WO2023092452A1 - T cell receptor for identifying ebv lmp2 antigen and use thereof - Google Patents

Abstract

The present invention provides a T cell receptor (TCR) for specifically identifying an EBV LMP2 antigen, a conjugate and a fusion protein comprising the TCR, an immune cell expressing the TCR, a T cell drug containing the TCR, and the use of the TCR in preventing or treating EBV-related diseases (such as EBV-positive tumors or EBV-related lymphoproliferative disorders).

Description

T cell receptor for recognizing EBV LMP2 antigen and its application technical field

The invention relates to the fields of immunology and tumor treatment. Specifically, the present invention relates to a T cell receptor (TCR) that specifically recognizes EBV LMP2 antigens, conjugates and fusion proteins comprising the TCR, immune cells expressing the TCR, and T cell drugs comprising them, and its use for preventing or treating diseases associated with EBV (for example, EBV LMP2 protein-positive tumors or EBV-associated lymphoproliferative diseases).

Background technique

Epstein-Barr virus (EBV), a gamma-1 herpes virus, was discovered in 1964 by Epstein, Achong and Barr in cells cultured from Burkitt lymphoma tissue, and is one of the most common viruses in humans. EBV is carried in the human population as a lifelong asymptomatic infection, and about 90% of adults have EBV antibodies in their blood.

In 1968, EBV was shown to be the causative agent of heterophile-positive infectious mononucleosis. In 1970, EBV DNA was detected in tissues of patients with nasopharyngeal carcinoma. In the 1980s, EBV was found to be associated with non-Hodgkin's lymphoma and acquired immunodeficiency syndrome (AIDS) in patients with oral hairy leukoplakia (Jeffrey I. Cohen, 2000). Since then, EBV DNA has been detected in the genomes of different cancer types, indicating the association of EBV with these cancers.

EBV is transmitted by bodily fluids or tissue transplants, which infect resting B cells or epithelial cells, which in turn infect B cells. EBV combines the gp350 protein on its surface with the CD21 protein on the B cell surface to introduce the viral genome into the B cell (Fingeroth JD, 1984). Infected B cells first enter the lytic phase, packaging the virus or expressing the complete nucleoprotein and latent protein. Then, under the intervention of immune cells such as T cells, the infected B cells enter a latent period, and there are three states in the latent period, which express different antigens respectively. See Kerr BM, 1992 for specific expression types. Most of the tumors caused by EBV are in the latent stage, expressing nucleoprotein EBNA and latent proteins LMP1 and LMP2.

At present, there is no good treatment plan for patients with EBV infection. Although the commonly used antiviral drug acyclovir can inhibit the shedding of EBV virus, it cannot play a therapeutic role in the treatment of severe mononucleosis. Oral hairy leukoplakia, which has a better effect on acyclovir, is also prone to recurrence (Resnick L, 1988). Corticosteroids are also used clinically, with limited curative effect and large side effects (Straus SE, 1993) (Cook RC, 1999).

Contents of the invention

The present invention provides a T cell receptor (TCR) specifically recognizing EBV LMP2 antigen, cells and pharmaceutical compositions comprising the TCR, nucleic acid encoding the TCR, vectors and host cells for preparing the TCR, expression The immune cells of the TCR, and the method of using the TCR to treat a subject. The TCR provided by the present invention can induce an immune response against EBV-positive cells (eg, tumor cells) and thus treat EBV-related diseases (eg, EBV-positive tumors) in a subject. In addition, the TCR provided by the present invention is MHC-I-restricted, and the MHC-I-restricted population accounts for nearly one-third of the total population in my country, and has broad application prospects. Therefore, the present invention provides a brand-new T cell-based immunotherapy for the treatment of EBV-related diseases, which has great clinical value.

Therefore, in one aspect, the application provides an isolated T cell receptor (TCR) or an antigen-binding fragment thereof capable of specifically recognizing an EBV LMP2 antigen, the TCR or an antigen-binding fragment thereof comprising an alpha chain variable region (Vα) and/or beta chain variable region (Vβ), wherein,

(a) said Vα includes CDR1α, CDR2α and CDR3α, wherein said CDR3α has a sequence as shown in AVX ₁ X ₂ NNDMR (SEQ ID NO: 26); wherein:

X _is selected from (i) amino acid residues L, I and (ii) amino acid residues that are conservatively substituted with respect to (i);

_X is selected from (i) amino acid residues N, Y, A, H, Q, L, S, F, W, G, D, I, M, V, R, K, P and (ii) relative to ( i) is a conservatively substituted amino acid residue;

and / or,

(b) the Vβ includes CDR1β, CDR2β and CDR3β, wherein the CDR3β has a sequence as shown in ASSX ₃ X ₄ X ₅ X ₆ YEQY (SEQ ID NO: 27); wherein:

X _is selected from (i) amino acid residues P, L, R, H, Q and (ii) amino acid residues that are conservatively substituted with respect to (i);

X _is selected from (i) amino acid residues G, A, N, D, S, H and (ii) amino acid residues that are conservatively substituted with respect to (i);

X _is selected from (i) amino acid residues R, K and (ii) amino acid residues that are conservatively substituted with respect to (i);

_X6 is selected from (i) amino acid residues W, F and (ii) amino acid residues that are conservatively substituted with respect to (i).

In certain embodiments, X _is selected from amino acid residues L, I; X _is selected from amino acid residues N, Y, A, H, Q, L, S, F, W, G, D, I, M , V, R, K, P; _X3 is selected from amino acid residues P, L, R, H, Q; _X4 is selected from amino acid residues G, A, N, D, S, H; _X5 is selected from amino acid residues Residues R, K; _X6 are selected from amino acid residues W, F.

In certain embodiments, the TCR is soluble or membrane bound.

In certain embodiments, the TCR is a full length TCR, a soluble TCR or a single chain TCR.

In certain embodiments, the TCR may be a full length TCR comprising a full length alpha chain and a full length beta chain.

In certain embodiments, the TCR is a soluble TCR lacking one or more transmembrane and/or cytoplasmic regions. In certain embodiments, solubility is generated by fusing the extracellular domain of the TCR of the present application to other protein domains (e.g., maltose-binding protein, thioredoxin, human constant kappa domain, or leucine zipper). TCR, see eg, et al., Front Oncol. 2014;4:378, which is hereby incorporated by reference in its entirety.

In certain embodiments, the TCR of the present application may also be a single-chain TCR (scTCR) comprising Vα and Vβ linked by a peptide linker. Such scTCRs may include Vα and Vβ, each linked to a TCR constant region. Alternatively, a scTCR may comprise Vα and Vβ, wherein Vα, Vβ, or both Vα and Vβ are not linked to a TCR constant region. Exemplary scTCRs are described in PCT Publication Nos. WO 2003/020763, WO 2004/033685, and WO 2011/044186, the disclosures of each of which are incorporated herein by reference in their entirety.

In certain embodiments, a TCR of the present application may comprise two polypeptide chains (e.g., an alpha chain and a beta chain), wherein the chains have been engineered to each have a cysteine that can form an interchain disulfide bond Residues. Accordingly, the TCR of the present application may comprise two polypeptide chains linked by an engineered disulfide bond. Exemplary TCRs with engineered disulfide bonds are described in US Patent Nos. 8,361,794 and 8,906,383, each of which is incorporated herein by reference in its entirety.

In certain embodiments, the TCR or antigen-binding fragment thereof has one or more of the following characteristics:

(i) the CDR1α has the sequence shown in SEQ ID NO: 11 or has one or several amino acid substitutions, deletions or additions thereto (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) the sequence of;

(ii) the CDR2α has the sequence shown in SEQ ID NO: 12 or has one or several amino acid substitutions, deletions or additions thereto (such as 1, 2 or 3 amino acid substitutions, deletions or additions) the sequence of;

(iii) the CDR1β has the sequence shown in SEQ ID NO: 14 or has one or several amino acid substitutions, deletions or additions thereto (such as 1, 2 or 3 amino acid substitutions, deletions or additions) the sequence of;

(iv) The CDR2β has the sequence shown in SEQ ID NO: 15 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) the sequence of.

In certain embodiments, the TCR or antigen-binding fragment thereof comprises:

(i) CDR1α as shown in SEQ ID NO:11; CDR2α as shown in SEQ ID NO:12; CDR3α as shown in any one of SEQ ID NOs:13, 28-44; and/or,

(ii) CDR1β as shown in SEQ ID NO:14; CDR2β as shown in SEQ ID NO:15; CDR3β as shown in any one of SEQ ID NOs:16-17, 45-54.

In certain embodiments, the TCR or antigen-binding fragment thereof comprises:

(1) CDR1α as shown in SEQ ID NO:11; CDR2α as shown in SEQ ID NO:12; CDR3α as shown in SEQ ID NO:13; and/or, CDR1β as shown in SEQ ID NO:14 ; CDR2β as shown in SEQ ID NO:15; CDR3β as shown in SEQ ID NO:16;

(2) CDR1α as shown in SEQ ID NO:11; CDR2α as shown in SEQ ID NO:12; CDR3α as shown in SEQ ID NO:13; and/or, CDR1β as shown in SEQ ID NO:14 ; CDR2β as shown in SEQ ID NO:15; CDR3β as shown in SEQ ID NO:17;

(3) CDR1α as shown in SEQ ID NO:11; CDR2α as shown in SEQ ID NO:12; CDR3α as shown in any one of SEQ ID NOs:28-44; and/or, as shown in SEQ ID NO: CDR1β as shown in 14; CDR2β as shown in SEQ ID NO:15; CDR3β as shown in SEQ ID NO:16;

(4) CDR1α as shown in SEQ ID NO:11; CDR2α as shown in SEQ ID NO:12; CDR3α as shown in any one of SEQ ID NOs:28-44; and/or, as shown in SEQ ID NO: CDR1β as set forth in 14; CDR2β as set forth in SEQ ID NO:15; CDR3β as set forth in SEQ ID NO:17; or

(5) CDR1α as shown in SEQ ID NO:11; CDR2α as shown in SEQ ID NO:12; CDR3α as shown in SEQ ID NO:13; and/or, CDR1β as shown in SEQ ID NO:14 ; CDR2β as shown in SEQ ID NO: 15; CDR3β as shown in any one of SEQ ID NOs: 45-54.

In certain embodiments, the CDRs described in any of the above embodiments are defined according to the IMGT numbering system.

