Modified T cell receptors for the 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 of related applications This application claims the benefits of U.S. Provisional Patent Application No. 63 / 227,195, filed on 29 July 2021. The entire disclosure of the said application is incorporated herein by reference.

[0002] Sequence listing reference This disclosure includes references to amino acid and nucleic acid sequences submitted concurrently with this specification as a sequence listing XML file named "17768IB-01-WO-POA_seq_listing.xml", with a file size of 107 kilobytes (KB) and created on July 25, 2022. The above sequence listing is incorporated herein by reference in its entirety in accordance with Section 1.52(e)(5) of the U.S. Patent Law Enforcement Regulations.

[0003] This disclosure relates to a modified T cell receptor (TCR) that may be administered to a subject for the prevention and / or treatment of viral infection and / or cancer. [Background technology]

[0004] The background information includes information that may be useful in understanding the compositions and methods described herein. This does not constitute an endorsement that any of the information provided herein is prior art or relating to the compositions and methods, nor does it constitute an endorsement 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 via T cell receptors in response to appropriate co-stimuli (e.g., helper CD4) + T cell release of pro-inflammatory cytokines or cytotoxic CD8 + This initiates a signaling pathway that activates T cells to respond to antigens (through the initiation of cell lysis by T cells).

[0006] Cancer and viruses can evade T-cell-mediated immune responses by reducing TCR signaling, thereby downmodulating T-cell responses. Modifying TCRs to enhance T-cell responses may improve the host immune response to cancer or viral infections.

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

[0008] Modified TCRs that may be used to treat and / or prevent viral infections and / or cancer are disclosed herein. The modified TCR is a heterodimer comprising two distinct 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 linking peptide, the variable and constant regions being engaged via a linker. The linking peptide is located between the constant region and the transmembrane domain. In some embodiments, the intracellular domain comprises a CD28 region and a CD3ζITAM region.

[0009] Disclosed herein is also a method for expressing functionally modified TCR in T cells, wherein the peptide fragments of the modified TCR self-assemble with each other on the cell surface and do not self-assemble with endogenous TCR peptides that are also expressed on the T cell surface. The modified TCR can be genetically engineered to express a variable region comprising α and β chains having specificity for an HLA-presented peptide. The modified TCR can be genetically engineered to express a variable region comprising an Ig variable domain having specificity for a tumor-specific antigen.

[0010] Cells comprising the modified TCR are also disclosed herein.

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

[0012] Disclosed herein is also a method for the prevention and / or treatment of cancer or viral infection in a patient in need of prevention and / or treatment of cancer or viral infection, comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising a modified TCR, a nucleic acid encoding the modified TCR, or a cell comprising the modified TCR.

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

[0014] [Figure 1] 1 illustrates one embodiment of the modified TCR of the present disclosure comprising a heterodimer of peptide chains P-NR-025 and P-NR-026. [Figure 2] 2 illustrates another embodiment of the modified TCR of the present disclosure comprising a heterodimer of peptide chains P-NR-027 and P-NR-028. [Figure 3A]The plasmid constructs encoding specific peptide chains of the modified TCRs of this disclosure are shown. Figure 3A shows the plasmid encoding peptide chain p-NR-025. Figure 3B shows the plasmid encoding peptide chain p-NR-026. Figure 3C shows the plasmid encoding peptide chain p-NR-027. Figure 3D shows the plasmid encoding peptide chain p-NR-028. [Figure 3B] The plasmid constructs encoding specific peptide chains of the modified TCRs of this disclosure are shown. Figure 3A shows the plasmid encoding peptide chain p-NR-025. Figure 3B shows the plasmid encoding peptide chain p-NR-026. Figure 3C shows the plasmid encoding peptide chain p-NR-027. Figure 3D shows the plasmid encoding peptide chain p-NR-028. [Figure 3C] The plasmid constructs encoding specific peptide chains of the modified TCRs of this disclosure are shown. Figure 3A shows the plasmid encoding peptide chain p-NR-025. Figure 3B shows the plasmid encoding peptide chain p-NR-026. Figure 3C shows the plasmid encoding peptide chain p-NR-027. Figure 3D shows the plasmid encoding peptide chain p-NR-028. [Figure 3D] The plasmid constructs encoding specific peptide chains of the modified TCRs of this disclosure are shown. Figure 3A shows the plasmid encoding peptide chain p-NR-025. Figure 3B shows the plasmid encoding peptide chain p-NR-026. Figure 3C shows the plasmid encoding peptide chain p-NR-027. Figure 3D shows the plasmid encoding peptide chain p-NR-028. [Figure 4-1] The results of killing assays of activated natural killer (aNK) cells transfected with plasmid constructs encoding the peptide chains of the modified TCRs of this disclosure are shown. Figures 4A and 4B show target cell lysis by aNK cells transfected with the 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] This shows the expression of chimeric TCRs in aNK(NK92) cells transfected with plasmid constructs encoding the peptide chains of the chimeric TCRs P-NR-025+P-NR-026, P-NR-025+PWH295, PWH305+PWH308, and PWH303+PWH308. [Figure 6] Figure 5 shows the results of the killing assay in aNK cells expressing wild-type and chimeric TCRs. [Figure 7] This shows chimeric TCR expression in aNK transfected with plasmid constructs encoding the peptide chains of the chimeric TCRs PWH305+PWH308 and PWH303+PWH308. [Figure 8] Figure 7 shows the results of the killing assay in aNK cells expressing wild-type and chimeric TCRs. [Modes for carrying out the invention]

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

[0016] The "T cell receptor" or "TCR" typically refers to a dimeric polypeptide found on the surface of T cells. Each peptide chain of the TCR generally contains an extracellular domain, a transmembrane domain, and an intracellular domain, including a variable region and a constant region. The variable region is the part of the TCR that interacts with antigens presented by the MHC. The constant region is the respective region of the two peptides, covalently linked by a disulfide bond. The intracellular domain generally contains 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] When used in relation to nucleic acids, "coding" refers to the process where transcription begins with a nucleic acid in a cell, and the resulting transcript is translated into a given protein. That is, a nucleic acid "codes" a peptide if its tRNA codon triplet produces a polypeptide from that nucleic acid in accordance with the normal operation of transcription and translation in a cell.

