the modified TCRs, cells comprising the modified TCRs, nucleic acids encoding the modified TCRs, and vectors comprising nucleic acids encoding the modified TCRs are administered, any suitable route of administration may be used, including, but not limited to, oral administration, intravenous injection, intramuscular injection, subcutaneous injection, and inhalation (e.g., aerosol inhalation). In certain embodiments, the TCRs are administered by modifying T cells or NK cells to express the TCR and then injecting the modified immune cells into the patient.

[0050] In a preferred embodiment, the modified TCR is transfected into autologous T cells from a patient with an infectious disease cancer. The T cells may be derived from whole blood, tumor, or draining lymph nodes. In one embodiment, donor T cells may be used. The modified TCR described herein may be transfected into primary T cells as a nucleic acid, which may be DNA or RNA in any suitable vector. The DNA vector may be an adenovirus. The nucleic acid may be RNA. The RNA may be in a nanoparticle format, such as that described in U.S. Pat. No. 11,141,377, which is incorporated herein by reference. Transfection may be performed by standard techniques, such as electroporation (e.g., as described in U.S. Pat. No. 11,377,652 and U.S. Patent Application Publication No. 2022 / 0025402, both of which are incorporated herein by reference), or by using the MaxCyte™ system (Rockville, USA). Autologous T cells transfected in this manner can be enriched and expanded ex vivo. For example, CD3-enriched T cells can be expanded in Immunocult™ (StemCell Technologies, Cambridge, USA) and IL-2. The T cells can be administered to a patient in a therapeutically effective amount. In some embodiments, a composition comprising T cells produced by the methods described herein can be administered in a therapeutically effective amount. 2 ~10 12 cells / kg body weight, 10 2 ~10 10 cells / kg body weight, 10 5 ~10 9 cells / kg body weight, 10 5 ~10 8 cells / kg body weight, 10 5 ~10 7 cells / kg body weight, 10 7 ~10 9 cells / kg body weight, or 10 7 ~10 8 The composition may be administered at a dose of up to 100 cells / kg body weight, including all integer values ​​within those ranges. The number of T cells will depend on the therapeutic use for which the composition is intended.

[0051] When a nucleic acid encoding a modified TCR is to be transfected into cells, e.g., T cells, any suitable amount can be transfected into the cells, including (but not limited to) 10 ng, 50 ng, 100 ng, 500 ng, 1 μg, 5 μg, 10 μg, 50 μg, 100 μg, 500 μg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, and 500 mg or more. To transfect subject cells with the polynucleotides described herein, it is useful to extract cells from the subject, transfect them according to known techniques, and then infuse the transfected cells back into the subject. Electroporation is a particularly suitable transfection method (see, e.g., WO 20 / 14264 and WO 21 / 07315, each of which is incorporated herein by reference in its entirety). Particularly suitable cells include cells that circulate throughout the body, such as circulating lymphocytes (eg, T cells, NK cells).

[0052] If a nucleic acid is to be transduced, the viral vector can be administered directly to the subject, or cells can be extracted for transduction and reinfusion. The viral vector can be administered to the subject by any suitable route of administration, including, but not limited to, intravenous injection, intramuscular injection, subcutaneous injection, and inhalation (e.g., aerosol inhalation).

[0053] The therapeutically effective viral dose is, but not limited to, 1×10 7 5 x 10 viral particles (VP) 7 VP, 1×10 8 VP, 5×10 8 VP, 1×10 9 VP, 5×10 9 VP, 1×10 10 VP or 1 x 10 10VP or more. Adenoviral vectors are particularly suitable for this purpose due to the large cargo capacity of adenovirus. Suitable adenoviral vectors include those disclosed in WO 98 / 17783, WO 02 / 27007, WO 09 / 6479, and WO 14 / 31178, each of which is incorporated herein by reference in its entirety. Suitable methods for administering these adenoviral vectors are disclosed in WO 16 / 112188, which is incorporated herein by reference in its entirety.