In certain embodiments, in the TCR or antigen-binding fragment thereof,

(a) said Vα also includes FR1α, FR2α, FR3α and FR4α, wherein:

The FR1α has the sequence shown in SEQ ID NO: 55 or 59 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared therewith sequence;

The FR2α has a sequence shown in SEQ ID NO:56 or a sequence having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;

The FR3α has a sequence shown in SEQ ID NO:57 or a sequence having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;

The FR4α has a sequence shown in SEQ ID NO: 58 or a sequence having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;

and / or,

(b) said Vβ also includes FR1β, FR2β, FR3β and FR4β, wherein:

The FR1β has a sequence shown in SEQ ID NO: 60 or a sequence having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;

The FR2β has a sequence shown in SEQ ID NO: 61 or a sequence having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;

The FR3β has a sequence shown in SEQ ID NO: 62 or a sequence having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;

The FR4β has the sequence shown in SEQ ID NO: 63 or a sequence with one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto.

In certain embodiments, the substitutions are conservative substitutions.

In some embodiments, the Vα comprises the sequence shown in SEQ ID NO:3 or a variant thereof, and the Vβ comprises the sequence shown in SEQ ID NO:4 or a variant thereof, the variant is derived from There is one or several amino acid substitutions, deletions or additions (eg, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence.

In some embodiments, the Vα comprises the sequence shown in SEQ ID NO: 3 or a variant thereof, wherein the variant is selected from one or more of the following (for example, 1, 2, 3 or 4) amino acid substitutions are included in the amino acid position, and the amino acid position is determined according to the IMGT TCR numbering system: 107, 108; the Vβ comprises the sequence shown in SEQ ID NO: 4 or a variant thereof, and the variant is selected from Amino acid substitutions are comprised in one or more (eg, 1, 2, 3, or 4) of the following amino acid positions, identified according to the IMGT TCR numbering system: 108, 109, 110, 111.

In some embodiments, the Vα comprises a variant of the sequence shown in SEQ ID NO: 3, the variant comprising one or more (for example, 1, 2, 3 or 4) amino acid substitutions, the amino acid positions are determined according to the IMGT TCR numbering system: the amino acid substitution at position 107 is I; the amino acid substitution at position 108 is Y, A, H, Q, L, S, F, W, G , D, I, M, V, R, K or P. In such embodiments, the Vβ preferably comprises the sequence shown in SEQ ID NO:4.

In certain embodiments, the Vβ comprises a variant of the sequence shown in SEQ ID NO: 4, the variant comprising 1 or more (for example, 1, 2, 3 or 4) amino acid substitutions, the amino acid positions are determined according to the IMGT TCR numbering system: amino acid substitutions at position 108 are L, R or H; amino acid substitutions at position 109 are A, N, D, S or H; amino acid substitutions at position 110 Amino acid substitution at position 111 was F. In such embodiments, said Vα preferably comprises the sequence shown in SEQ ID NO:3.

In some embodiments, the Vα comprises the sequence shown in SEQ ID NO: 7 or a variant thereof, and the Vβ comprises the sequence shown in SEQ ID NO: 8 or a variant thereof, the variant is derived from There is one or several amino acid substitutions, deletions or additions (eg, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence.

In some embodiments, the Vα comprises the sequence shown in SEQ ID NO: 7 or a variant thereof, wherein the variant is selected from one or more of the following (for example, 1, 2, 3 or 4) amino acid substitutions are included in the amino acid position, and the amino acid position is determined according to the IMGT TCR numbering system: 107, 108; the Vβ comprises the sequence shown in SEQ ID NO: 8 or a variant thereof, and the variant is selected from Amino acid substitutions are comprised in one or more (eg, 1, 2, 3, or 4) of the following amino acid positions, identified according to the IMGT TCR numbering system: 108, 109, 110, 111.

In certain embodiments, said Vα comprises a variant of the sequence shown in SEQ ID NO: 7, said variant comprising 1 or several (for example, 1, 2, 3 or 4) amino acid substitutions, the amino acid positions are determined according to the IMGT TCR numbering system: the amino acid substitution at position 107 is I; the amino acid substitution at position 108 is Y, A, H, Q, L, S, F, W, G , D, I, M, V, R, K or P. In such embodiments, the Vβ preferably comprises the sequence shown in SEQ ID NO:8.

In certain embodiments, the Vβ comprises a variant of the sequence shown in SEQ ID NO: 8, the variant comprising 1 or more (for example, 1, 2, 3 or 4) amino acid substitutions, the amino acid positions are determined according to the IMGT TCR numbering system: amino acid substitutions at position 108 are L, R or H; amino acid substitutions at position 109 are A, N, D, S or H; amino acid substitutions at position 110 Amino acid substitution at position 111 was F. In such embodiments, said Vα preferably comprises the sequence shown in SEQ ID NO:7.

In certain embodiments, the TCR or the antigen-binding fragment thereof can specifically recognize an epitope peptide presented by MHC-I molecules, and the epitope peptide comprises amino acid residues 340-349 of the EBV LMP2 antigen.

In certain embodiments, the 340-349 amino acid residues of the EBV LMP2 antigen have the sequence shown in SEQ ID NO: 1 or 2.

In certain embodiments, the MHC class I molecule is HLA-A.

In certain embodiments, the MHC class I molecule is HLA-A11. In certain embodiments, the MHC class I molecule is HLA-A*11:01.

In certain embodiments, when the TCR or antigen-binding fragment thereof is expressed on the surface of a T cell, the T cell interacts with a second cell displaying the epitope peptide (e.g., a cell expressing an HLA class I molecule). cells) were activated during co-culture.

In another aspect, the present application also provides a conjugate comprising a TCR or an antigen-binding fragment thereof as described above and an effector moiety conjugated thereto.

In this context, the term "effector moiety" refers to a component or functional group capable of modulating (eg increasing or decreasing) a natural activity of a molecule to which it is linked or conferring a novel activity on that molecule. In some embodiments, the effector moiety is a biologically active compound or polypeptide (eg, a compound or polypeptide having an effect on a cell targeted by the TCR), or a detectable label.

In this context, the term "conjugation" refers to any method known in the art for functionally linking protein domains, including but not limited to: recombinant fusion with or without linker, intein-mediated fusion, non- Covalent bonding and covalent bonding, such as disulfide bonding, peptide bonding, hydrogen bonding, electrostatic bonding, and conformational bonding, such as biotin-avidin bonding. In certain embodiments, conjugation to an effector moiety can be performed chemically or recombinantly by forming a covalent bond between two molecules to form one molecule.

In certain embodiments, the effector moiety may be a therapeutic moiety. A therapeutic moiety refers to a compound or polypeptide that is useful as a therapeutic agent. The conjugates take advantage of the targeting of the TCR to allow the therapeutic moiety to exert a therapeutic effect on cells targeted by the TCR.

In certain embodiments, said TCR or antigen-binding fragment thereof in said conjugate is soluble.

In certain embodiments, a TCR or antigen-binding fragment thereof of the present application is optionally conjugated to an effector moiety via a linker (eg, a peptide linker). In certain embodiments, the effector moiety is linked to the N-terminus or C-terminus of a TCR of the present application or an antigen-binding fragment thereof.

In another aspect, the present application also provides a fusion protein comprising the above-mentioned TCR or an antigen-binding fragment thereof and another peptide or protein.

In certain embodiments, a TCR or antigen-binding fragment thereof of the invention is fused to another peptide or protein, optionally via a peptide linker. In certain embodiments, the additional peptide or protein is linked to the N- or C-terminus of a TCR of the invention or an antigen-binding fragment thereof.

In certain embodiments, the additional peptide or protein is an effector portion of the peptide or protein described in the above aspect.

In certain embodiments, said TCR or antigen-binding fragment thereof in said fusion protein is soluble.

In another aspect, the application also provides an isolated nucleic acid molecule, which comprises a TCR or an antigen-binding fragment thereof encoding the above-mentioned TCR or its α chain variable region and/or a β chain variable region or its α chain and/or The nucleotide sequence of the beta chain, or comprises the nucleotide sequence encoding the fusion protein as described above.

In certain embodiments, said isolated nucleic acid molecule comprises a first nucleotide sequence encoding said TCR alpha chain variable region and a second nucleotide sequence encoding said TCR beta chain variable region.

In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the alpha chain of the TCR or an antigen-binding fragment thereof and a second core encoding the beta chain of the TCR or an antigen-binding fragment thereof nucleotide sequence.

In certain embodiments, said first nucleotide sequence and said second nucleotide sequence are present on different isolated nucleic acid molecules.

In certain embodiments, said first nucleotide sequence and said second nucleotide sequence are present in any order on the same isolated nucleic acid molecule; in certain embodiments, said first nucleotide sequence The acid sequence and said second nucleotide sequence are linked in any order by a nucleotide sequence encoding a self-cleaving peptide (eg P2A).

In another aspect, the present application also provides a vector comprising the isolated nucleic acid molecule as described above.

In certain embodiments, the vector comprises nucleotides encoding a TCR as described above or an antigen-binding fragment thereof or an α-chain variable region and/or a β-chain variable region or an α-chain and/or a β-chain thereof sequence.

In certain embodiments, the vector comprises a first nucleotide sequence encoding the variable region of the TCR alpha chain and a second nucleotide sequence encoding the variable region of the TCR beta chain.

In certain embodiments, the vector comprises a first nucleotide sequence encoding the alpha chain of the TCR or an antigen-binding fragment thereof and a second nucleotide sequence encoding the beta chain of the TCR or an antigen-binding fragment thereof .

In certain embodiments, said first nucleotide sequence and said second nucleotide sequence are present on different vectors.

In certain embodiments, said first nucleotide sequence and said second nucleotide sequence are present in any order on the same vector. In certain embodiments, the first nucleotide sequence and the second nucleotide sequence are linked in any order by a nucleotide sequence encoding a self-cleaving peptide (eg, P2A).

In certain embodiments, the vector comprises a nucleotide sequence encoding a fusion protein as described above.

In certain embodiments, the vector is a viral vector, such as a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a baculoviral vector.

In another aspect, the present application also provides a host cell comprising the isolated nucleic acid molecule as described above, or the vector as described above.