[0018] "Effective dose" or "therapeutic effective dose" refers to the amount and / or dosage and / or administration plan of one or more drugs necessary to produce a desired outcome, for example, 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 dose or therapeutic effective dose refers to an amount and / or dosage and / or administration plan 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 medical conditions, including the development of a tumor, neoplasm, or otherwise unwanted, abnormal and / or disordered cell proliferation in a patient's body, tissue, or organ. In some embodiments, cancer includes bladder cancer, bone cancer, brain cancer (including medulloblastoma, meningioma, and neuroblastoma), breast cancer, central nervous system cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, eye cancer, gallbladder cancer, head and neck cancer, and gastric cancer. The cancer is selected from the group consisting of 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 lymphoma and Hodgkin lymphoma), melanoma, multiple myeloma, nasopharyngeal cancer, 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, cancer is bladder cancer, breast cancer, colon cancer, or pancreatic cancer.

[0020] In the context of two or more nucleic acid or polypeptide sequences, "identical" or "identical" percentage 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 across a comparison window and aligned for maximum correspondence. For the 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 satisfy a positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the adjacent word score threshold (Altschul et al., op. cit.). Initial adjacent word hits act as seeds to initiate a search for longer HSPs containing them. Next, word hits are extended in both directions along each sequence as long as the cumulative alignment score can increase. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for pairs of matching residues; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of word hits in each direction stops when the cumulative alignment score falls by an amount X from its maximum achieved value; when the cumulative score becomes zero or less due to the accumulation of one or more negative scoring residue alignments; or when the end of either sequence is reached. Default parameters of the BLAST program may be used to determine the identity percentage of an amino acid sequence or nucleic acid sequence. For amino acid sequence analysis, the BLASTP defaults are word length (W), 3; expected value (E), 10; and the BLOSUM62 scoring matrix.For nucleic acid sequence analysis, the BLASTN program defaults to word length (W), 11; expected value (E), 10; M=5; N=-4; and comparison of both strands. The TBLASTN program (which queries nucleotide sequence databases using protein sequences) uses word length (W) 3, expected value (E) 10, and the BLOSUM62 scoring matrix as defaults (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

[0021] In addition to calculating the sequence identity percentage, 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 indicator of the probability that the match between two nucleotide or amino acid sequences occurs by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum propability in the comparison between the test nucleic acid and the reference nucleic acid is less than approximately 0.01.

[0022] "Viral infection" refers to a condition in which a virus invades and replicates in a host, such as a patient. A viral infection does not necessarily require the host to exhibit symptoms of the 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 viruses, and are not limited to, but include those belonging to the herpesviridae, adenoviridae, and polyomaviridae families. rDNA viruses may belong to any family, including the families (idae) and poxviridae. Specific embodiments of DNA viruses include human herpesviruses and varicella-zoster virus. RNA viruses may also be single-stranded or double-stranded and may belong to any family, though not limited to, the families of RNA viruses, including reoviridae, coronaviruses, picornaviridae, flaviviridae, hepeviridae, togaviridae, filoviridae, paramyxoviridae, pneumoviridae, rhabdoviridae, hantaviridae, and orthomyxoviridae. Specific 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 human immunodeficiency virus.

[0023] "Subject," "individual," and "patient" are interchangeable terms for mammals, preferably humans or non-human primates, but also for domestic mammals (e.g., dogs or cats), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs), and agricultural mammals (e.g., horses, cattle, pigs, sheep). In some embodiments, the subject may be a human being (e.g., an adult male, an adult female, a young male, a young female, a boy, a girl) under the care of a physician or other healthcare professional. In some embodiments, the subject may not be under the care of a physician or other healthcare professional.

[0024] "Treat" and "treatment" refer, respectively, to a method of reducing, inhibiting, or otherwise improving an infection by administering a therapeutic agent to a subject requiring treatment. In some embodiments, subjects requiring treatment may include subjects who have, are diagnosed with, or are suspected of having an infection, such as 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 viral infection). In some embodiments, the subject may be asymptomatic. Treatment includes the administration of a modified TCR, cells containing a modified TCR, nucleic acids encoding a modified TCR, and / or vectors containing nucleic acids encoding a modified TCR.

[0025] "Combined administration" or "in combination with" includes administering a drug (e.g., modified TCR, cells containing modified TCR, and / or nucleic acids encoding modified TCR) in the presence of an additional drug. Examples of combined administration in therapeutic procedures include the simultaneous administration of the first, second, third, or additional drug. Examples of combined administration include administering the first or additional drug in the presence of the second or additional drug, the second or additional drug may, for example, be administered prior to the administration. Combined therapeutic procedures may be carried out stepwise by different agents. For example, one agent may administer the first drug to a subject, and a second agent may administer the second drug to the subject, and the administration steps may be carried out simultaneously or nearly simultaneously. The agents and the subject may be the same entity (e.g., a human). Therefore, this term encompasses both simultaneous administration and substantially simultaneous administration, i.e., nearly simultaneous administration.

[0026] II. Modified 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 comprising an extracellular domain (including a variable region, a constant region, and a linking peptide), a transmembrane domain, and an intracellular domain. In a specific embodiment, the variable region and the constant region are engaged via a linker. In another specific embodiment, the linking 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 disulfide bonds between the linking peptides of each peptide chain.