[0054] The above proteins, peptides, cells, nucleic acids, and vectors can be used to treat and / or prevent and / or reduce the occurrence of cancer in a patient. Cancers include bladder cancer, bone cancer, brain cancer (including medulloblastoma, meningioma, neuroblastoma), breast cancer, cancer of the central nervous system, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, gallbladder cancer, head and neck cancer, gastric cancer, HIV / AIDS-related cancer, kidney cancer, leukemia (acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), B-cell leukemia (BCL), chronic lymphocytic carcinoma (CLL), chronic myeloid leukemia (CML), and other cancers. The cancer may be T-cell lymphocytic leukemia (CTLL), liver cancer, lung cancer (including non-small cell and small cell), lymphoma (including non-Hodgkin's lymphoma and Hodgkin's lymphoma), melanoma, multiple myeloma, nasopharyngeal carcinoma, oral cancer (including cancer of the mouth, tongue, salivary glands, or gums), neuroendocrine cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and vaginal cancer. In certain embodiments, the patient may have bladder cancer, breast cancer, colon cancer, or pancreatic cancer.

[0055] The above proteins, peptides, cells, nucleic acids, and vectors can be used to treat and / or prevent viral infections and / or reduce the occurrence of such infections in patients. The virus can be either a DNA or RNA virus. The patient may be suffering from an infection from a DNA virus, e.g., a single-stranded or double-stranded virus. The DNA virus may belong to any family of DNA viruses, including, but not limited to, herpesviridae, adenoviridae, polyomavididae, and poxviridae. Alternatively, the patient may be suffering from an infection from an RNA virus, e.g., a single-stranded or double-stranded virus. The RNA virus can belong to any family of RNA viruses, including, but not limited to, reoviridae, coronaviridae, picornaviridae, flaviviridae, hepeviridae, togaviridae, filoviridae, paramyxoviridae, pneumoviridae, rhabdoviridae, hantaviridae, and orthomyxoviridae. In particular, the patient may be infected with rotavirus, coronavirus, SARS virus, poliovirus, rhinovirus, hepatitis A virus, yellow fever virus, West Nile virus, hepatitis C virus, dengue virus, Zika virus, rubella virus, Sindbis virus, chikungunya virus, Ebola virus, Marburg virus, measles virus, mumps virus, respiratory syncytial virus, rabies virus, influenza A virus, influenza B virus, influenza C virus, influenza D virus, and human immunodeficiency virus.

[0056] Embodiment Embodiment 1: An engineered T cell receptor (TCR) comprising a first peptide chain and a second peptide chain, each peptide chain comprising an extracellular domain; a transmembrane domain; and an intracellular domain, wherein the extracellular domain comprises a variable region, a constant region, and a connecting peptide, wherein the variable region and the constant region are engaged via a linker, wherein the constant region of the first peptide chain comprises an Ig-Cκ domain and the constant region of the second peptide chain comprises an Ig-CH-1 domain, and wherein 1) the transmembrane domain of the first peptide chain comprises an HLA-DRA domain and the transmembrane domain of the second peptide chain comprises an HLA-DRB domain, or 2) the transmembrane domain of the first peptide chain comprises an HLA-DRB domain and the transmembrane domain of the second peptide chain comprises an HLA-DRA domain.

[0057] Embodiment 2: The TCR of embodiment 1, wherein the linker is a flexible linker.

[0058] Embodiment 3: The TCR of embodiment 1 or 2, wherein the Ig-CH-1 domain is IgG-CH-1a, IgG-CH-1b, or IgM-CH-1.

[0059] Embodiment 4: The TCR of any one of embodiments 1 to 3, wherein the HLA-DRB domain is HLA-DRB1 or HLA-DRB2.

[0060] Embodiment 5: The TCR of any one of embodiments 1 to 4, wherein the variable region on each of the peptide chains is the same variable region.

[0061] Embodiment 6: The TCR of any one of embodiments 1 to 5, wherein the variable regions on each peptide chain are different from each other.

[0062] Embodiment 7: The TCR of any one of embodiments 1 to 6, wherein the intracellular domain comprises a CD28 region and a CD3ζ ITAM region.

[0063] Embodiment 8: The TCR of any one of embodiments 1 to 7, wherein the first peptide chain comprises Ig-Cκ as the constant region; HLA-DRA as the transmembrane domain; and CD28 and CD3ζ as the intracellular domain.