In certain embodiments, the host cell comprises nucleosides encoding a TCR as described above or an antigen-binding fragment thereof or an alpha chain variable region and/or a beta chain variable region or an alpha chain and/or a beta chain thereof acid sequence.

In certain embodiments, the host cell comprises a nucleotide sequence encoding a fusion protein as described above.

In certain embodiments, the host cell comprises E. coli, yeast, insect cells, or mammalian cells.

In another aspect, the present application also provides a method for preparing the above-mentioned TCR or its antigen-binding fragment, or the above-mentioned fusion protein, which includes culturing the above-mentioned host cell under conditions that allow protein expression , and recovering said TCR or antigen-binding fragment thereof, or fusion protein from cultured host cell culture.

In another aspect, the present application also provides engineered immune cells expressing the above-mentioned TCR or antigen-binding fragments thereof on the cell surface.

In certain embodiments, the engineered immune cell comprises a nucleotide sequence encoding a TCR as described above, or an antigen-binding fragment thereof.

Said immune cells may be isolated or obtained from any tissue in which such cells are found. For example, APCs can be found in mammalian peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, ascitic fluid, pleural effusion, spleen tissue or tumors, and then optionally in culture medium containing appropriate cytokines cultured and sorted in order to obtain the desired immune cells. Alternatively, the immune cells can also be cultured and provided in other ways, such as obtained from precursor cells of immune cells (for example, precursors of T lymphocytes) through induction and differentiation, and the precursor cells can be, for example, pluripotent Stem cells (eg, embryonic stem cells, induced pluripotent stem cells), hematopoietic stem cells or lymphocyte progenitor cells, hematopoietic stem cells or lymphocyte progenitor cells are isolated and/or enriched from, for example, bone marrow, umbilical cord blood or peripheral blood.

In certain embodiments, the immune cells are lymphocytes.

In certain embodiments, the immune cells are selected from T cells (such as αβ T cells, γδ T cells, or iPSC-induced T cells), tumor infiltrating lymphocytes (TIL), natural killer (NK) cells, natural killer T (NKT ) cells, or any combination thereof.

The immune cells may be self/autologous ("self") or non-self ("non-self", eg allogeneic). "Autologous" refers to cells from the same subject; "allogeneic" refers to cells of the same species that are genetically different from the compared cells.

It is understood that the engineered immune cells of the present application may be contained in an isolated cell population. The cell population may be a heterogeneous population, for example, the cell population contains, in addition to the engineered immune cells of the present application, at least one other cell that does not have antigen specificity for EBV LMP2, or for example, the The cell population contains more than one type of immune cells, but these types of immune cells all express the TCR of the application so as to have antigen specificity to EBV LMP2. Furthermore, the population of cells can also be a substantially homogeneous population, e.g., wherein the population consists essentially of (e.g., consists essentially of) T cells having antigen specificity for EBV LMP2.

In another aspect, the present application also provides a method for preparing the above-mentioned modified immune cells, which includes: (1) providing immune cells from a test; (2) using the above-mentioned isolated nucleic acid molecules or The above-mentioned vector is introduced into the immune cell described in step (1), and the nucleic acid molecule or vector comprises a nucleotide sequence encoding the above-mentioned TCR or its antigen-binding fragment, so as to obtain the expression of the TCR or its antigen-binding Fragments of immune cells.

In some embodiments, in step (1), the immune cells are pretreated; the pretreatment includes sorting, activation and/or proliferation of immune cells.

In some embodiments, the pretreatment comprises contacting the immune cells with one or more selected from anti-CD3 antibody, anti-CD28 antibody, IL-2 and IL-15, thereby stimulating the immune cells and inducing They proliferate, thereby generating preconditioned immune cells.

In some embodiments, in step (2), the nucleic acid molecules or vectors can be introduced into immune cells by various suitable methods, such as calcium phosphate transfection, DEAE-dextran mediated transfection, display Microinjection, electroporation, TALEN approach, ZFN approach, non-viral vector-mediated transfection (e.g. liposomes) or viral vector-mediated transfection (e.g. lentiviral infection, retroviral infection, adenoviral infection), And other physical, chemical or biological means for transferring into host cells, such as transposon technology, CRISPR-Cas9 and other technologies.

In some embodiments, after step (2), the method further includes: expanding the immune cells obtained in step (2).

In another aspect, the present application also provides a pharmaceutical composition, which comprises the above-mentioned TCR or its antigen-binding fragment, conjugate, fusion protein, nucleic acid molecule, vector, host cell, or engineered immune cell; and Pharmaceutically acceptable carrier and/or excipient.

In certain embodiments, the pharmaceutical composition further comprises an additional therapeutic agent, such as an antineoplastic agent or an immunopotentiator.

In certain embodiments, the antineoplastic agent is selected from the group consisting of alkylating agents, mitotic inhibitors, antineoplastic antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, radiation Sensitizers, anti-angiogenic agents, cytokines, immune checkpoint inhibitors (eg, antibodies to PD-1, antibodies to PD-L1, antibodies to CTLA-4, antibodies to LAG-3, or antibodies to TIM3).

In certain embodiments, the immunopotentiator is selected from immunostimulatory antibodies (such as anti-CD3 antibodies, anti-CD28 antibodies, anti-CD40L (CD154) antibodies, anti-41BB (CD137) antibodies, anti-OX40 antibodies, anti-GITR antibodies or any combination thereof) or immunostimulatory cytokines (such as IL-2, IL-3, IL-12, IL-15, IL-18, IFN-γ, IL-10, TGF-β, GM-CSF, or random combination).

In another aspect, the present application also provides the above-mentioned TCR or its antigen-binding fragment, conjugate, fusion protein, nucleic acid molecule, vector, host cell, transformed immune cell, or pharmaceutical composition, when preparing a drug The purposes in, described medicament is used for inducing the immune response against EBV positive cell in experimenter, and/or preventing or treating the disease associated with EBV in experimenter; Wherein, said nucleic acid molecule, carrier or host The cell comprises a nucleotide sequence encoding said TCR or an antigen-binding fragment thereof, or a fusion protein.

In certain embodiments, the EBV positive cells express the EBV LMP2 antigen.

In certain embodiments, the EBV positive cells are tumor cells.

In certain embodiments, the EBV-associated disease is selected from EBV-positive tumors or EBV-associated lymphoproliferative disorders.

In certain embodiments, the EBV-positive tumor is selected from: B-cell tumors (Burkitt lymphoma, Hodgkin lymphoma, diffuse large B-cell lymphoma, etc.), T-cell lymphoma, NK-cell lymphoma, nasopharyngeal carcinoma , gastric cancer, leiomyosarcoma.

In certain embodiments, the EBV-associated lymphoproliferative disorder is selected from post transplant lymphoproliferative disorder (PTLD).

In certain embodiments, the subject is a human.

In certain embodiments, the subject is HLA-A11 positive; in certain embodiments, the subject is HLA-A*11:01 positive.

In certain embodiments, the TCR or antigen-binding fragment thereof, conjugate, fusion protein, nucleic acid molecule or vector or host cell, engineered immune cell, or pharmaceutical composition is administered in combination with an additional therapeutic agent, such as Simultaneous, separate or sequential administration; in certain embodiments, the additional therapeutic agent is an immunostimulatory agent or an antineoplastic agent.

In another aspect, the present application also provides a method for inducing an immune response against EBV-positive cells in a subject and/or preventing or treating a disease associated with EBV in a subject, the method comprising: A subject in need thereof is administered an effective amount of a TCR or antigen-binding fragment thereof, conjugate, fusion protein, nucleic acid molecule, vector, host cell, engineered immune cell, or pharmaceutical composition as described above.

In certain embodiments, the EBV positive cells express the EBV LMP2 antigen.

In certain embodiments, the EBV positive cells are tumor cells.

In certain embodiments, the EBV-associated disease is selected from EBV-positive tumors or EBV-associated lymphoproliferative disorders.

In certain embodiments, the EBV-positive tumor is selected from: B-cell tumors (Burkitt lymphoma, Hodgkin lymphoma, diffuse large B-cell lymphoma, etc.), T-cell lymphoma, NK-cell lymphoma, nasopharyngeal carcinoma , gastric cancer, leiomyosarcoma.

In certain embodiments, the EBV-associated lymphoproliferative disorder is selected from post transplant lymphoproliferative disorder (PTLD).

In certain embodiments, the subject is a human.

In certain embodiments, the subject is HLA-A11 positive; in certain embodiments, the subject is HLA-A*11:01 positive.

In certain embodiments, the method further comprises administering to the subject an additional therapeutic agent, such as an immune potentiating agent or an antineoplastic agent.

In some embodiments, the method includes: (1) providing the immune cells required by the subject; (2) introducing the nucleotide sequence encoding the above-mentioned TCR or its antigen-binding fragment into step (1) For the immune cells, obtain immune cells expressing the TCR or an antigen-binding fragment thereof on the surface; (3) administer the immune cells obtained in step (2) to the subject.

In certain embodiments, the immune cells are lymphocytes.

In certain embodiments, the immune cells are selected from T cells (such as αβ T cells, γδ T cells, or iPSC-induced T cells), tumor infiltrating lymphocytes (TIL), natural killer (NK) cells, natural killer T (NKT ) cells, or any combination thereof.

In certain embodiments, a TCR or antigen-binding fragment thereof, conjugate, fusion protein, nucleic acid molecule, vector, host cell, engineered immune cell, or pharmaceutical composition described herein may be combined with an additional therapy Administration, for example simultaneously, separately or sequentially. This additional therapy can be any therapy known to be used on tumors, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy or palliative care.

The TCR or its antigen-binding fragment, conjugate, fusion protein, nucleic acid molecule, vector, host cell, transformed immune cell, or pharmaceutical composition of the present application can be formulated into any dosage form known in the medical field, for example, a tablet , pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injections and concentrated solutions for injections), inhalation Sprays, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceuticals of the present application should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the necessary doses of the TCR or antigen-binding fragments thereof, conjugates, fusion proteins, engineered immune cells, or drug combinations of the present application in an appropriate solvent and, optionally, simultaneously incorporate other desired ingredients (including but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof ), followed by filter sterilization. In addition, sterile injectable solutions can be prepared as sterile lyophilized powder (eg, by vacuum drying or freeze-drying) for ease of storage and use. Such sterile lyophilized powders can be dispersed in suitable carriers before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (such as 0.9% (w/v) NaCl), Dextrose solution (eg 5% glucose), surfactant containing solution (eg 0.01% polysorbate 20), pH buffer solution (eg phosphate buffer solution), Ringer's solution and any combination thereof.