[0027] The extracellular domain includes a variable region, a constant region, and a linked peptide. The exact sequence of the variable region is not particularly limited, except that it is capable of recognizing an antigen presented on an MHC molecule. By convention, the variable region on one modified TCR peptide chain may be called "Vα," and the variable region on the other peptide chain may be called "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 includes the sequence of SEQ ID NO: 15 (Vα-1) or SEQ ID NO: 30 (Vα-2). In other embodiments, Vβ includes the sequence of SEQ ID NO: 16 (Vβ-1) or SEQ ID NO: 31 (Vβ-2). Alternatively, the variable region includes a sequence having at least 70% sequence identity with sequence number 15, sequence number 16, sequence number 30, or sequence number 31 (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). In one embodiment, the variable region includes a sequence that has at least 70% sequence identity with sequence number 15 or sequence number 30 (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), but has 100% identity with any or all of the three complementarity determination regions (CDRs) of sequence number 15 or sequence number 30.In one embodiment, the variable region includes a sequence that has at least 70% sequence identity with sequence number 16 or sequence number 31 (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), but has 100% identity with any or all of the three complementarity determination regions (CDRs) of sequence number 16 or sequence number 31.

[0028] The constant region represents the peptide sequence between the variable region and the linked peptide. In specific embodiments, the constant region includes 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 peptide chain are different.

[0029] In certain embodiments, the constant region is an Ig domain. The Ig domain is not particularly limited and may include IgA, IgD, IgE, IgG, and IgM. The constant region may include Ig-Cκ, IgG-CH-1, or IgM-CH-1. In a specific embodiment, Ig-Cκ includes the sequence of SEQ ID NO: 17. In another embodiment, IgG-CH-1 includes 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 includes the sequence of SEQ ID NO: 33. In certain embodiments, the constant region of one peptide chain of the modified TCR includes the sequence of Ig-Cκ, and the constant region of the other peptide chain of the modified TCR includes the sequences of Ig-CH-1, e.g., IgG-CH-1a, IgG-CH-1b, and IgM-CH-1. Alternatively, the constant region includes a sequence having at least 70% sequence identity with sequence number 17, sequence number 18, sequence number 32, or sequence number 33 (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).

[0030] In alternative, specific embodiments, the constant region is a coiled-coil domain, for example, a WinZip domain. The WinZip domain is described; see, for example, U.S. Patent No. 6,897,017, which is incorporated herein by reference in its entirety. In specific embodiments, 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 specific 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 region and the constant region may be a flexible linker. In certain embodiments, the linker includes the amino acid sequence GGSGG (SEQ ID NO: 2).

[0032] The linked peptide ligates its constant region to the transmembrane domain. In some embodiments, the linked peptide contains 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 linked peptide) is covalently engaged with the 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 includes HLA-DRA, HLA-DRB1, or HLA-DRB2. In specific embodiments, the transmembrane domain includes 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 includes a sequence having at least 70% sequence identity with SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 34 (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). In one embodiment, the transmembrane domain for one peptide chain includes HLA-DRA, and the transmembrane domain for the other peptide chain of the modified TCR includes HLA-DRB, e.g., HLA-DRB1 or HLA-DRB2. In another specific embodiment, the transmembrane domain for one peptide chain of the modified TCR includes the amino acid sequence of SEQ ID NO: 21, and the transmembrane domain for the other peptide chain of the modified TCR includes the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 34.

[0034] The peptide chain of the modified TCR further comprises an intracellular domain, each comprising a CD28 region and a CD3ζITAM region. In certain embodiments, the CD28 region comprises the amino acid sequence of SEQ ID NO: 23. In another embodiment, the CD3ζITAM region comprises the amino acid sequence of SEQ ID NO: 24. Alternatively, the constant region comprises a sequence having at least 70% sequence identity with SEQ ID NO: 23 or SEQ ID NO: 24 (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). In yet another 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 in the modified TCR is identical to the others. In another embodiment, each of the two peptide chains in the modified TCR is different from the others.

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

[0037] [Table 1]

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

[0039] [Table 2]

[0040] The peptide chains in Table 1 or Table 2 can 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) can dimerize to form a modified TCR (Figure 1). Alternatively, P-NR-027 (SEQ ID NO: 3) and P-NR-028 (SEQ ID NO: 4) can dimerize to form another modified TCR (Figure 2). Additional peptide chain combinations for forming chimeric TCRs are shown in Table 3 below.

[0041] [Table 3]

[0042] Sequence IDs 15-24, 31-34, and 39-46 are provided merely as examples of preferred portions of peptide chains constituting modified TCRs (i.e., the specified variable region, constant region, linked peptide, transmembrane domain, CD28 region, and CD3ζITAM region sequences), but 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) with any one of sequence numbers 15-24, 31-34, and 39-46 are also useful for therapeutic purposes, provided that the molecule broadly retains the overall binding sites, structure, and / or orientation of the individual sequence numbers 15-24, 31-34, and 39-46.

[0043] III. Polynucleotides and Vectors Current molecular biologists understand methods for producing nucleic acids that express the peptide chains described herein, and methods for expressing such nucleic acids in cells to obtain the associated proteins. Further embodiments provided herein include nucleic acids or polynucleotides that encode the peptide chains constituting the modified TCR. For example, nucleic acids encoding the modified TCR described herein are presented herein as SEQ ID NOs: 7-14 and 35-38. Typical molecular biologists understand techniques for modifying the nucleotide sequences of SEQ ID NOs. 8, 10, 12, 14, and 35–38 to encode the peptide chains of SEQ ID NOs. 5, 6, 3, 4, and 26–29, respectively, and appropriate variants thereof (e.g., variants having at least 70% identity with any one of SEQ ID NOs. 5, 6, 3, and 4 (e.g., variants having 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%)). Non-limiting examples of nucleic acids encoding the 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 some embodiments, the nucleic acids described above may 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 modified for intracellular retention. The proteins thus produced can be purified from the culture medium. If desired, the proteins may be tagged with (e.g.) polyhistidine tags or other commercially available tags to facilitate purification. The proteins thus produced and purified can then be administered to subjects where they are needed, as described below.