[0064] Embodiment 9: The TCR of embodiment 8, wherein the first peptide chain comprises SEQ ID NO: 39.

[0065] Embodiment 10: The TCR of embodiment 9, wherein the first peptide chain comprises SEQ ID NO:5.

[0066] Embodiment 11: The TCR of embodiment 8, wherein the first peptide chain comprises SEQ ID NO: 43.

[0067] Embodiment 12: The TCR of embodiment 11, wherein the first peptide chain comprises SEQ ID NO:26.

[0068] Embodiment 13: The TCR of any one of embodiments 1 to 7, wherein the second peptide chain comprises Ig-CH-1 as the constant region; HLA-DRB as the transmembrane domain; and CD28 and CD3ζ as the intracellular domain.

[0069] Embodiment 14: The TCR of embodiment 13, wherein the second peptide chain comprises SEQ ID NO: 40.

[0070] Embodiment 15: The TCR of embodiment 14, wherein the second peptide chain comprises SEQ ID NO:6.

[0071] Embodiment 16: The TCR of embodiment 13, wherein the second peptide chain comprises SEQ ID NO: 44.

[0072] Embodiment 17: The TCR of embodiment 16, wherein the second peptide chain comprises SEQ ID NO: 27.

[0073] Embodiment 18: The TCR of embodiment 13, wherein the second peptide chain comprises SEQ ID NO: 45.

[0074] Embodiment 19: The TCR of embodiment 18, wherein the second peptide chain comprises SEQ ID NO: 28.

[0075] Embodiment 20: The TCR of embodiment 13, wherein the second peptide chain comprises SEQ ID NO: 46.

[0076] Embodiment 21: The TCR of embodiment 20, wherein the second peptide chain comprises SEQ ID NO: 29.

[0077] Embodiment 22: The TCR of any one of embodiments 1 to 21, wherein the first peptide chain comprises Ig-Cκ as the constant region, HLA-DRA as the transmembrane domain; and CD28 and CD3ζ as the intracellular domain; and the second peptide chain comprises Ig-CH-1 as the constant region, HLA-DRB as the transmembrane domain, and CD28 and CD3ζ as the intracellular domain.

[0078] Embodiment 23: The TCR of embodiment 22, wherein the first peptide chain comprises SEQ ID NO: 39 and the second peptide chain comprises SEQ ID NO: 40.

[0079] Embodiment 24: The TCR of embodiment 23, wherein the first peptide chain further comprises SEQ ID NO: 15 and the second peptide chain further comprises SEQ ID NO: 16.

[0080] Embodiment 25: The TCR of embodiment 24, wherein the first peptide comprises SEQ ID NO:5 and the second peptide chain comprises SEQ ID NO:6.

[0081] Embodiment 26: The TCR of embodiment 22, wherein the first peptide chain comprises SEQ ID NO: 43 and the second peptide chain comprises SEQ ID NO: 44.

[0082] Embodiment 27: The TCR of embodiment 26, wherein the first peptide chain further comprises SEQ ID NO: 30 and the second peptide chain further comprises SEQ ID NO: 31.

[0083] Embodiment 28: The TCR of embodiment 27, wherein the first peptide comprises SEQ ID NO: 26 and the second peptide chain comprises SEQ ID NO: 27.

[0084] Embodiment 29: The TCR of embodiment 22, wherein the first peptide chain comprises SEQ ID NO: 43 and the second peptide chain comprises SEQ ID NO: 44.

[0085] Embodiment 30: The TCR of embodiment 29, wherein the first peptide chain further comprises SEQ ID NO: 30 and the second peptide chain further comprises SEQ ID NO: 31.

[0086] Embodiment 31: The TCR of embodiment 30, wherein the first peptide comprises SEQ ID NO: 26 and the second peptide chain comprises SEQ ID NO: 28.

[0087] Embodiment 32: The TCR of embodiment 22, wherein the first peptide chain comprises SEQ ID NO: 43 and the second peptide chain comprises SEQ ID NO: 44.

[0088] Embodiment 33: The TCR of embodiment 32, wherein the first peptide chain further comprises SEQ ID NO: 30 and the second peptide chain further comprises SEQ ID NO: 31.