Accordingly, in certain exemplary embodiments, the pharmaceutical compositions described herein comprise sterile injectable liquids (eg, aqueous or non-aqueous suspensions or solutions). In certain exemplary embodiments, such sterile injectable liquids are selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (eg 0.9% (w/v) NaCl), dextrose solutions (eg, 5% dextrose), solutions containing surfactants (eg, 0.01% polysorbate 20), pH buffered solutions (eg, phosphate buffered saline), Ringer's solutions, and any combination thereof.

The TCR or its antigen-binding fragment, conjugate, fusion protein, nucleic acid molecule, vector, host cell, engineered immune cell, or pharmaceutical composition of the present application can be administered by any suitable method known in the art, including But not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (eg, powder, ointment, or drops), or nasal route . However, for many therapeutic uses, the preferred route/mode of administration is parenteral (eg, intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will understand that the route and/or manner of administration will vary depending on the intended purpose. In certain embodiments, the TCR or antigen-binding fragment thereof, conjugate, fusion protein, nucleic acid molecule, vector, host cell, engineered immune cell, or pharmaceutical composition of the present application is administered by intravenous injection or bolus injection.

Definition of Terms

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the laboratory operation steps of virology, biochemistry and immunology used in this paper are all routine steps widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

When the terms "for example", "such as", "such as", "including", "comprises" or variations thereof are used herein, these terms will not be considered as terms of limitation, but will be construed to mean "but not limited to ” or “not limited to”.

Unless otherwise indicated herein or clearly contradicted by context, the terms "a" and "an" and "the" and similar designations in the context of describing the present invention (especially in the context of the following claims) are to be construed to cover singular and plural.

As used herein, the term "MHC" refers to a group of genes that determine whether transplanted tissues are identical, are closely related to immune responses, and are closely linked, mainly including MHC-I molecules and MHC-II molecules. "MHC class I molecule" refers to a dimer of an MHC class I alpha chain and a beta2 microglobulin chain, and "MHC class II molecule" refers to a dimer of an MHC class II alpha chain and an MHC class II beta chain. The human MHC is called the human leukocyte antigen (HLA) complex. In some cases, the MHC molecule can be an membrane-bound protein expressed on the surface of a cell. In other cases, MHC molecules may be soluble proteins lacking transmembrane or cytoplasmic regions.

As used herein, the terms "T cell receptor" and "TCR" are used interchangeably and refer to a molecule comprising a CDR or variable region from an αβ or γδ T cell receptor. Examples of TCRs include, but are not limited to, full-length TCRs, antigen-binding fragments of TCRs, soluble TCRs lacking transmembrane and cytoplasmic regions, single-chain TCRs containing TCR variable regions attached by flexible linkers, Key-linked TCR chains, etc.

As used herein, the term "full-length TCR" refers to a TCR comprising a dimer of a first polypeptide chain and a second polypeptide chain, each of which comprises a TCR variable region and comprises a TCR The TCR constant region of the transmembrane region and the TCR cytoplasmic region. In certain embodiments, the full-length TCR includes a mature full-length TCR alpha chain and a mature full-length TCR beta chain. In certain embodiments, the full-length TCR includes a mature full-length TCR gamma chain and a mature full-length TCR delta chain.

As used herein, the term "variable region" refers to the portion of a mature TCR polypeptide chain (e.g., TCR alpha chain or beta chain) that is not composed of the TRAC gene of the TCR alpha chain, the TRBC1 gene or the TRBC2 gene of the TCR beta chain, The TRDC gene of TCR δ chain or the TRGC1 gene or TRGC2 gene of TCR γ chain encodes. In certain embodiments, the TCR variable region of the TCR α chain encompasses all amino acids of the mature TCR α chain polypeptide encoded by the TRAV gene and/or TRAJ gene, and the TCR variable region of the TCR β chain encompasses all amino acids of the mature TCR α chain polypeptide encoded by the TRBV gene, TRBD gene and/or or all amino acids of the mature TCRβ chain polypeptide encoded by the TRBJ gene (see for example, "T cell receptor Factsbook (T cell receptor Factsbook)", (2001), LeFranc and LeFranc, Academic Press (Academic Press), ISBN0-12 -441352-8, which is incorporated herein by reference in its entirety). TCR variable regions typically include framework regions (FRs) 1, 2, 3 and 4 and complementarity determining regions (CDRs) 1, 2 and 3. Herein, the terms "α chain variable region" and "Vα" are used interchangeably and refer to the variable region of the TCRα chain. The terms "beta chain variable region" and "Vβ" are used interchangeably and refer to the variable region of the TCR beta chain.

As used herein, the term "CDR" or "complementarity determining region" in reference to a TCR refers to the non-contiguous antigen binding sites found within the variable region of a TCR chain (eg, an alpha chain or a beta chain). These regions have been described in Lefranc, (1999) "The Immunologist" 7:132-136; Lefranc et al., (1999) "Nucleic Acids Res" 27:209-212; LeFranc (2001) " T Cell Receptor Sourcebook", Academic Press, ISBN 0-12-441352-8; Lefranc et al., (2003) "Dev Comp Immunol" 27(1):55-77; and in Kabat et al., (1991) "Sequences of proteins of immunological interest," each of which is hereby incorporated by reference in its entirety. In certain embodiments, CDRs are defined according to the IMGT numbering system described in Lefranc (1999), supra. In certain embodiments, the CDRs are defined according to the Kabat numbering system described in Kabat, supra.

As used herein, the term "FR" or "framework region" in reference to a TCR refers to those amino acid residues in the variable region of a TCR chain (eg, alpha chain or beta chain) other than the CDRs as defined above.

As used herein, the term "constant region" with respect to a TCR refers to the TRAC gene (for the TCR α chain), the TRBC1 or TRBC2 gene (for the TCR β chain), the TRDC gene (for the TCR δ chain), or the TRGC1 or TRGC2 gene (for the TCR δ chain) TCR gamma chain) encodes a portion of the TCR, optionally lacking all or a portion of the transmembrane region and/or all or a portion of the cytoplasmic region. In certain embodiments, the TCR constant region lacks transmembrane and cytoplasmic regions. The TCR constant region does not contain amino acids encoded by the TRAV, TRAJ, TRBV, TRBD, TRBJ, TRDV, TRDD, TRDJ, TRGV, or TRGJ genes (see, e.g., T Cell Receptor Sourcebook, (2001), LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8, which is hereby incorporated by reference in its entirety).

As used herein, the term "antigen-binding fragment" with respect to a TCR refers to any part or fragment of a TCR that retains the biological activity of the TCR (the parental TCR) as part of the TCR. The biological activity may include the ability to specifically bind to the same antigen (e.g. EBV LMP2 antigen) or MHC-antigen complex to which the parental TCR binds.

As used herein, the term "specific binding" or "specific recognition" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen. The strength or affinity of a specific binding interaction can be expressed in terms of the equilibrium dissociation constant ( _KD ) for that interaction. In the present application, the term " _KD " refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. The specific binding properties between two molecules can be determined using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation. Both the "association rate constant" ( _ka or k _on ) and the "dissociation rate constant" (k _dis or k _off ) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature , 1993, 361:186-187). The ratio kdis/kon is equal to the dissociation constant _KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). _KD , _kon and _kdis values can be measured by any effective method, for example by surface plasmon resonance (SPR) in Biacore, or using bioluminescent interferometry or Kinexa.

In the context of a TCR, the term "specific binding" or "specific recognition" refers to the ability of a TCR to preferentially bind a specific antigen (eg, a specific peptide or a specific peptide-MHC complex combination). Typically, a TCR that specifically binds an antigen does not bind or binds with lower affinity to other antigens. For example, an antigen-specific TCR at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1,000-fold, 5,000-fold, or 10,000-fold higher in association constant (K _a ) compared to other antigens that are not specific A fold of _Ka binds to the target antigen. In certain embodiments, a TCR disclosed herein, or an antigen-binding fragment thereof, specifically binds to EBV LMP2. In certain embodiments, a TCR disclosed herein or an antigen-binding fragment thereof specifically binds to the epitope peptide of the first aspect or a variant thereof. In certain embodiments, a TCR disclosed herein or an antigen-binding fragment thereof specifically binds to the sequence shown in SEQ ID NO: 1 or 2 (in particular, SEQ ID NO: 20).

As used herein, the term "immune cell" refers to any cell of the immune system that has one or more effector functions. Immune cells typically include cells that play a role in the immune response, usually of hematopoietic origin. The term "effector function" refers to a specialized function of an immune cell, such as a function or response that enhances or promotes an immune attack on a target cell (eg, killing the target cell, or inhibiting its growth or proliferation). For example, an effector function of a T cell, eg, may be cytolytic activity or helper or activity including secretion of cytokines. Examples of immune cells include T cells (such as α/β T cells and γ/δ T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived macrophages, among others.

The immune cells described herein may be self/autologous ("self") or non-self ("non-self", eg, allogeneic, syngeneic or allogeneic). As used herein, "self" refers to cells from the same subject; "allogeneic" refers to cells of the same species that are genetically different from the comparative cell; The same cell from a different subject; "allogeneic" refers to a cell from a different species than the compared cell. In certain embodiments, the immune cells of the present application are autologous or allogeneic.

As used herein, the term "cytotoxic agent" includes any agent that is detrimental to (eg, kills) cells, such as chemotherapeutic drugs, bacterial toxins, plant toxins, or radioisotopes, among others.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector is capable of achieving expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phage such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, papillomaviruses, Polyoma vacuolar virus (eg SV40). A vector can contain a variety of elements that control expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication.

As used herein, the term "host cell" refers to cells that can be used to introduce vectors, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells, immune cells (such as T lymphocytes) cells, NK cells, monocytes, macrophages or dendritic cells, etc.). A host cell can include a single cell or a population of cells.

As used herein, the term "isolated" means that it has been separated or purified from components that naturally accompany it, such as nucleic acids, proteins, or other naturally occurring biological or organic molecules.

As used herein, the expression "the XX amino acid residue of the EBV LMP2 antigen" means that when the target sequence is compared with the EBV LMP2 amino acid sequence (for example, the sequence shown in Genbank: M24212.1) to generate At the time of maximum identity, the amino acid residue at the same position as the amino acid residue at position XX of the EBV LMP2 amino acid sequence in the target sequence.