[0045] Alternatively, the nucleic acids may be expressed in primary T cells, for example, T cells obtained from peripheral blood, tumors, and / or lymph nodes. Primary T cells may be recovered and manipulated in accordance with the conventions of the art. Primary T cells may come from subjects having a condition treatable by the modified TCR described herein. Alternatively, primary T cells may come from another subject having primary T cells that are immunocompatible with the subject to be treated.

[0046] Furthermore, or alternatively, the nucleic acids described herein may be incorporated into a vector (e.g., a translocation vector or a viral transduction vector). Such a vector can then be translocated or transduced into the cells of the subject itself. In this way, the cells of the subject itself produce a modified TCR. Non-limiting examples of vectors containing the above nucleic acids are provided herein as SEQ ID NOs: 7-14. The interaction 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 taken up into a cell. Such a cell can translate the nucleic acid encoding the modified TCR and express the TCR. The cell may be the subject's own cell (e.g., autologous cell) or a cell from a suitable donor (e.g., xenocellular cell).

[0048] IV. Methods of prevention or treatment The proteins, peptides, cells, nucleic acids, and vectors described above may be used to treat and / or prevent viral infections and / or cancer, and / or reduce their occurrence. To treat and / or prevent viral infections and / or cancer, modified TCRs, cells containing modified TCRs, nucleic acids encoding modified TCRs, and vectors containing nucleic acids encoding modified TCRs described herein may be administered in therapeutically effective doses to subjects in need. Subjects may be symptomatic or asymptomatic. Therapeutic effective doses of these modified TCRs include, but are not limited to, 1 μg of 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 modified TCRs, cells containing modified TCRs, nucleic acids encoding modified TCRs, and vectors containing nucleic acids encoding modified TCRs are administered, any preferred route of administration may be used, but is not limited to oral administration, intravenous injection, intramuscular injection, subcutaneous injection, and inhalation (e.g., aerosol inhalation). In certain embodiments, TCRs are administered by modifying T cells or NK cells to express TCRs, and then injecting the modified immune cells into the patient.

[0050] In a preferred embodiment, the modified TCR is transfected into autologous T cells derived from a patient having cancer or an infectious disease. T cells may be derived from whole blood, a tumor, or a draining lymph node. In one embodiment, donor T cells may be used. The modified TCR described herein can be transfected as a nucleic acid into primary T cells, and the nucleic acid can be DNA or RNA of any suitable vector. The DNA vector can be an adenovirus. The nucleic acid can be RNA. The RNA can be in a nanoparticle format, for example, as described in U.S. Pat. No. 11,141,377, which is incorporated herein by reference. Transfection can be performed by standard techniques, such as electroporation (e.g., those 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). The autologous T cells transfected in this way 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 is 10 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 cells / kg body weight (including all integer values within these ranges). The number of T cells depends on the therapeutic use for which the composition is intended.

[0051] When nucleic acids encoding modified TCRs are to be transfused into cells, for example, T cells, any suitable amount 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 may be transfused into the cells. To transfuse target cells with the polynucleotides described herein, it is useful to extract cells from the target, transfuse them according to known techniques, and then infuse the transfused cells back into the target. Electroporation is a particularly preferred transfuse method (see, for example, International Publication No. 20 / 14264 and International Publication No. 21 / 07315, each of which is incorporated herein by reference as a whole). Particularly suitable cells include those that circulate throughout the body, such as circulating lymphocytes (e.g., T cells and NK cells).

[0052] When nucleic acids are to be transduced, the viral vector may be administered directly to the subject, or cells may be extracted for transduction and reinfusion. The viral vector may be administered to the subject by any preferred 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 load is not limited to 1 × 10⁻⁶. 7 Individual virus particles (VP), 5 x 10 7 VP, 1 x 10 8 VP, 5×10 8 VP, 1 x 10 9 VP, 5×10 9 VP, 1 x 10 10 VP, or 1 × 10 10Examples include VP vectors. Adenovirus vectors are particularly suitable for this purpose due to the large cargo capacity of the adenovirus. Suitable adenovirus vectors are disclosed in International Publication Nos. 98 / 17783, 02 / 27007, 09 / 6479, and 14 / 31178, each of which is incorporated herein by reference as a whole. Suitable methods for administering these adenovirus vectors are disclosed in International Publication No. 16 / 112188, which is incorporated herein by reference as a whole.

[0054] The proteins, peptides, cells, nucleic acids, and vectors described above may be used to treat and / or prevent cancer in patients and / or reduce its occurrence. Cancers include bladder cancer, bone cancer, brain cancer (including medulloblastoma, meningioma, neuroblastoma), breast cancer, central nervous system cancer, 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 chronic This may include T-cell lymphocytic leukemia (CTLL), liver cancer, lung cancer (including non-small cell and small cell), lymphoma (including non-Hodgkin lymphoma and Hodgkin lymphoma), melanoma, multiple myeloma, nasopharyngeal cancer, oral cancer (including cancer of the mouth, tongue, salivary glands, or gums), neuroendocrine cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, sarcoma, skin cancer, gastric 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 proteins, peptides, cells, nucleic acids, and vectors described above may be used to treat and / or prevent viral infections in patients and / or to reduce their occurrence. Viruses may be either DNA or RNA viruses. Patients may have been infected with DNA viruses, such as single-stranded or double-stranded viruses. DNA viruses include, but are not limited to, herpesviridae, adenoviridae, and polyomaviridae. r The patient may belong to any family of DNA viruses, including the families (idae) and poxviridae. Alternatively, the patient may have been infected with an RNA virus, such as a single-stranded or double-stranded virus. RNA viruses may belong to any family of RNA viruses, including, but are not limited to, the families (reoviridae), coronaviruses, picornaviridae, flaviviridae, hepeviridae, togaviridae, filoviridae, paramyxoviridae, pneumoviridae, rhabdoviridae, hantaviridae, and orthomyxoviridae). In particular, patients 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: A modified 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 linked peptide, the variable region and the constant region being engaged via a linker, the constant region of the first peptide chain comprising an Ig-Cκ domain, and the constant region of the second peptide chain comprising an Ig-CH-1 domain, and either 1) the transmembrane domain of the first peptide chain comprising an HLA-DRA domain and the transmembrane domain of the second peptide chain comprising an HLA-DRB domain, or 2) the transmembrane domain of the first peptide chain comprising an HLA-DRB domain and the transmembrane domain of the second peptide chain comprising an HLA-DRA domain.