[0089] Embodiment 34: The TCR of embodiment 33, wherein the first peptide comprises SEQ ID NO: 26 and the second peptide chain comprises SEQ ID NO: 29.

[0090] Embodiment 35: A cell comprising a TCR of any one of embodiments 1 to 34.

[0091] Embodiment 36: A nucleic acid encoding the TCR of any one of embodiments 1 to 34.

[0092] Embodiment 37: A vector comprising the nucleic acid of embodiment 36.

[0093] Embodiment 38: A method of reducing the occurrence of or treating cancer or a viral infection in a patient in need thereof, comprising administering to the patient a pharmaceutical composition comprising a therapeutically effective amount of a modified TCR of any one of claims 1 to 34 or a nucleic acid encoding a modified TCR of any one of embodiments 1 to 34.

[0094] Embodiment 39: The method of embodiment 38, wherein the medicament comprises a vector comprising the nucleic acid.

[0095] Embodiment 40: The method of embodiment 38 or 39, wherein the pharmaceutical composition comprises a cell comprising the modified TCR or a nucleic acid encoding the modified TCR.

[0096] Embodiment 41: The method of any one of claims 38 to 40, wherein the cancer is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, cancer of the central nervous system, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, gallbladder cancer, head and neck cancer, gastric cancer, HIV / AIDS-related cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, multiple myeloma, nasopharyngeal cancer, oral cancer, neuroendocrine cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and vaginal cancer.

[0097] Embodiment 42: The method of any one of claims 38 to 40, wherein the viral infection is caused by a virus from a viral family selected from the group consisting of herpesviridae, adenoviridae, polyomavididae, poxviridae, reoviridae, coronaviridae, picornaviridae, flaviviridae, hepeviridae, togaviridae, filoviridae, paramyxoviridae, pneumoviridae, rhabdoviridae, hantaviridae, and orthomyxoviridae.

[0098] Embodiment 43: Use of the TCR of claim 1, the cell of claim 35, the nucleic acid of claim 36 or the vector of claim 37 for preventing or treating cancer or a viral infection in a patient in need thereof. [Example]

[0099] The following examples are provided to further illustrate the invention disclosed herein, but should not be construed as in any way limiting its scope.

[0100] Example 1: Cloning of TCR constructs HLA-DRA or HLA-DRB1 linking peptide plus transmembrane (CP-TM) region DNA templates were constructed by annealing partially overlapping long oligonucleotides (IDTs). Each of these CP-TM sequences was fused to the CD28 intracellular (IC) plus CD3ζ (IC) sequence obtained from a previously cloned template by overlap extension PCR (OE-PCR). DNA templates encoding the extracellular constant domains from Ig-CK or Ig-CH-1 were obtained by PCR from a previously cloned pAO156 template. Extracellular constant domains composed of linker-WinZip-B1 or linker-WinZip-A2 coiled-coil domains were obtained as gBlock DNA fragments (IDTs) encoding either linker-WinZip-B1 or linker-WinZip-A2. Each of these extracellular constant domains was fused to either the HLA-DRA or HLA-DRB1 CP-TM plus CD28-CD3ζ intracellular sequence by OE-PCR. These invariant CP-TM-IC sequences were cloned into a multipurpose expression vector (pRNi). The invariant CP-TM-IC insert was cloned by blunt ligation at the 5' end to recreate an EcoRV site and a PacI overlap at the 3' end. The resulting invariant CP-TM-IC construct in pRNi has an NcoI restriction site immediately upstream of the coding sequence. Therefore, any TCR Vα or Vβ sequence with a BsaI or Esp3I restriction site at the 5' end and a blunt phosphorylated 3' end can be designed or amplified and cloned into one of the invariant CP-TM-IC vectors digested with NcoI and EcoRV. Figures 3A-3D illustrate specific embodiments of the cloned modified TCR constructs.