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by an adenine, or both a position in each of the polypeptides is occupied by lysine), then the molecules are identical at that position. "Percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions being compared x 100. For example, two sequences are 60% identical if 6 out of 10 positions match. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, comparisons are made when two sequences are aligned for maximum identity. Such alignments can be achieved using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453 which can be conveniently performed by computer programs such as the Align program (DNAstar, Inc.). The algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been integrated into the ALIGN program (version 2.0), can also be used, using the PAM120 weight residue table , a gap length penalty of 12, and a gap penalty of 4 to determine the percent identity between two amino acid sequences. In addition, the algorithm of Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) in the GAP program that has been incorporated into the GCG software package (available at www.gcg.com) can be used, using the Blossum 62 matrix or PAM250 matrix with a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 to determine percent identity between two amino acid sequences .

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the expected properties of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions for amino acid residues with amino acid residues that have similar side chains, e.g., are physically or functionally similar (e.g., have similar size, shape, charge, chemical properties, including Substitution of residues with the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains have been defined in the art. These families include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine) , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (such as alanine, valine, leucine, isoleucine amino acid, proline, phenylalanine, methionine), beta branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine) amino acids. Therefore, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying amino acid conservative substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999) and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

The writing of the twenty conventional amino acids referred to herein follows conventional usage. See, e.g., Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this application, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in this application, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient compatible with the subject and the active ingredient pharmacologically and/or physiologically, These are well known in the art (see e.g. Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers agents, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include but are not limited to cationic, anionic or nonionic surfactants such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents to maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (eg, buffered saline), alcohols and polyols (eg, glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizer has the meaning generally understood by those skilled in the art, and it can stabilize the desired activity of the active ingredient in the medicine, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose , lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (eg, aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (eg 0.9% (w/v) NaCl), dextrose solutions (eg, 5% dextrose), solutions containing surfactants (eg, 0.01% polysorbate 20), pH buffered solutions (eg, phosphate buffered saline), Ringer's solutions, and any combination thereof.

As used herein, the term "prevention" refers to methods performed to prevent or delay the occurrence of a disease or disorder or symptom (eg, a tumor) in a subject. As used herein, the term "treatment" refers to a method performed to obtain a beneficial or desired clinical result. For the purposes of this application, a beneficial or desired clinical outcome includes, but is not limited to, relief of symptoms, reduction of the extent of the disease, stabilization (i.e., no longer worsening) of the disease state, delay or slowing of the progression of the disease, amelioration or alleviation of the disease state status, and relief of symptoms (whether partial or total), whether detectable or not. In addition, "treating" can also refer to prolonging survival as compared to expected survival if not receiving treatment.

As used herein, the term "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing a disease (for example, an EBV-positive tumor or an EBV-related lymphoproliferative disease) is sufficient to prevent, arrest, or delay the occurrence of a disease (for example, an EBV-positive tumor or an EBV-related lymphoproliferative disease) Amount; an effective amount for treating a disease is an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capability of those skilled in the art. For example, amounts effective for therapeutic use will depend on the severity of the disease being treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered concomitantly etc.

As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the term "subject" is meant to include living organisms in which an immune response can be elicited. In certain embodiments, the subject (eg, a human) has, or is at risk of having, an EBV-positive tumor or an EBV-associated lymphoproliferative disorder.

Beneficial Effects of the Invention

The present invention provides a T cell receptor (TCR) specifically recognizing EBV LMP2 antigen, cells and pharmaceutical compositions comprising the TCR, nucleic acid encoding the TCR, vectors and host cells for preparing the TCR, expression The immune cells of the TCR, and the method of using the TCR to treat a subject. The TCR provided by the present invention can induce an immune response against EBV-positive cells (eg, tumor cells) and thus treat EBV-related diseases (eg, EBV-positive tumors) in a subject. In addition, the TCR provided by the present invention is MHC-I-restricted, and the MHC-I-restricted population accounts for nearly one-third of the total population in my country, and has broad application prospects. Therefore, the present invention provides a brand-new T cell-based immunotherapy for the treatment of EBV-related diseases, which has great clinical value.

Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention, rather than limiting the scope of the present invention. Various objects and advantages of this invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiment.

Description of drawings

Figure 1 shows the release of IL2 after co-culture of T cells expressing recombinant D051.1 TCR or D051.2 TCR with CLC cells loaded with pSSC antigen peptide 01.

Figure 2 shows the release of IFNγ after co-culture of T cells expressing recombinant D051.1 TCR or D051.2 TCR with CLC cells loaded with pSSC antigen peptide 01.

Figure 3 shows the release of IFNγ after co-culture of T cells expressing recombinant D051.1 TCR or D051.2 TCR with Cos7-A11-LMP2 cells, Cos7 cells loaded with LMP2 pSSC antigen peptide 01, and Cos7-A11 cells, respectively.

Figure 4 shows the release of IL2 after co-culture of T cells expressing recombinant D051.1 TCR or D051.2 TCR with Cos7-A11 cells loaded with pSSC antigen peptide 01.

Figure 5 shows the release of IFNγ after co-culture of T cells expressing recombinant D051.1 TCR or D051.2 TCR with Cos7-A11 cells loaded with pSSC antigen peptide 01.

Figure 6 shows the results of lysis of HNE1-A11 tumor cells loaded with pSSC antigen peptide 01 by T cells expressing recombinant D051.1 TCR or D051.2 TCR.

Figure 7 shows the release of IFNγ after co-culture of healthy human CD3 T cells expressing D051TCR mutant and LCL cells loaded with pSSC antigen peptide 01.

Figure 8 shows the release of IFNγ after co-culture of T cells expressing recombinant D051.1 TCR or D051.2 TCR with Cos7 cells loaded with LMP2 pSSC antigenic peptide 01 or pSSC antigenic peptide 02, and Cos7-A11 cells, respectively.

Figure 9 shows the results of lysis of HNE1-A11 tumor cells loaded with pSSC antigenic peptide 01 or pSSC antigenic peptide 01 by T cells expressing recombinant D051.1 TCR.

Figure 10 shows the results of lysis of HNE1-A11 tumor cells loaded with pSSC antigenic peptide 01 or pSSC antigenic peptide 01 by T cells expressing recombinant D051.2TCR.

sequence information

A description of the sequences referred to in this application is provided in the table below.

Table 1: Sequence information

Detailed ways

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Unless otherwise specified, the molecular biology experiment methods and immunoassay methods used in the present invention are basically with reference to J.Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M.Ausubel et al., Compiled Molecular Biology Experimental Guide, 3rd Edition, John Wiley & Sons, Inc., 1995 by the method described; restriction endonucleases were used in accordance with the conditions recommended by the product manufacturer. Those skilled in the art understand that the examples describe the present invention by way of example and are not intended to limit the scope of the claimed invention.

Example 1: Peripheral blood T cell screening

Using LMP2-A11 Tetramer (that is, HLA-A*11:01 complex LMP2 pSSC antigen peptide 01 (SEQ ID NO:1) tetramer protein) and healthy Chinese peripheral blood PBMC cells (HLA-A*11:01+ ), incubated at 2-8°C for 30 minutes, washed with FACS buffer washing solution, and flow cytometrically detected whether there are T cells that can bind Tetramer in the peripheral blood. The test results showed that 0.9% Tetramer binding could be detected in the donor number D051 PBMC cells.

5×10 ⁷ PBMC cells (derived from donor D051), washed with FACS buffer, added Tetramer, incubated at 2-8°C for 30min, washed with FACS buffer, and sorted Tetramer positive by BD FACS AriaIII cells, the results showed that Tetramer positive T cells accounted for 1.1%. The sorted Tetramer-positive cells were expanded in vitro using REP (Rapid Expansion Protocol: PBMCs from 3 healthy people were mixed, irradiated with gamma rays at 5000cGy, and co-cultured with the selected T cells), and autologous D051 The derived LCL presenting pSSC was given to the sorted and expanded Tetramer positive CTL to detect its IFNγ-specific release (LCL cell induction method: peripheral blood mononuclear cells (5×10 ⁶ ) of the patient were treated with 10% fetal calf serum The RPMI1640 medium was resuspended, and B95.8 cell culture medium containing EBV was added, and half of the medium was replaced every 7 days during the induction period, and the induced LCL cell line was expanded and frozen). Tetramer-positive CTLs were washed once with DPBS and subjected to 10×Genomics single-cell sequencing.

Example 2: Recombination verification of T cell receptors

The TCR coding sequences of the positive CTL clones (D051.1 and D051.2) obtained in Example 1 were gene-synthesized according to the sequence of VαmCa-P2A-VβmCb (wherein Vα is the α-chain variable region of the above-mentioned TCR clone, and mCa is the mouse TCRα constant region, its amino acid sequence is shown in SEQ ID NO: 18, its nucleotide sequence is shown in SEQ ID NO: 20; Vβ is the β chain variable region of the above TCR clone, mCb is the mouse TCRβ constant region, its amino acid The sequence is shown in SEQ ID NO:19, the nucleotide sequence is shown in SEQ ID NO:21), and cloned into the lentiviral shuttle vector (GV401). The amino acid sequences of the TCR variable regions of D051.1 and D051.2 are shown in Table 2-1.

Table 2-1: TCR variable region sequences of D051.1 and D051.2

The shuttle vector was transiently transfected into 293T cells according to the standard lentiviral vector packaging method, and the culture supernatant was collected, which contained the lentiviral vector expressing TCR.

T cells from healthy donors were activated on OKT3/15E8 antibody-coated 6-well plates for 24 hours, transduced with TCR-containing lentiviral vectors and cultured for 6-8 days for TCR screening (the transduced T cells were collected, washed with FACS buffer, and 1×10 ⁶ modified T cells were stained with an antibody recognizing the mouse TCRβ constant region or Tetramer to detect the expression of recombinant TCR and the combination with Tetramer). The detection results of recombinant TCR expression efficiency and Tetramer binding efficiency are shown in Table 2-2.

Table 2-2: Detection of recombinant TCR expression efficiency

serial number	mTCR%	Tetramer%
D051.1	66.6	86.9
D051.2	69.9	86.1
MockT	0.0	7.8

The results in Table 2-2 show that D051.1TCR and D051.2TCR can be recombinantly expressed on the surface of T cells and can be combined with LMP2-A11 Tetramer.