[0057] Embodiment 2: The linker is a flexible linker, as in Embodiment 1's TCR.

[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: A TCR from any one of Embodiments 1 to 3, wherein the HLA-DRB domain is either HLA-DRB1 or HLA-DRB2.

[0060] Embodiment 5: A TCR from any one of Embodiments 1 to 4, wherein each variable region on the peptide chain is the same variable region.

[0061] Embodiment 6: Each variable region on the peptide chain is a TCR from any one of Embodiments 1 to 5, which are different from each other.

[0062] Embodiment 7: The intracellular domain is one of the TCRs from Embodiments 1 to 6, comprising a CD28 region and a CD3ζITAM region.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[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 containing any one of the TCRs from Embodiments 1 to 34.

[0091] Embodiment 36: A nucleic acid encoding any one of the TCRs from Embodiments 1 to 34.

[0092] Embodiment 37: A vector containing the nucleic acid of Embodiment 36

[0093] Embodiment 38: A method for reducing or treating cancer or viral infection in a patient in whom reduction or treatment of such occurrence is required, comprising administering a pharmaceutical composition to a patient, wherein the pharmaceutical composition comprises a therapeutically effective amount of any one modified TCR of claims 1 to 34 or a nucleic acid encoding any one modified TCR of embodiments 1 to 34.

[0094] Embodiment 39: The method of Embodiment 38, wherein the pharmaceutical agent comprises a vector containing nucleic acid.

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

[0096] Embodiment 41: The method according to 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, central nervous system cancer, 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, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, and vaginal cancer.

[0097] Embodiment 42: Viral infections are caused by herpesviridae, adenoviridae, and polyomaviridae. r A method according to any one of claims 38 to 40, caused by a virus from a viridae selected from the group consisting of poxviridae, reoviridae, coronavirusidae, picornaviridae, flaviviridae, hepeviridae, togaviridae, filoviridae, paramyxoviridae, pneumoviridae, rhabdoviridae, hantaviridae, and orthomyxoviridae.

[0098] Embodiment 43: Use of the TCR of claim 1, the cells of claim 35, the nucleic acid of claim 36, or the vector of claim 37 for the prevention or treatment of cancer or viral infection in a patient requiring prevention or treatment of cancer or viral infection. [Examples]

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

[0100] Example 1: Cloning of TCR constructs HLA-DRA or HLA-DRB1 linked 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 a CD28 intracellular (IC) plus CD3ζ (IC) sequence obtained from a pre-cloned template by overlap extension PCR (OE-PCR). DNA templates encoding extracellular constant domains from Ig-Cκ or Ig-CH-1 were obtained by PCR from a pre-cloned pAO156 template. Extracellular constant domains consisting 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 HLA-DRA or HLA-DRB1 CP-TM plus CD28-CD3ζ intracellular sequences 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 regenerate the EcoRV site and the PacI overlap at the 3' end. The resulting invariant CP-TM-IC construct in the pRNi has an NcoI restriction site immediately upstream of the coding sequence. Therefore, any TCR Vα or Vβ sequence having 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 by 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 construct from Example 1 was inserted into the expression vector. The construct was 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 TCR 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 transcribed in vitro and polyadenylated mRNA (NEB catalog numbers E2040S and M0276S) using a BIO-RAD Gene Pulser II with a 2 mm gap cuvette, resulting in 3 × 10¹⁶ cells in 50 μL. 6 Cells were electroporated with 3 μg of mRNA per cell. The electroporated aNK cells were then placed in the wells of a 6-well TC-treated plate in complete RPMI medium (Corning RPMI 1640 with L-Glu supplemented with 10% FBS and 1× PSA) at a concentration of 1 × 10⁶ cells per mL. 6 Individual cells were incubated overnight at 37°C with 5% CO2.

[0102] Example 3: Killing Assay Target D3C6 cells that stably express HLA-A2 (i.e., KG-1 cells in which all MHC-I alleles are knocked out) were treated with 4 μg / mL of hCMV pp65 NLV peptide-HLA-A2. * Either 0201 restrictive (NLVPMVATV (SEQ ID NO: 25), in DMSO) or an equal volume of DMSO, is present in 4.4 mL, containing 6.4 × 10⁴ units. 5 Each cell was pulsed at 10⁴ cells / mL. The pulsed target cells were incubated overnight in a T-25 flask in complete IMDM medium (ATCC Iskov IMDM supplemented with 10% FBS and 1× PSA) at 37°C and 5% CO2. 20-24 hours after electroporation, the aNK effector cells from Example 2 were washed in PBS (calcium and magnesium-free) and resuspended in complete RPMI 1640 medium. The effector aNKs were then counted, with 1×10⁴ cells per well. 5From 6.25 × 10 4 The target cells were serially diluted to a maximum of 10⁶ viable effector cells and deposited in 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 containing 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, resuspended in 10 mL of complete RPMI, and counted. The target cells were placed in the wells of the 96-well round-bottom plate at a rate of 5 × 10⁶ per well. 3 The killer assay plates were loaded with viable target cells along with their effector cells. The killing assay plates were centrifuged at 400×g for 5 minutes and incubated at 37°C and 5% CO2 for 4 hours. After incubation, 22 μL of 9% (v / v) Triton X-100 (Sigma Aldrich) was added to the largest lysis control well, and the plates were incubated at room temperature for 5 minutes. The killing plates were centrifuged again, and 100 μL of the supernatant was transferred to a 96-well immunoassay plate for reading excitation at 485±20 nm and emission at 528±20 nm. Each sample was plated in triplicate.