[0101] Example 2: mRNA synthesis and effector cell electroporation The modified TCR constructs of Example 1 were inserted into an expression vector. The constructs were flanked upstream by a T7 promoter and 5' UTR, and downstream by a 3' UTR adopted from mouse hemoglobin alpha and a short poly(A) followed by an AarI linearization site. This allows for T7-based in vitro transcription of the modified TCRs after linearization of the final vectors P-NR-025, P-NR-026, P-NR-027, and P-NR-028 with AarI. Activated natural killer (aNK) cells were cultured at 3 x 10 6 cells / ml in 50 μL using a BIO-RAD Gene Pulser II with a 2 mm gap cuvette using in vitro transcribed and polyadenylated mRNA (NEB catalog numbers E2040S and M0276S). 6 Electroporated aNK cells were electroporated with 3 μg of mRNA per cell. Electroporated aNK cells were diluted to 1 × 10 per mL in complete RPMI medium (Corning RPMI 1640 with L-Glu supplemented with 10% FBS and 1 × PSA) in wells of a 6-well TC-treated plate. 6 The cells were incubated overnight at 37°C and 5% CO2.

[0102] Example 3: Killing assay Target D3C6 cells stably expressing HLA-A2 (i.e., KG-1 cells in which all MHC-I alleles were knocked out) were transfected with 4 μg / mL of hCMV pp65 NLV peptide-HLA-A * 6.4 x 10 β-Lysine monophosphate (NLVPMVATV (SEQ ID NO: 25) in DMSO) or an equal volume of DMSO in 4.4 mL 5 The aNK effector cells were pulsed at 1 x 10 cells / mL each. The pulsed target cells were incubated overnight in a T-25 flask in complete IMDM medium (ATCC Iscove's IMDM supplemented with 10% FBS and 1x PSA) at 37°C and 5% CO2. 20-24 hours after electroporation, the aNK effector cells from Example 2 were washed in PBS (without calcium or magnesium) and resuspended in complete RPMI 1640 medium. Effector aNK cells were then counted and plated at 1 x 10 cells / mL per well. 56.25 x 10 pieces 4 Viable effector cells were serially diluted and deposited into a round-bottom 96-well plate. Unbound peptide or DMSO was removed from the pulsed target cells by washing once with complete RPMI medium and twice with PBS. The washed target cells were each resuspended in 2 mL of PBS with 20 μL of calcein-AM (Fisher Scientific catalog number C3099) and incubated at 37°C and 5% CO2 for 20 minutes with gentle shaking. The calcein-loaded target cells were washed with PBS, then with complete RPMI, before being resuspended in 10 mL of complete RPMI and counted. Target cells were plated at 5 x 10 per well into a 96-well round-bottom plate. 3 Viable target cells were loaded along with their effector cells. Killing assay plates were centrifuged at 400 × g for 5 minutes and incubated at 37°C and 5% CO for 4 hours. After incubation, 22 μL of 9% (v / v) Triton X-100 (Sigma-Aldrich) was added to the maximum lysis control wells, and the plates were incubated at room temperature for 5 minutes. Killing plates were centrifuged again, and 100 μL of supernatant was transferred to a 96-well immunoassay plate for reading at excitation wavelengths of 485 ± 20 nm and emission wavelengths of 528 ± 20 nm. Each sample was plated in triplicate.

[0103] Figures 4A-4E demonstrate the percent specific target cell lysis of HLA-A2-positive target cells pulsed with pp65-NLV or DMSO (control) and exposed to either aNK cells electroporated with TCRαβ-ITAM fusions or control aNK cells. In Figure 4A, "P-NR-025 + P-NR-026 aNK" represents the TCRαβ-ITAM fusion shown in Figure 1. In Figure 4B, "P-NR-027 + P-NR-028 aNK" represents the TCRαβ-ITAM fusion shown in Figure 2. In Figure 4C, "P-NR-002 + P-NR-016 + CD3γδ aNK" represents wild-type TCRαβ containing the same anti-pp65-NLV-HLA-A2 variable domain as the other constructs and co-electroporated with CD3γδ to serve as a positive killing control. "P-WT-173" in Figure 4D is also a positive killing control, but stably expresses the same wild-type anti-pp65-NLV-HLA-A2 TCRαβ and CD3γδ. A negative control is shown by aNK electroporated with GFP alone (Figure 4E). P-NR-025 + P-NR-026, P-NR-027 + P-NR-028, P-NR-002 + P-NR-016, and P-WT-173 all express HLA-A2 TCRαβ. * Each antibody contained the same variable TCRαβ sequence pair previously determined to bind to the NLV peptide on 02. Error bars only represent ± standard deviation of technical replicates.