Example 3: Functional validation of T cell receptors

The T cells expressing the recombinant TCR obtained in Example 2 and the D051LCL cells loaded with pSSC antigen peptide 01 were mixed and spread into 96-well plates according to the cell volume of 2×10 ⁴ and 2×10 ⁴ , and co-cultured at 37°C for 24 hours . Take 50 μl of the supernatant, and use a cytokine detection kit (purchased from BD biosciences, product number: 551809) to detect the release of cytokines IL2 and IFNγ. The detection results are shown in Figure 1 and Figure 2 .

The results in Figure 1 and Figure 2 show that T cells expressing two recombinant TCRs, D051.1 and D051.2, can specifically release IL2 and IFNγ.

Example 4: HLA Restriction Assay

Cos7-A11-LMP2 (Cos7 cells expressing HLA-A*11:01 and EBV LMP2a full-length protein (SEQ ID NO:64), which was constructed by lentiviral vector transduction), loaded with LMP2 pSSC antigen peptide 01 Cos7 cells, or Cos7-A11 cells (that is, Cos7 cells expressing HLA-A*11:01, which are constructed by lentiviral vector transduction), according to the expression obtained in Example 2 respectively with 2×10 ⁴ and 2×10 ⁴ The T cells with the recombinant TCR were mixed, placed in a 96-well plate, and incubated together for 16-24 hours, and the culture supernatant was taken to measure the release of specific IFNγ. The results are shown in Figure 3.

The results in Fig. 3 show that D051.1TCR and D051.2TCR can specifically recognize the LMP2 antigenic peptide pSSC-1 (SSSCSCPLTK) and antigenic peptide pSSC-2 (SSCSSCPLSK) restricted by HLA-A*11:01. At the same time, the full-length LMP2 protein can be presented inside Cos7 cells and specifically recognized by D051.1TCR and D051.2TCR.

Example 5: Determination of D051TCR functional affinity (Functional Avidity)

Mix Cos7-A11 cells with LMP2 pSSC antigen peptide 01 at a final concentration of 10 μg/ml, 1 μg/ml, 0.1 μg/ml, 0.01 μg/ml, 0.001 μg/ml, and 0 μg/ml, and incubate at 37°C for 1 hour. Wash once with phosphate buffer as antigen presenting cells. The T cells expressing the recombinant TCR obtained in Example 2 were mixed with the presenting cells according to the cell volume of 2×10 ⁴ +2×10 ⁴ , spread into a 96-well plate, and co-cultured at 37° C. for 24 hours. Take 50 μl of the supernatant, and use a cytokine detection kit to detect the release of cytokines in the cell supernatant. The recognition ability (Functional Avidity) of the recombinant TCR to the LMP2 pSSC antigen peptide was measured, and the results are shown in Fig. 4 and Fig. 5 .

The results in Fig. 4 and Fig. 5 show that D051.1 and D051.2 TCR can recognize the LMP2 antigen peptide pSSC presented by HLA-A*11:01 in a concentration gradient-dependent manner.

Example 6: D051TCRT Tumor Cell Lysis Assay

Mix HNE1-A11 tumor cells (purchased from Shanghai Chuanqiu Biotechnology, product number: H096) with pSSC antigen peptide 01 at a final concentration of 1 μg/ml, incubate at 37°C for 1 hour, wash once with phosphate buffer, and use as antigen-presenting cells . Spread 8000 cells/well into a 96-well plate. The T cells expressing the recombinant TCR obtained in Example 2 and the presenting cells were spread into 96-well plates according to the ratio of 30:1, 10:1, 3:1, 1:1, and 0.3:1 according to the number of cells, and each ratio Set up two replicate wells. Co-cultivate at 37°C for 24 hours. Remove the supernatant, add 200 μl DPBS to wash once, add CCK8 (Cell Counting Kit8 kit purchased from Tongren Chemical, article number: CK04) to detect the lysis of tumor cells by T cells expressing D051TCR, the results are shown in Figure 6.

The results in Figure 6 show that compared with MockT, T cells expressing D051.1TCR and D051.2TCR both showed lysis of HNE1-A11 cell line loaded with LMP2 pSSC antigen peptide.

Example 7: Recombinant TCR affinity maturation screening

The corresponding amino acids of D051.1TCR CDR3 were subjected to site-directed full mutation (except Cys mutation) to construct a TCR mutant lentiviral vector shuttle plasmid. The positions to be mutated are shown in Table 3, wherein the amino acid positions are determined according to IMGT TCR numbering.

Table 3: Amino acid sites to be mutated

TCR variable region	mutant amino acid
Vα	107L, 108N
Vβ	108P, 109G, 110R, 111W

The lentiviral shuttle vector encoding the D051.1 TCR mutant was transiently transfected into 293T cells according to the standard lentiviral vector packaging method, and the culture supernatant was collected, and the culture supernatant contained the lentiviral vector encoding TCR. The Jurkat-NFAT-GFP cell line (purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) was transduced with a lentiviral vector, and flow cytometry was performed using anti-mTCRβ and LMP2-A11 Tetramer, and the correct pairing rate of TCR was calculated by the staining ratio and the ability to bind LMP2 Tetramer; according to the TCR mutant αβ chain pairing rate ≥ 40%, 48 clones were selected from 86 clones to enter the second round of screening. The results of the mutant recombinant TCR expression and αβ chain pairing rate determination are shown in Table 4 .

Table 4: Mutant recombinant TCR expression and αβ chain pairing rate determination results

serial number	TCR mutant	Tetramer+%	mTCRβ+%	αβ match%
1	a107L-A	0.07%	75.67%	0.10%
2	a107L-D	6.40%	99.60%	6.40%
3	a107L-F	0.09%	96.69%	0.10%
4	a107L-G	1.37%	99.27%	1.40%
5	a107L-H	0.12%	99.12%	0.10%
6	a107L-I	97.80%	99.57%	98.20%
7	a107L-N	82.30%	82.38%	99.90%
8	a107L-P	84.10%	98.00%	85.80%
9	a107L-R	0.20%	99.20%	0.20%
10	a107L-S	39.20%	67.50%	58.10%
11	a107L-T	92.40%	99.07%	93.30%
12	a107L-Y	0.12%	98.12%	0.10%
13	a108N-A	99.50%	99.79%	99.70%
14	a108N-D	89.60%	96.74%	92.60%
15	a108N-F	97.30%	99.00%	98.30%
16	a108N-G	86.20%	95.67%	90.10%
17	a108N-H	94.40%	98.01%	96.30%
18	a108N-I	96.80%	98.92%	97.90%
19	a108N-K	88.00%	97.17%	90.60%
20	a108N-L	97.50%	99.24%	98.20%
twenty one	a108N-M	98.30%	99.38%	98.90%
twenty two	a108N-P	73.70%	95.90%	76.90%
twenty three	a108N-Q	90.50%	97.25%	93.10%
twenty four	a108N-R	89.30%	96.63%	92.40%
25	a108N-S	95.60%	98.58%	97.00%
26	a108N-T	6.06%	93.96%	6.40%
27	a108N-V	96.00%	98.76%	97.20%
28	a108N-W	88.10%	96.68%	91.10%
29	a108N-Y	98.20%	99.42%	98.80%
30	b108P-H	81.00%	96.10%	84.30%

31	b108P-L	85.00%	95.60%	88.90%
32	b108P-R	90.40%	97.60%	92.60%
33	b109G-A	97.60%	99.28%	98.30%
34	b109G-D	92.70%	98.12%	94.50%
35	b109G-E	2.98%	97.48%	3.10%
36	b109G-F	71.50%	97.50%	73.30%
37	b109G-H	85.80%	99.30%	86.40%
38	b109G-I	0.17%	98.57%	0.20%
39	b109G-K	12.10%	98.60%	12.30%
40	b109G-L	0.24%	96.24%	0.20%
41	b109G-M	1.58%	99.18%	1.60%
42	b109G-N	94.10%	98.34%	95.70%
43	b109G-P	0.26%	98.66%	0.30%
44	b109G-Q	1.99%	99.59%	2.00%
45	b109G-R	0.53%	95.63%	0.60%
46	b109G-S	92.00%	98.08%	93.80%
47	b109G-T	80.30%	99.30%	80.90%
48	b109G-V	0.47%	99.50%	0.50%
49	b109G-W	82.90%	97.00%	85.50%
50	b109G-Y	85.20%	99.20%	85.90%
51	b110R-A	67.10%	95.10%	70.60%
52	b110R-D	0.06%	87.46%	0.10%
53	b110R-E	0.14%	85.54%	0.20%
54	b110R-F	70.20%	99.20%	70.80%
55	b110R-G	0.30%	98.50%	0.30%
56	b110R-H	93.20%	99.70%	93.50%
57	b110R-I	85.70%	99.00%	86.60%
58	b110R-K	93.70%	98.68%	95.00%
59	b110R-L	95.00%	99.19%	95.80%
60	b110R-M	97.00%	99.66%	97.30%
61	b110R-N	76.40%	98.20%	77.80%
62	b110R-P	44.70%	99.40%	45.00%
63	b110R-Q	98.70%	99.71%	99.00%
64	b110R-S	44.30%	98.00%	45.20%
65	b110R-T	86.90%	97.20%	89.40%
66	b110R-V	98.90%	99.92%	99.00%
67	b110R-W	0.09%	90.99%	0.10%
68	b110R-Y	12.70%	99.50%	12.80%

69	b111W-A	0.16%	94.36%	0.20%
70	b111W-D	0.08%	81.38%	0.10%
71	b111W-E	0.07%	81.07%	0.10%
72	b111W-F	90.90%	97.59%	93.10%
73	b111W-G	0.05%	83.25%	0.10%
74	b111W-H	0.12%	96.12%	0.10%
75	b111W-I	0.14%	99.34%	0.10%
76	b111W-K	0.09%	97.69%	0.10%
77	b111W-L	0.10%	96.10%	0.10%
78	b111W-M	0.15%	99.85%	0.20%
79	b111W-N	0.11%	97.81%	0.10%
80	b111W-P	0.14%	97.54%	0.10%
81	b111W-Q	0.19%	95.69%	0.20%
82	b111W-R	0.02%	49.62%	0.00%
83	b111W-S	0.01%	53.51%	0.00%
84	b111W-T	0.17%	97.37%	0.20%
85	b111W-V	0.21%	99.21%	0.20%
86	b111W-Y	67.10%	99.00%	67.80%

Example 8: Functional assay of TCR mutants

The 48 TCR mutant clones selected in Example 7 were expressed in Jurkat-NFAT-EGFP (NFAT RE-MiniP-EGFP), using autologous D051-LCL cells as antigen-presenting cells, loaded with 1ug/ml LMP2 pSSC antigen peptide 01 Finally, according to the effect-to-target ratio=1:1 (2E5+2E5), the Jurkat-NFAT-GFP cell line expressing TCR and the presenting cells loaded with antigen peptides were co-cultured in a 24-well plate, and the percentage of GFP expression was detected after 24 hours And MFI (mean fluorescence intensity), the results are shown in Table 5.