[0103] Figures 4A–4E demonstrate % specific target cell lysis of HLA-A2-positive target cells exposed to either aNK cells pulsed with pp65-NLV or DMSO (control) and electroporated with the TCRαβ-ITAM fusion, or control aNK cells. "P-NR-025+P-NR-026 aNK" in Figure 4A represents the TCRαβ-ITAM fusion shown in Figure 1. "P-NR-027+P-NR-028 aNK" in Figure 4B represents the TCRαβ-ITAM fusion shown in Figure 2. "P-NR-002+P-NR-016+CD3γδ aNK" in Figure 4C represents wild-type TCRαβ co-electroporated with CD3γδ to function as a positive killing control, containing the same anti-pp65-NLV-HLA-A2 variable domain as the other constructs. "P-WT-173" in Figure 4D is also a positive killing control, but it stably expresses the same wild-type anti-pp65-NLV-HLA-A2 TCRαβ and CD3γδ. The 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-A * It contained the same variable TCRαβ sequence pair that was pre-determined to bind to the NLV peptide on 02. The error bars only indicate the ± standard deviation of technical repeats.

[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 then immersed in RPMI for 10 minutes. 7 The cells were resuspended at a concentration of 1 cell / 50 μL. 5 μg of mRNA encoding the first and second peptide chains was added to 10 μg of RPMI per 2 mm cuvette. 7Individual aNK cells were combined. The cuvettes were subjected to three 20ms pulses at 200V using a BioRad GenePulser II. The electroporated cells were transferred to a culture medium containing IL-2 (Corning RPMI 1640 with L-Glu supplemented with 10% FBS and 1× PSA) and incubated overnight.

[0105] After overnight incubation, 2 × 10⁻¹⁰ samples were taken from each sample. 5 Individual cells were obtained and washed with PBS / BSA / EDTA buffer. The cells were resuspended in 100 μL of washing buffer. 5 μL of PE-HLA-A * Each sample was treated with either 0201, NLVPMVATV-PE, or an HLA-A2 dextramer-negative control. 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 electroporation and overnight incubation, aNK cells were washed three times and incubated with HLA-A2 stable KG-1 cells stained with calcein AM in a 10:1 ratio (effector:target). After 4 hours of incubation, the supernatant was obtained and analyzed for the fluorescence of calcein AM. Figure 6 shows the % specific killing of target cells by aNK cells expressing the modified TCR described herein.

[0107] Example 5: Expression of PWH308+PWH305 and chimeric TCRs of PWH308+PWH305 and cytotoxicity in primary T cells Human primary T cells were obtained from donor-derived leukopack (Charles River, Wilmington, USA). Peripheral blood mononuclear cells (PBMCs) were separated via a ficoll gradient, washed with K100 buffer, and sterilized in K100 for 10 minutes. 7The cells were resuspended at a concentration of 100 μL / 100 μL. CD3-rich T cells were then expanded and cultured in ImmunoCult® (StemCell Technologies, Cambridge, USA) and IL-2. 10 μg of mRNA encoding the first and second peptide chains was added to 10 μL of K100 buffer per 2 mm cuvette. 7 The cells were combined with individual T cells. Both cells were electroporated according to the electroporation protocols described herein, both incorporated herein by reference: U.S. Patent No. 11,377,652 and U.S. Patent Application Publication No. 20220025402. The electroporated cells were transferred to culture medium and incubated overnight.

[0108] After overnight incubation, 2 × 10⁻¹⁰ samples were taken from each sample. 5 Individual cells were obtained and washed with PBS / BSA / EDTA buffer. The cells were resuspended in 100 μL of washing buffer. 5 μL of PE-HLA-A * Each sample was treated with either 0201, NLVPMVATV-PE, or an HLA-A2 dextramer-negative control. 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] Following electroporation and overnight incubation, primary T cells were washed three times and incubated with HLA-A2 stable KG-1 cells stained with calcein AM in a 10:1 ratio (effector:target). After 4 hours of incubation, the supernatant was obtained and analyzed for calcein AM fluorescence. Figure 8 shows the % specific killing of target cells by primary T cells expressing the modified TCR described herein.

[0110] Example 6: Transfusion of patient-derived T cells using a modified TCR Patient T cells are derived from whole peripheral blood or isolated from tumor or aspiration area lymph nodes. The T cells are electroporated using a modified TCR as described herein. The electroporated T cells are proliferated ex vivo to a clinically effective number of cells, and a treatment-relevant number of cells are administered to the patient.

[0111] All references cited herein, including publications, patent applications, and patents, are incorporated herein by reference to the same extent as they are incorporated herein by reference, with each reference indicated to be incorporated individually and specifically.

