Combination of NY-ESO-1 specific T cell receptor and chimeric costimulatory receptor

Abstract

The present invention relates to immune cells expressing a TCR and a costimulatory receptor. In particular, the present invention relates to immune cells expressing (i) a T cell receptor (TCR) specific for a TCR specific for the NY-ESO-1 peptide SLLMWITQC, and (ii) a chimeric costimulatory receptor comprising an extracellular domain derived from PD-1 (CD279) and an intracellular domain derived from 4-1BB (CD137).

Description

[Technical Field]

[0001] The present invention relates to immune cells expressing a TCR and a costimulatory receptor. In particular, the present invention relates to immune cells expressing (i) a T cell receptor (TCR) specific for a TCR specific for the NY-ESO-1 peptide SLLMWITQC, and (ii) a chimeric costimulatory receptor comprising an extracellular domain derived from PD-1 (CD279) and an intracellular domain derived from 4-1BB (CD137). [Background technology]

[0002] NY-ESO-1 and LAGE-1 are important immunotherapy targets belonging to the cancer / testis antigen family, which are expressed in various malignant tumors and in germ cells of the testis, but not in other adult tissues.

[0003] The success of immunotherapy using TCR-modified T cells depends not only on the selection of target antigens but also on the selection of TCRs with high antigen specificity and sensitivity. An additional challenge, particularly in the treatment of solid tumors, is the immunosuppressive tumor microenvironment (TME), which adversely affects the efficacy, compatibility, and persistence of TCR-modified T cells. In addition to being deprived of inhibitory cytokines and essential metabolic factors, T cells face the inhibitory checkpoint PD-1 / PD-L1 axis in the TME, which reduces T cell infiltration and exhausts them. As a result, new strategies are needed to equip TCR-modified T cells with the properties to overcome the suppressive immunosuppressive TME. More specifically, TCR-modified T cells targeting specific antigens, such as NY-ESO-1, with high specificity and enhanced proliferation, cytokine release, and cytotoxicity are desired. Summary of the Invention

[0004] OBJECTS AND SUMMARY OF THE INVENTION To overcome these needs, the present invention provides a combination of a high-avidity TCR and a chimeric costimulatory receptor that enables the generation of highly specific T cells targeting antigens, such as NY-ESO-1, with enhanced cytokine release, proliferation, and cytotoxicity, particularly multifunctional immune cells secreting two or more cytokines.

[0005] The present invention provides (A) Antigen-specific TCR, and (B) Chimeric costimulatory receptor a target-specific immune cell expressing It concerns target-specific immune cells that secrete at least two proteins.

[0006] Thus, one embodiment of the present invention comprises: (A) NY-ESO-1 / LAGE-1-specific TCR containing: - a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 35, a CDR2 having the amino acid sequence of SEQ ID NO: 36, and a CDR3 having the amino acid sequence of SEQ ID NO: 37; and - a TCR beta chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 38, a CDR2 having the amino acid sequence of SEQ ID NO: 39 and a CDR3 having the amino acid sequence of SEQ ID NO: 40, (B) a chimeric costimulatory receptor comprising: - an extracellular domain containing polypeptide derived from PD-1; - a transmembrane domain, and - an intracellular domain containing a polypeptide derived from 4-1BB The present invention provides a cell comprising the

[0007] The NY-ESO-1-specific TCR used is capable of binding to the NY-ESO-1 peptide having the amino acid sequence SLLMWITQC (SEQ ID NO: 34) or a portion thereof, or its HLA-A2-binding form. It provides high functional avidity and advantageous tumor cell recognition and killing properties. The costimulatory receptor reverses the inhibitory checkpoint axis PD-1 / PD-L1 to improve T cell functionality, particularly in the inhibitory TME. Thus, the combination of the TCR and chimeric costimulatory receptor of the present invention enables improved targeting of NY-ESO-1 with high specificity and enhanced proliferation, cytokine release, and cytotoxicity.

[0008] The chimeric costimulatory receptor can comprise a transmembrane domain derived from PD-1. In certain embodiments, the sequence of the chimeric costimulatory receptor can comprise the sequence of SEQ ID NO:26.