Table 5: GFP expression percentage and MFI detection results

The above results show that both D051TCR Vα and Vβ mutants can maintain the ability to bind to LMP2-A11 Tetramer, and the active TCR mutants are shown in Table 6.

Table 6: Active TCR mutants

TCR mutation location	active mutant
α107L	I
α108N	Y, A, H, Q, L, S, F, W, G, D, I, M, V, R, K, P

β108P	L, R, H
β109G	A, N, D, S, H
β110R	K
β111W	f

Embodiment 9: Determination of TCR mutant functional affinity (Functional Avidity)

D051LCL cells were loaded with LMP2 pSSC antigen peptide 01 for concentration gradient dilution (initial concentration 10ug/ml, 7-fold dilution), and co-cultured with healthy human CD3T cells expressing D051TCR mutants (2×10 ⁴ antigen-loaded D051LCL and 2×10 ⁴ human CD3 T cells expressing the D051TCR mutant), placed in RPMI1640 medium containing 2% fetal bovine serum for 16-24 hours, and measured the release of IFNγ in the supernatant, and determined its functional affinity, the results are shown in Figure 7 Show.

The results in Fig. 7 show that the recombinant TCR mutants can recognize the LCL cell line loaded with LMP2 pSSC antigen peptide in a concentration gradient dependence.

Example 10: Recognition of LMP2 variants by D051TCR

1. Determination of Cytokine Release

Cos7-A11 cells (i.e., Cos7 cells expressing HLA-A*11:01, which were constructed by lentiviral vector transduction) or Cos7-A11 cells loaded with LMP2 pSSC antigenic peptide 01 or pSSC antigenic peptide 02, according to 2×10 ⁴ and 2×10 ⁴ were respectively mixed with T cells expressing recombinant TCR obtained in Example 2, placed in a 96-well plate, incubated together for 16-24 hours, and the culture supernatant was taken to measure the release of specific IFNγ, the results are shown in Figure 8 shown.

The results in Fig. 8 show that D051.1TCR and D051.2TCR can specifically recognize LMP2 pSSC antigen peptide 01 (SSSCSCPLTK) and pSSC antigen peptide 02 (SSCSSCPLSK) restricted by HLA-A*11:01.

2. Determination of Tumor Cell Lysis

HNE1-A11 tumor cells were mixed with pSSC antigenic peptide 01 or pSSC antigenic peptide 02 at a final concentration of 1 μg/ml, incubated at 37°C for 1 hour, washed once with phosphate buffer, and used as antigen-presenting cells. Spread 8000 cells/well into a 96-well plate. The T cells expressing the recombinant TCR obtained in Example 2 and the presenting cells were spread into 96-well plates according to the ratio of the cell number of 30:1, 10:1, 3:1, 1:1, and 0.3:1, and each ratio Repeat for both holes. Co-cultivate at 37°C for 24 hours. Remove the supernatant, add 200 μl DPBS to wash once, and add CCK8 to detect the lysis of tumor cells by T cells expressing D051TCR (Cell Counting Kit 8). The results are shown in Figure 9 and Figure 10.

The results in Figures 9-10 show that, compared with MockT, both D051.1 and D051.2 showed lysis of HNE1-A11 cell lines loaded with LMP2 pSSC antigenic peptide 01 and pSSC antigenic peptide 02.

Although the specific implementation of the present invention has been described in detail, those skilled in the art will understand that: according to all the teachings that have been published, various modifications and changes can be made to the details, and these changes are all within the protection scope of the present invention . The full scope of the invention is given by the claims appended hereto and any equivalents thereof.

Claims (17)

1. An isolated T cell receptor (TCR) or an antigen-binding fragment thereof capable of specifically recognizing the EBV LMP2 antigen, said TCR or an antigen-binding fragment thereof comprising an alpha chain variable region (Vα) and/or a beta chain variable region Region (Vβ), where,

   (a) said Vα includes CDR1α, CDR2α and CDR3α, wherein said CDR3α has a sequence as shown in AVX ₁ X ₂ NNDMR (SEQ ID NO: 26); wherein:

   X _is selected from (i) amino acid residues L, I and (ii) amino acid residues that are conservatively substituted with respect to (i);

   _X is selected from (i) amino acid residues N, Y, A, H, Q, L, S, F, W, G, D, I, M, V, R, K, P and (ii) relative to ( i) is a conservatively substituted amino acid residue;

   and / or,

   (b) the Vβ includes CDR1β, CDR2β and CDR3β, wherein the CDR3β has a sequence as shown in ASSX ₃ X ₄ X ₅ X ₆ YEQY (SEQ ID NO: 27); wherein:

   X _is selected from (i) amino acid residues P, L, R, H, Q and (ii) amino acid residues that are conservatively substituted with respect to (i);

   X _is selected from (i) amino acid residues G, A, N, D, S, H and (ii) amino acid residues that are conservatively substituted with respect to (i);

   X _is selected from (i) amino acid residues R, K and (ii) amino acid residues that are conservatively substituted with respect to (i);

   X _is selected from (i) amino acid residues W, F and (ii) amino acid residues that are conservatively substituted with respect to (i);

   Preferably, X _is selected from amino acid residues L, I; X _is selected from amino acid residues N, Y, A, H, Q, L, S, F, W, G, D, I, M, V, R , K, P; _X3 is selected from amino acid residues P, L, R, H, Q; _X4 is selected from amino acid residues G, A, N, D, S, H; _X5 is selected from amino acid residues R, K; X _is selected from amino acid residues W, F;

   Preferably, said TCR is soluble or membrane bound;

   Preferably, the TCR is a full length TCR, a soluble TCR or a single chain TCR.
2. The TCR or antigen-binding fragment thereof according to claim 1, which has one or more of the following characteristics:

   (i) the CDR1α has the sequence shown in SEQ ID NO: 11 or has one or several amino acid substitutions, deletions or additions thereto (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) the sequence of;

   (ii) the CDR2α has the sequence shown in SEQ ID NO: 12 or has one or several amino acid substitutions, deletions or additions thereto (such as 1, 2 or 3 amino acid substitutions, deletions or additions) the sequence of;

   (iii) the CDR1β has the sequence shown in SEQ ID NO: 14 or has one or several amino acid substitutions, deletions or additions thereto (such as 1, 2 or 3 amino acid substitutions, deletions or additions) the sequence of;

   (iv) The CDR2β has the sequence shown in SEQ ID NO: 15 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) the sequence of.
3. The TCR or antigen-binding fragment thereof of claim 1 or 2, comprising:

   (i) CDR1α as shown in SEQ ID NO:11; CDR2α as shown in SEQ ID NO:12; CDR3α as shown in any one of SEQ ID NOs:13, 28-44; and/or,

   (ii) CDR1β as shown in SEQ ID NO:14; CDR2β as shown in SEQ ID NO:15; CDR3β as shown in any one of SEQ ID NOs:16-17, 45-54.
4. The TCR or antigen-binding fragment thereof according to any one of claims 1-3, wherein,

   (a) said Vα also includes FR1α, FR2α, FR3α and FR4α, wherein:

   The FR1α has the sequence shown in SEQ ID NO: 55 or 59 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared therewith sequence;

   The FR2α has a sequence shown in SEQ ID NO:56 or a sequence having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;

   The FR3α has a sequence shown in SEQ ID NO:57 or a sequence having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;

   The FR4α has a sequence shown in SEQ ID NO: 58 or a sequence having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared therewith;

   and / or,

   (b) said Vβ also includes FR1β, FR2β, FR3β and FR4β, wherein:

   The FR1β has a sequence shown in SEQ ID NO: 60 or a sequence having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared therewith;

   The FR2β has a sequence shown in SEQ ID NO: 61 or a sequence having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared therewith;

   The FR3β has a sequence shown in SEQ ID NO: 62 or a sequence having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared therewith;

   The FR4β has a sequence shown in SEQ ID NO: 63 or a sequence having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared therewith;

   Preferably, said substitution is a conservative substitution.
5. The TCR or antigen-binding fragment thereof according to any one of claims 1-4, wherein said Vα comprises the sequence shown in SEQ ID NO:3 or 7 or a variant thereof, and said Vβ comprises SEQ ID NO:4 or 8 The sequence shown or a variant thereof having one or several amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions) compared to the sequence from which it is derived , missing or added);

   Preferably, said Vα comprises a variant of the sequence shown in SEQ ID NO: 3 or 7, said variant comprising 1 or several (for example, 1, 2, 3 or 4) selected from the following ) amino acid substitution, the amino acid position is determined according to the IMGT TCR numbering system: the amino acid substitution at position 107 is I; the amino acid substitution at position 108 is Y, A, H, Q, L, S, F, W, G, D , I, M, V, R, K or P;

   Preferably, said Vβ comprises a variant of the sequence shown in SEQ ID NO: 4 or 8, said variant comprising 1 or several (for example, 1, 2, 3 or 4) selected from the following ) amino acid substitutions, the amino acid positions are determined according to the IMGT TCR numbering system: amino acid substitution at position 108 is L, R or H; amino acid substitution at position 109 is A, N, D, S or H; amino acid substitution at position 110 Substitution was K; amino acid substitution at position 111 was F.
6. The TCR or its antigen-binding fragment according to any one of claims 1-5, wherein the TCR or its antigen-binding fragment can specifically recognize an epitope peptide presented by an MHC-I molecule, and the epitope peptide comprises EBV LMP2 340-349 amino acid residues of the antigen;