[0104] Example 4: Chimeric TCR expression and cytotoxicity of P-NR-025+P-NR-026, P-NR-025+PWH295, PWH308+PWH305, or PWH308+PWH305 in activated NK cells aNK (NK92) cells were washed with RPMI buffer and cultured in RPMI for 10 min. 7 Five micrograms of mRNA encoding the first and second peptide chains were resuspended at a concentration of 10 cells / 50 μL in 50 μL of RPMI for a 2 mm cuvette. 7The cuvette was subjected to three 20 ms pulses at 200 V using a BioRad GenePulser II. The electroporated cells were transferred to culture medium (Corning RPMI 1640 with L-Glu supplemented with 10% FBS and 1x PSA) containing IL-2 and incubated overnight.

[0105] After overnight incubation, 2 x 10 cells from each sample 5 The cells were harvested and washed with PBS / BSA / EDTA buffer. The cells were resuspended in 100 μL of wash buffer. 5 μL of PE-HLA-A * 0201, NLVPMVATV-PE, or an HLA-A2 dextramer negative control was added to each sample. Samples were incubated at 4°C for 20 minutes, washed with PBS / BSA / EDTA, and resuspended in 200 μL. Cells were analyzed via flow cytometry. Figure 5 shows the expression of various chimeric TCRs in electroporated aNK cells.

[0106] After overnight incubation after electroporation, aNK cells were washed three times and incubated with HLA-A2 stable KG-1 cells stained with calcein AM at a 10:1 (effector:target) ratio. After 4 hours of incubation, supernatants were obtained and analyzed for calcein AM fluorescence. Figure 6 shows the % specific killing of target cells by aNK cells expressing the modified TCRs described herein.

[0107] Example 5: Chimeric TCR expression and cytotoxicity of PWH308+PWH305 and PWH308+PWH305 in primary T cells Human primary T cells were obtained from donor-derived leukopacks (Charles River, Wilmington, USA). Peripheral blood mononuclear cells (PBMCs) were separated through a ficoll gradient, washed with K100 buffer, and diluted to 100% T cells in K100. 7The CD3-enriched T cells were then expanded in ImmunoCult™ (StemCell Technologies, Cambridge, USA) and IL-2. 10 μg of mRNA encoding the first and second peptide chains was diluted to 100 μL in 100 μL of K100 buffer for a 2 mm cuvette. 7 The cells were combined with 100 T cells. The cells were electroporated according to the electroporation protocol described in U.S. Pat. No. 11,377,652 and U.S. Patent Application Publication No. 20220025402, both of which are incorporated herein by reference. The electroporated cells were transferred to culture medium and incubated overnight.

[0108] After overnight incubation, 2 x 10 cells from each sample 5 The cells were harvested and washed with PBS / BSA / EDTA buffer. The cells were resuspended in 100 μL of wash buffer. 5 μL of PE-HLA-A * 0201, NLVPMVATV-PE, or an HLA-A2 dextramer negative control was added to each sample. Samples were incubated at 4°C for 20 minutes, washed twice with PBS / BSA / EDTA, and resuspended in 200 μL. Cells were analyzed via flow cytometry. Figure 7 shows the expression of various chimeric TCRs in electroporated primary T cells.

[0109] After overnight incubation after electroporation, primary T cells were washed three times and incubated with HLA-A2 stable KG-1 cells stained with calcein AM at a 10:1 (effector:target) ratio. After 4 hours of incubation, supernatants were obtained and analyzed for calcein AM fluorescence. Figure 8 shows the % specific killing of target cells by primary T cells expressing the modified TCRs described herein.

[0110] Example 6: Transfection of patient-derived T cells with modified TCRs Patient T cells are derived from whole peripheral blood or isolated from tumors or draining lymph nodes. The T cells are electroporated with the modified TCRs described herein. The electroporated T cells are expanded ex vivo to a clinically effective number of cells, and a therapeutically relevant number of cells are administered to the patient.