[0009] Thus, a further aspect is - a nucleic acid encoding a NY-ESO-1-specific T cell receptor (TCR), comprising: - a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 35, a CDR2 having the amino acid sequence of SEQ ID NO: 36 and a CDR3 having the amino acid sequence of SEQ ID NO: 37, and - a TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 38, a CDR2 having the amino acid sequence of SEQ ID NO: 39, and a CDR3 having the amino acid sequence of SEQ ID NO: 40, and - a nucleic acid encoding a chimeric costimulatory receptor comprising: - an extracellular domain containing polypeptide derived from PD-1; - a transmembrane domain, and - an intracellular domain containing a polypeptide derived from 4-1BB The present invention relates to a composition comprising:

[0010] Furthermore, one aspect is - a nucleic acid encoding a NY-ESO-1-specific T cell receptor (TCR), comprising: - a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 35, a CDR2 having the amino acid sequence of SEQ ID NO: 36 and a CDR3 having the amino acid sequence of SEQ ID NO: 37, and - a TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 38, a CDR2 having the amino acid sequence of SEQ ID NO: 39, and a CDR3 having the amino acid sequence of SEQ ID NO: 40; and - a nucleic acid encoding a chimeric costimulatory receptor comprising: - an extracellular domain containing polypeptide derived from PD-1; - a transmembrane domain, and - an intracellular domain containing a polypeptide derived from 4-1BB The present invention relates to a nucleic acid comprising:

[0011] Another embodiment relates to a vector comprising a nucleic acid comprising sequences for a NY-ESO-1 specific TCR and a chimeric costimulatory receptor. Cells comprising the nucleic acid compositions and / or vectors are also encompassed.

[0012] Typically, the cells are peripheral blood lymphocytes (PBLs) or peripheral blood mononuclear cells (PBMCs). In certain embodiments, the cells are T cells.

[0013] multifunctional immune cells One of the objects of the present invention is to (A) Antigen-specific TCR; (B) Chimeric costimulatory receptor A cell population comprising cells expressing The present invention relates to a cell population comprising cells that secrete at least two proteins.

[0014] In some embodiments, the chimeric costimulatory receptor is - an extracellular domain containing polypeptide derived from PD-1; - a transmembrane domain derived from PD-1, and - an intracellular domain containing a polypeptide derived from 4-1BB; wherein the cell population comprises cells that secrete at least two proteins.

[0015] A further aspect of the present invention is (A) Antigen-specific TCR, and (B) A chimeric costimulatory receptor described herein. a target-specific immune cell expressing It concerns target-specific immune cells that secrete at least two proteins.

[0016] In some embodiments, the cells secrete at least three proteins, such as at least four or at least five proteins.

[0017] Further aspects relate to pharmaceutical compositions comprising the cells, compositions, nucleic acids, and vectors defined herein. Further aspects relate to the cells, compositions, nucleic acids, and vectors defined herein for the treatment of cancer.

[0018] Surprisingly, the inventors have found that TCR-T cells expressing a chimeric costimulatory receptor as defined herein exhibit greater polyfunctionality compared to TCR-T cells lacking a chimeric costimulatory receptor as defined herein. Greater polyfunctionality in transgenic T cells indicates greater functionality and anti-tumor activity in vivo and correlates with clinical outcome.

[0019] In particular, the inventors have been able to show that cell populations expressing both the target-specific NY-ESO-2-specific TCR and the chimeric costimulatory receptor defined herein comprise cells secreting at least two proteins, e.g., at least three proteins, at least four proteins, at least five proteins, at least six proteins. [Brief explanation of the drawings]