   Preferably, the 340-349 amino acid residues of the EBV LMP2 antigen have the sequence shown in SEQ ID NO: 1 or 2;

   Preferably, said MHC-I class molecule is HLA-A;

   Preferably, the MHC-I class molecule is HLA-A11; preferably, the MHC-I class molecule is HLA-A*11:01;

   Preferably, when said TCR or an antigen-binding fragment thereof is expressed on the surface of a T cell, said T cell is co-cultured with a second cell displaying said epitope peptide (e.g. a cell expressing an HLA class I molecule) time activation.
7. A conjugate comprising the TCR or antigen-binding fragment thereof of any one of claims 1-6 and an effector moiety conjugated thereto.
8. A fusion protein comprising the TCR or antigen-binding fragment thereof of any one of claims 1-6 and an additional peptide or protein.
9. An isolated nucleic acid molecule comprising nucleosides encoding the TCR of any one of claims 1-6 or an antigen-binding fragment thereof or an alpha chain variable region and/or a beta chain variable region or an alpha chain and/or a beta chain acid sequence, or comprise the nucleotide sequence encoding the fusion protein described in claim 8;

   Preferably, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the variable region of the TCR α chain and a second nucleotide sequence encoding the variable region of the TCR β chain;

   Preferably, said isolated nucleic acid molecule comprises a first nucleotide sequence encoding the alpha chain of said TCR or an antigen-binding fragment thereof and a second nucleotide sequence encoding the beta chain of said TCR or an antigen-binding fragment thereof;

   Preferably, said first nucleotide sequence and said second nucleotide sequence are present on different isolated nucleic acid molecules;

   Preferably, said first nucleotide sequence and said second nucleotide sequence are present in any order on the same isolated nucleic acid molecule; preferably, said first nucleotide sequence and said second The nucleotide sequences are linked in any order by a nucleotide sequence encoding a self-cleaving peptide (eg, P2A).
10. A carrier comprising the isolated nucleic acid molecule of claim 9;

    Preferably, the vector comprises nucleosides encoding the TCR of any one of claims 1-6 or its antigen-binding fragment or its α-chain variable region and/or β-chain variable region or its α-chain and/or β-chain acid sequence;

    Preferably, the vector comprises a first nucleotide sequence encoding the variable region of the TCRα chain and a second nucleotide sequence encoding the variable region of the TCRβ chain;

    Preferably, the vector comprises a first nucleotide sequence encoding the α-chain of the TCR or an antigen-binding fragment thereof and a second nucleotide sequence encoding the β-chain of the TCR or an antigen-binding fragment thereof;

    Preferably, said first nucleotide sequence and said second nucleotide sequence are present on different vectors;

    Preferably, the first nucleotide sequence and the second nucleotide sequence are present on the same carrier in any order; preferably, the first nucleotide sequence and the second nucleotide sequence Linked by a nucleotide sequence encoding a self-cleaving peptide (eg, P2A) in any order;

    Preferably, the vector comprises a nucleotide sequence encoding the fusion protein of claim 8;

    Preferably, the vector is a viral vector, such as a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector or a baculoviral vector.
11. A host cell comprising the isolated nucleic acid molecule of claim 9, or the vector of claim 10;

    Preferably, the host cell comprises a TCR or an antigen-binding fragment thereof encoding any one of claims 1-6 or its α-chain variable region and/or β-chain variable region or its α-chain and/or β-chain the nucleotide sequence;

    Preferably, the host cell comprises a nucleotide sequence encoding the fusion protein of claim 8;

    Preferably, the host cells comprise Escherichia coli, yeast, insect cells, or mammalian cells.
12. The method for preparing the TCR or antigen-binding fragment thereof according to any one of claims 1-6, or the fusion protein according to claim 8, comprising culturing the host according to claim 11 under conditions that allow protein expression cells, and recovering said TCR or antigen-binding fragment thereof, or fusion protein from cultured host cell culture.
13. A modified immune cell that expresses the TCR or an antigen-binding fragment thereof according to any one of claims 1-6 on the cell surface;

    Preferably, the engineered immune cell comprises a nucleotide sequence encoding the TCR or an antigen-binding fragment thereof according to any one of claims 1-6;

    Preferably, the immune cells are lymphocytes;

    Preferably, the immune cells are selected from T cells (such as αβT cells, γδT cells or iPSC-induced T cells), tumor infiltrating lymphocytes (TIL), natural killer (NK) cells, natural killer T (NKT) cells, or any combination thereof.
14. The method for preparing the modified immune cell of claim 13, which comprises: (1) providing immune cells from a test; (2) separating the nucleic acid molecule of claim 9 or the nucleic acid molecule of claim 10 The vector is introduced into the immune cell described in step (1), and the nucleic acid molecule or vector comprises a nucleotide sequence encoding the TCR or an antigen-binding fragment thereof according to any one of claims 1-6, so as to obtain the expression of the TCR or Immune cells of antigen-binding fragments thereof;

    Preferably, in step (1), the immune cells are pretreated; the pretreatment includes sorting, activation and/or proliferation of immune cells;

    Preferably, the pretreatment comprises contacting the immune cells with one or more selected from anti-CD3 antibody, anti-CD28 antibody, IL-2 and IL-15, thereby stimulating the immune cells and inducing their proliferation, by This produces preconditioned immune cells.
15. A pharmaceutical composition comprising the TCR or antigen-binding fragment thereof according to any one of claims 1-6, the conjugate according to claim 7, the fusion protein according to claim 8, and the nucleic acid according to claim 9 The molecule, the carrier of claim 10, the host cell of claim 11, or the transformed immune cell of claim 13; and a pharmaceutically acceptable carrier and/or excipient;

    Preferably, the pharmaceutical composition further comprises another therapeutic agent, such as an antineoplastic agent or an immunopotentiator;

    Preferably, the antitumor agent is selected from the group consisting of alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, radiosensitizers, Anti-angiogenic agents, cytokines, immune checkpoint inhibitors (eg, PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG-3 antibody, or TIM3 antibody);

    Preferably, the immunopotentiator is selected from immunostimulatory antibodies (such as anti-CD3 antibody, anti-CD28 antibody, anti-CD40L (CD154) antibody, anti-41BB (CD137) antibody, anti-OX40 antibody, anti-GITR antibody or any combination thereof) or an immunostimulatory cytokine (eg, IL-2, IL-3, IL-12, IL-15, IL-18, IFN-γ, IL-10, TGF-β, GM-CSF, or any combination thereof).
16. The TCR or antigen-binding fragment thereof according to any one of claims 1-6, the conjugate according to claim 7, the fusion protein according to claim 8, the nucleic acid molecule according to claim 9, or the nucleic acid molecule according to claim 10 The vector described in claim 11, the host cell described in claim 11, the modified immune cell described in claim 13, or the pharmaceutical composition described in claim 15 are used in the preparation of medicines, and the medicines are used in subjects Inducing an immune response against EBV-positive cells, and/or preventing or treating EBV-related diseases in a subject; wherein, the nucleic acid molecule, vector or host cell comprises encoding the TCR or an antigen-binding fragment thereof, or the nucleotide sequence of the fusion protein;

    Preferably, the EBV positive cells express EBV LMP2 antigen;

    Preferably, the EBV positive cells are tumor cells;

    Preferably, the EBV-related disease is selected from EBV-positive tumors or EBV-related lymphoproliferative diseases;

    Preferably, the EBV-positive tumor is selected from: B cell tumors (Burkitt lymphoma, Hodgkin lymphoma, diffuse large B cell lymphoma, etc.), T cell lymphoma, NK cell lymphoma, nasopharyngeal carcinoma, gastric cancer, smooth muscle sarcoma;

    Preferably, the EBV-associated lymphoproliferative disorder is selected from post-transplantation lymphoproliferative disorder (PTLD);

    Preferably, the subject is a human;

    Preferably, the subject is HLA-A11 positive; Preferably, the subject is HLA-A*11:01 positive;

    Preferably, the TCR or antigen-binding fragment thereof, conjugate, fusion protein, nucleic acid molecule or vector or host cell, engineered immune cell, or pharmaceutical composition is administered in combination with an additional therapeutic agent, e.g. simultaneously, separately or Administered sequentially; preferably, the additional therapeutic agent is an immunostimulant or antineoplastic agent.
17. A method for inducing an immune response against EBV-positive cells in a subject and/or preventing or treating a disease associated with EBV in a subject, the method comprising administering to a subject in need thereof an effective amount of The TCR or antigen-binding fragment thereof according to any one of claims 1-6, the conjugate according to claim 7, the fusion protein according to claim 8, the nucleic acid molecule according to claim 9, or the nucleic acid molecule according to claim 10 The vector, the host cell of claim 11, the transformed immune cell of claim 13, or the pharmaceutical composition of claim 15;

    Preferably, the EBV positive cells express EBV LMP2 antigen;

    Preferably, the EBV positive cells are tumor cells;

    Preferably, the EBV-related disease is selected from EBV-positive tumors or EBV-related lymphoproliferative diseases;

    Preferably, the EBV-positive tumor is selected from: B cell tumors (Burkitt lymphoma, Hodgkin lymphoma, diffuse large B cell lymphoma, etc.), T cell lymphoma, NK cell lymphoma, nasopharyngeal carcinoma, gastric cancer, smooth muscle sarcoma;

    Preferably, the EBV-associated lymphoproliferative disorder is selected from post-transplantation lymphoproliferative disorder (PTLD);

    Preferably, the subject is a human;

    Preferably, the subject is HLA-A11 positive; Preferably, the subject is HLA-A*11:01 positive;

    Preferably, the method further comprises administering to the subject an additional therapeutic agent, such as an immunopotentiator or an antineoplastic agent;

    Preferably, the method comprises: (1) providing immune cells required by the subject; (2) introducing the nucleotide sequence encoding the TCR or antigen-binding fragment thereof according to any one of claims 1-6 into the step (1) The immune cells obtained by obtaining the immune cells expressing the TCR or an antigen-binding fragment thereof on the surface; (3) administering the immune cells obtained in step (2) to the subject;

    Preferably, the immune cells are lymphocytes;

    Preferably, the immune cells are selected from T cells (such as αβT cells, γδT cells or iPSC-induced T cells), tumor infiltrating lymphocytes (TIL), natural killer (NK) cells, natural killer T (NKT) cells, or any combination thereof.

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