[0112] In the context describing the present invention (particularly in the context of the following claims), the terms “a,” “an,” “the,” and “at least one,” as well as similar references, should be interpreted as covering both singular and plural forms, unless otherwise specified herein or unless clearly inconsistent with the context. 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 interpreted as meaning one item (A or B) selected from the listed items or any combination of two or more listed items (A and B), unless otherwise specified herein or unless clearly inconsistent with the context. The terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., “including, but not limited to.”) unless otherwise noted. Unless otherwise specified herein, the descriptions of value ranges are intended only as an abbreviation for each distinct value falling within that range, and each distinct value is incorporated herein as if it were described separately herein. All methods described herein may be carried out in any preferred order unless otherwise specified herein or unless it is particularly obviously inconsistent with the context. Any and all examples or exemplary words (e.g., “for example”) provided herein are intended only to better illustrate the invention and do not limit the scope of the invention unless specifically claimed. Words herein should not be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0113] Specific embodiments of the Invention, including the best mode understood by the inventors for carrying out the Invention, are described herein. Modifications of those specific embodiments may become apparent to those skilled in the art by reading the above detailed description. The inventors anticipate that those skilled in the art will use such modifications as appropriate, and the inventors intend that the Invention may be carried out in ways other than those specifically described herein. Accordingly, the Invention includes all modifications and equivalents of the subject matter described in the claims appended herein, as permitted by applicable law. Furthermore, any combination of the above elements in all conceivable modifications is encompassed by the Invention unless otherwise specifically noted herein or unless it is particularly incongruous with the context. This specification includes the following embodiments. [1] A modified T cell receptor (TCR) comprising a first peptide chain and a second peptide chain, wherein each peptide chain is Extracellular domain; Transmembrane domain; and intracellular domain Includes, The extracellular domain comprises a variable region, a constant region, and a linked peptide. The variable region and the steady-state 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 linker is a flexible linker, as described in [1]. [3] The TCR according to [1] or [2], wherein the Ig-CH-1 domain is IgG-CH-1a, IgG-CH-1b, or IgM-CH-1. [4] The TCR according to any one of [1] to [3], wherein the HLA-DRB domain is HLA-DRB1 or HLA-DRB2. [5] The TCR according to any one of [1] to [4], wherein the variable region on each of the peptide chains is the same variable region. [6] The variable regions on each peptide chain are different from each other, as described in any of [1] to [5]. [7] The intracellular domain comprises a CD28 region and a CD3ζITAM region, as described in any of [1] to [6]. [8] The first peptide chain is The aforementioned constant region is Ig-Cκ; HLA-DRA as the aforementioned transmembrane domain; and CD28 and CD3ζ as the intracellular domains mentioned above. A TCR, including any of the TCRs listed in [1] to [7]. [9] The first peptide chain comprises the TCR described in [8], including SEQ ID NO: 39.

[10] The first peptide chain comprises the TCR described in [8], including SEQ ID NO: 43.

[11] The second peptide chain is Ig-CH-1 as the constant region; HLA-DRB as the aforementioned transmembrane domain; and CD28 and CD3ζ as the intracellular domains mentioned above. A TCR, including any of the TCRs listed in [1] to [7].

[12] The second peptide chain comprises the TCR described in

[11] , including SEQ ID NO: 40.

[13] The second peptide chain comprises the TCR described in

[11] , including SEQ ID NO: 44.

[14] The second peptide chain comprises the TCR described in

[11] , including SEQ ID NO: 45.

[15] The second peptide chain comprises the TCR described in

[11] , including SEQ ID NO: 46.

[16] The TCR according to any one of [1] to

[15] , 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 domains; 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 domains.

[17] The TCR according to

[16] , wherein the first peptide chain comprises SEQ ID NO: 39 and the second peptide chain comprises SEQ ID NO: 40.

[18] The TCR according to

[17] , wherein the first peptide chain further comprises SEQ ID NO: 15, and the second peptide chain further comprises SEQ ID NO: 16.

[19] The TCR according to

[16] , wherein the first peptide chain comprises SEQ ID NO: 43 and the second peptide chain comprises SEQ ID NO: 44.

[20] The TCR according to

[19] , wherein the first peptide chain further comprises SEQ ID NO: 30, and the second peptide chain further comprises SEQ ID NO: 31.

[21] The TCR according to

[16] , wherein the first peptide chain comprises SEQ ID NO: 43 and the second peptide chain comprises SEQ ID NO: 44.

[22] The TCR according to

[21] , wherein the first peptide chain further comprises SEQ ID NO: 30, and the second peptide chain further comprises SEQ ID NO: 31.

[23] The TCR according to

[16] , wherein the first peptide chain comprises SEQ ID NO: 43 and the second peptide chain comprises SEQ ID NO: 44.

[24] The TCR according to

[23] , wherein the first peptide chain further comprises SEQ ID NO: 30, and the second peptide chain further comprises SEQ ID NO: 31. Cells containing a TCR as described in any of

[25] [1] to

[24] . A nucleic acid encoding a TCR as described in any of

[26] [1] to

[24] . A vector containing the nucleic acids described in

[27]

[26] .

[28] A method for reducing the occurrence of cancer or viral infection in a patient in whom reduction of the occurrence of cancer or viral infection or treatment thereof is required, comprising administering a pharmaceutical composition to the patient, wherein the pharmaceutical composition comprises a therapeutically effective amount of a modified TCR according to any of [1] to

[24] or a nucleic acid encoding a modified TCR according to any of [1] to

[24] .

[29] The method according to

[28] , wherein the pharmaceutical product comprises a vector containing the nucleic acid.

[30] The method according to

[28] or

[29] , comprising the pharmaceutical composition and cells comprising the modified TCR or nucleic acid encoding the modified TCR.

[31] The cancer is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, central nervous system cancer, 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, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, and vaginal cancer, according to any one of

[28] to

[30] .

[32] The method according to any one of

[28] to

[30] , wherein the viral infection is caused by a virus from a viridae selected from the group consisting of herpesviridae, adenoviridae, polyomavididae, poxviridae, reoviridae, coronavirusidae, picornaviridae, flaviviridae, hepeviridae, togaviridae, filoviridae, paramyxoviridae, pneumoviridae, rhabdoviridae, hantaviridae, and orthomyxoviridae.

[33] Use of the TCR described in [1], the cells described in

[25] , the nucleic acid described in

[26] , or the vector described in

[27] for the prevention or treatment of cancer or viral infection in patients who require the prevention or treatment of cancer or viral infection.