[0111] All references cited in this specification, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0112] Use of the terms "a," "an," "the," and "at least one" and similar referents in the context of describing the invention (particularly in the context of the claims below) should be construed to cover both the singular and the plural unless otherwise stated herein or clearly contradicted by context. Use of the term "at least one" followed by a list of one or more items (e.g., "at least one of A and B") should be construed to mean one item (A or B) selected from the listed items or any combination of two or more of the listed items (A and B) unless otherwise stated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted. Recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within that range, unless otherwise stated herein, and each separate value is incorporated herein as if it were individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise stated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "for example") provided herein is intended merely to better clarify the invention and does not limit the scope of the invention unless specifically claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0113] Certain embodiments of this invention, including the best mode known to the inventors for carrying out the invention, are described herein. Variations on those specific embodiments may become apparent to those skilled in the art upon reading the foregoing detailed description. The inventors anticipate that such variations will be employed by those skilled in the art, and the inventors intend that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0114] TIFF2025124736000005.tif241170TIFF2025124736000006.tif240170TIFF2025124736000007.tif240170TIFF2025124736000008.tif240170TIFF2025124736000009.tif241170TIFF2025124736000010.tif242170TIFF2025124736000011.tif242170TIFF2025124736000012.tif241170TIFF2025124736000013.tif241170TIFF2025124736000014.tif241170TIFF2025124736000015.tif242170TIFF2025124736000016.tif241170TIFF2025124736000017.tif240170TIFF2025124736000018.tif240170TIFF2025124736000019.tif241170TIFF2025124736000020.tif240170TIFF2025124736000021.tif241170TIFF2025124736000022.tif241170TIFF2025124736000023.tif241170TIFF2025124736000024.tif242170TIFF2025124736000025.tif241170TIFF2025124736000026.tif240170TIFF2025124736000027.tif241170TIFF2025124736000028.tif241170TIFF2025124736000029.tif240170TIFF2025124736000030.tif240170TIFF2025124736000031.tif241170TIFF2025124736000032.tif239170TIFF2025124736000033.tif242170TIFF2025124736000034.tif241170TIFF2025124736000035.tif241170TIFF2025124736000036.tif157170

Claims

1. 1. A modified T cell receptor (TCR) comprising a first peptide chain and a second peptide chain, each peptide chain comprising: extracellular domain; a transmembrane domain; and Intracellular domain Including, the extracellular domain comprises a variable region, a constant region, and a connecting peptide; the variable region and the constant region are engaged via a linker; the constant region of the first peptide chain comprises an Ig-Cκ domain and the constant region of the second peptide chain comprises an Ig-CH-1 domain; 1) the transmembrane domain of the first peptide chain comprises an HLA-DRA domain and the transmembrane domain of the second peptide chain comprises an HLA-DRB domain, or 2) the transmembrane domain of the first peptide chain comprises an HLA-DRB domain and the transmembrane domain of the second peptide chain comprises an HLA-DRA domain.

2. The TCR of claim 1, wherein the linker is a flexible linker.

3. The TCR of claim 1 or 2, wherein the Ig-CH-1 domain is IgG-CH-1a, IgG-CH-1b, or IgM-CH-1.

4. The TCR of any one of claims 1 to 3, wherein the HLA-DRB domain is HLA-DRB1 or HLA-DRB2.

5. The TCR of any one of claims 1 to 4, wherein the variable regions on each of the peptide chains are the same variable region.

6. The TCR of any one of claims 1 to 5, wherein the variable regions on each peptide chain are different from each other.

7. The TCR of any one of claims 1 to 6, wherein the intracellular domain comprises a CD28 region and a CD3ζITAM region.

8. The first peptide chain comprises: Ig-Cκ as the constant region; HLA-DRA as the transmembrane domain; and CD28 and CD3ζ as the intracellular domains The TCR of any one of claims 1 to 7, comprising:

9. The TCR of claim 8, wherein the first peptide chain comprises SEQ ID NO:

39.

10. The TCR of claim 8, wherein the first peptide chain comprises SEQ ID NO:

43.

11. The second peptide chain comprises: Ig-CH-1 as the constant region; HLA-DRB as the transmembrane domain; and CD28 and CD3ζ as the intracellular domains The TCR of any one of claims 1 to 7, comprising:

12. The TCR of claim 11, wherein the second peptide chain comprises SEQ ID NO:

40.

13. The TCR of claim 11, wherein the second peptide chain comprises SEQ ID NO:

44.

14. The TCR of claim 11, wherein the second peptide chain comprises SEQ ID NO:

45.

15. The TCR of claim 11, wherein the second peptide chain comprises SEQ ID NO:

46.

16. the first peptide chain comprises Ig-Cκ as the constant region, HLA-DRA as the transmembrane domain, and CD28 and CD3ζ as the intracellular domain; and the second peptide chain comprises Ig-CH-1 as the constant region, HLA-DRB as the transmembrane domain, and CD28 and CD3ζ as the intracellular domain. A TCR according to any one of claims 1 to 15.

17. 17. The TCR of claim 16, wherein the first peptide chain comprises SEQ ID NO: 39 and the second peptide chain comprises SEQ ID NO:

40.

18. 18. The TCR of claim 17, wherein the first peptide chain further comprises SEQ ID NO: 15 and the second peptide chain further comprises SEQ ID NO:

16.

19. 17. The TCR of claim 16, wherein the first peptide chain comprises SEQ ID NO: 43 and the second peptide chain comprises SEQ ID NO:

44.

20. 20. The TCR of claim 19, wherein the first peptide chain further comprises SEQ ID NO: 30 and the second peptide chain further comprises SEQ ID NO:

31.

21. 17. The TCR of claim 16, wherein the first peptide chain comprises SEQ ID NO: 43 and the second peptide chain comprises SEQ ID NO:

44.

22. 22. The TCR of claim 21, wherein the first peptide chain further comprises SEQ ID NO: 30 and the second peptide chain further comprises SEQ ID NO:

31.

23. 17. The TCR of claim 16, wherein the first peptide chain comprises SEQ ID NO: 43 and the second peptide chain comprises SEQ ID NO:

44.

24. 24. The TCR of claim 23, wherein the first peptide chain further comprises SEQ ID NO: 30 and the second peptide chain further comprises SEQ ID NO:

31.

25. A cell comprising a TCR according to any one of claims 1 to 24.

26. A nucleic acid encoding the TCR of any one of claims 1 to 24.

27. A vector comprising the nucleic acid of claim 26.

28. 1. A method of reducing the incidence of or treating cancer or a viral infection in a patient in need thereof, comprising administering to said patient a pharmaceutical composition comprising: A method in which the pharmaceutical composition comprises a therapeutically effective amount of a modified TCR according to any one of claims 1 to 24 or a nucleic acid encoding the modified TCR according to any one of claims 1 to 24.

29. 29. The method of claim 28, wherein the medicament comprises a vector comprising the nucleic acid.

30. 30. The method of claim 28 or 29, wherein the pharmaceutical composition comprises a cell comprising the modified TCR or a nucleic acid encoding the modified TCR.

31. 31. The method of any one of claims 28 to 30, wherein the cancer is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, cancer of the central nervous system, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, gallbladder cancer, head and neck cancer, gastric cancer, HIV / AIDS-related cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, multiple myeloma, nasopharyngeal cancer, oral cancer, neuroendocrine cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and vaginal cancer.

32. The viral infection may be caused by a virus such as herpesviridae, adenoviridae, polyomaviridae, poxviridae, reoviridae, coronaviruses, picornaviridae, flaviviridae, hepeviridae, togavirus, or the like.

31. The method of any one of claims 28 to 30, wherein the disease is caused by a virus from a viral family selected from the group consisting of Togaviridae, Filoviridae, Paramyxoviridae, Pneumoviridae, Rhabdoviridae, Hantaviridae, and Orthomyxoviridae.

33. Use of the TCR of claim 1, the cell of claim 25, the nucleic acid of claim 26 or the vector of claim 27 for preventing or treating cancer or viral infection in a patient in need of such prevention or treatment.

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