[0114] TIFF0007923864000004.tif241170TIFF0007923864000005.tif240170TIFF0007923864000006.tif240170TIFF0007923864000007.tif240170TIFF0007923864000008.tif241170TIFF0007923864000009.tif242170TIFF0007923864000010.tif242170TIFF0007923864000011.tif241170TIFF0007923864000012.tif241170TIFF0007923864000013.tif241170TIFF0007923864000014.tif242170TIFF0007923864000015.tif241170TIFF0007923864000016.tif240170TIFF0007923864000017.tif240170TIFF0007923864000018.tif241170TIFF0007923864000019.tif240170TIFF0007923864000020.tif241170TIFF0007923864000021.tif241170TIFF0007923864000022.tif241170TIFF0007923864000023.tif242170TIFF0007923864000024.tif241170TIFF0007923864000025.tif240170TIFF0007923864000026.tif241170TIFF0007923864000027.tif241170TIFF0007923864000028.tif240170TIFF0007923864000029.tif240170TIFF0007923864000030.tif241170TIFF0007923864000031.tif239170TIFF0007923864000032.tif242170TIFF0007923864000033.tif241170TIFF0007923864000034.tif241170TIFF0007923864000035.tif157170

Claims

1. A method for expressing a functionally modified T cell receptor (TCR) in T cells, comprising transtransferring at least one nucleic acid encoding first and second TCR peptide chains, wherein the first and second peptide chains of the modified TCR are combined with each other on the cell surface but not with the endogenous TCR peptide expressed on the T cell surface. The first peptide chain and the second peptide chain are, respectively, It includes an extracellular domain; a transmembrane domain; and an intracellular domain. The extracellular domain comprises a variable region, a constant region, and a linked peptide. The intracellular domain includes the CD28 region and the CD3ζITAM region. The variable region and the steady-state region are engaged via a linker. a) The constant region of the first peptide chain contains an Ig-Cκ domain, and the constant region of the second peptide chain contains an Ig-CH-1 domain, or b) The constant region of the first peptide chain includes the WinZip-B1 domain, and the constant region of the second peptide chain includes the WinZip-A2 domain. i) The transmembrane domain of the first peptide chain contains an HLA-DRA domain, and the transmembrane domain of the second peptide chain contains an HLA-DRB domain, or ii) The transmembrane domain of the first peptide chain contains an HLA-DRB domain, and the transmembrane domain of the second peptide chain contains an HLA-DRA domain. method.

2. The method according to claim 1, wherein the HLA-DRB domain is HLA-DRB1 or HLA-DRB2.

3. The method according to claim 1, wherein the variable region of the extracellular domain of the first peptide chain comprises a TCRα chain, and the variable region of the extracellular domain of the second peptide chain comprises a TCRβ chain, and the α and β chains have specificity for the HLA-presented peptide.

4. The method according to claim 1, wherein the variable region of the extracellular domain of the first peptide chain comprises a TCRβ chain, and the variable region of the extracellular domain of the second peptide chain comprises a TCRα chain, and the α and β chains are specific to the HLA-presented peptide.

5. The method according to claim 1, wherein the variable region of at least one of the extracellular domains of the first peptide chain and the second peptide chain includes an Ig variable domain, and the Ig variable domain has specificity for a tumor antigen.

6. The method according to claim 1, wherein the first peptide chain comprises the sequences of SEQ ID NO: 17 and SEQ ID NO: 21, and the second peptide chain comprises the sequences of SEQ ID NO: 18 and SEQ ID NO:

22.

7. The method according to claim 1, wherein the first peptide chain comprises the sequences of SEQ ID NO: 17 and SEQ ID NO: 21, and the second peptide chain comprises the sequences of SEQ ID NO: 18 and SEQ ID NO:

34.

8. The method according to claim 1, wherein the first peptide chain comprises the sequence of SEQ ID NO:

39.

9. The method according to claim 8, wherein the second peptide chain comprises the sequence of SEQ ID NO:

40.

10. The method according to claim 8, wherein the second peptide chain comprises the sequence of SEQ ID NO:

44.

11. The method according to claim 8, wherein the second peptide chain comprises the sequence of SEQ ID NO:

45.

12. The method according to claim 8, wherein the second peptide chain comprises the sequence of SEQ ID NO:

46.

13. The method according to claim 1, wherein the first peptide chain comprises Ig-Cκ as a constant region and HLA-DRA as a transmembrane domain, and the second peptide chain comprises Ig-CH-1 or Ig-CH-1b as a constant region and HLA-DRB1 or HLA-DRB2 as a transmembrane domain.

14. The method according to claim 13, wherein the first peptide chain further comprises a TCR variable α domain, and the second peptide chain further comprises a TCR variable β domain.

15. The method according to claim 14, wherein the first peptide chain further comprises the sequence of SEQ ID NO: 30, and the second peptide chain further comprises the sequence of SEQ ID NO:

31.

16. The method according to claim 13, wherein the first peptide chain further comprises a TCR variable β domain and the second peptide chain further comprises a TCR variable α domain.

17. The method according to claim 16, wherein the first peptide chain further comprises the sequence of SEQ ID NO: 31, and the second peptide chain further comprises the sequence of SEQ ID NO:

30.

18. T cells produced by the method according to any one of claims 1 to 17.

19. A composition used to reduce the occurrence of cancer or viral infection in patients who require reduction of the occurrence of cancer or viral infection or treatment thereof, comprising a therapeutically effective amount of T cells according to claim 18.

20. The composition according to claim 19, wherein the cancer is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, central nervous system cancer, 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, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, and vaginal cancer.

21. The aforementioned viral infections include those from the families Herpesviridae, Adenoviridae, Polyomaviridae, Poxviridae, Reoviridae, Coronaviridae, Picornaviridae, Flaviviridae, and Hepeviridae. The composition according to claim 19, caused by a virus from a viridae selected from the group consisting of Togaviridae, Filoviridae, Paramyxoviridae, Pneumoviridae, Rhabdoviridae, Hantaviridae, and Orthomyxoviridae.

Images