[0020] [Figure 1-1]Figure 1 shows that NY-ESO-1-targeting TCR-T cells expressing PD1-41BB exhibit higher polyfunctionality compared with NY-ESO-1-targeting TCR-T cells lacking PD1-41BB. TCR-transgenic T cells with or without PD1-41BB were analyzed for their single-cell polyfunctionality (release of two or more cytokines) using IsoLight® technology (IsoPlexis). After coculture with PD-L1-overexpressing NY-ESO-1-positive MelA375 and Mel624.38 tumor cells, single CD8+ T cells were assessed for secretion of 32 T cell cytokines / proteins compared with untransduced TCR-T cells. (A) TCR-T cells expressing PD1-41BB exhibited a higher percentage of polyfunctional T cells compared with TCR-T cells lacking PD1-41BB. The various grey fills indicate how many cytokines were simultaneously released by a single T cell. [Figure 1-2] (B) The polyfunctional strength index (PSI) was calculated by multiplying the intensity of various secreted cytokines by the percentage of polyfunctional T cells. TCR-T cells expressing PD1-41BB exhibited a higher polyfunctional strength index (PSI) compared with TCR-T cells lacking PD1-41BB. Classification of the various cytokines / lytic proteins released revealed a high contribution of effector (Gzm-B, IFN-γ, MIP-1a, perforin, TNF-α, TNF-β) and stimulatory (GM-CSF, IL-2, IL-5, IL-8, IL-9, IL-12) cytokines / lytic proteins to superior PSI, followed by chemoattractant (IP-10, MIP-1β, RANTES) cytokines. Regulatory (IL-4, IL-10, IL-22, sCD137, TGF-β1) and pro-inflammatory (IL-6, IL-17F, MCP-1) cytokines were released to a lesser extent. [Figure 1-3](C) Detailed analysis of single-cell multicytokine release using multifunctional heatmaps showed that TCR-T cells with and without PD1-41BB differed in their multicytokine signatures. Notably, PD1-41BB-expressing TCR-T cells contained a higher proportion of single cells secreting two to six cytokines simultaneously. [Figure 2-1] Figure 1 shows improved effector function of NY-ESO-1 TCR- T cells co-expressing the chimeric PD1-41BB costimulatory receptor. (A) Flow cytometry analysis of untransduced (UT), NY-ESO-1 TCR-expressing (TCR), and NY-ESO-1 TCR-coexpressing PD1-41BB (TCR+PD1-41BB) T cells. [Figure 2-2] (B) NY-ESO-1 TCR transgenic CD8+ T cells with or without PD1-41BB were cultured with tumor cell lines expressing different levels of NY-ESO-1 and PD-L1, and the levels of released IFN-γ were determined by ELISA (t-test, ** indicates p>0.01, *** indicates p<0.001). [Figure 2-3] (C) NY-ESO-1 expression levels were measured by qPCR and quantified against a standard calibration curve. PD-L1 levels summarized here were assessed by both qPCR and FACS analysis. [Figure 2-4] (D) NY-ESO-1 TCR transgenic CD8+ T cells with or without PD1-41BB were stained with a dye membrane tracker and co-cultured with NY-ESO-1+PD-L1+ tumor cell lines for 5 days. Proliferation was analyzed by flow cytometry, and data were evaluated using FlowJo. [Figure 3-1]Figure 1. Enhanced polyfunctionality of NY-ESO-1 TCR-T cells by coexpression of the chimeric costimulatory receptor PD1-41BB. NY-ESO-1-specific TCR-T cells with (TCR+PD1-41BB) or without (TCR) coexpression of the chimeric costimulatory receptor PD1-41BB were analyzed using single-cell proteomic analysis of a panel of 32 secreted cytokines, chemokines, and cytotoxic molecules after 24 hours of coculture with the tumor-targeting cell lines Mel624.38_PD-L1 and MelA375_PD-L1 (IsoLight technology, IsoPlexis). Coculture with untransduced T cells (UT) from the same donor served as a control. Cells secreting two or more cytokines are considered polyfunctional. (A) Polyfunctionality of single TCR T cells shown as % of samples, categorized into % of cells simultaneously expressing 2, 3, 4, and 5+ analytes (orange fill). [Figure 3-2] (B) The polyfunctional intensity index (PSI) of the indicated sample is defined as the number of T cells secreting three or more effector molecules per cell (polyfunctional T cells in a) multiplied by the mean fluorescence intensity (MFI) of the proteins secreted by each cell, categorized into effector, stimulatory, chemoattractant, regulatory, and inflammatory-related proteins (grey fill). [Figure 3-3] (C) Single-cell polyfunctionality heatmap showing the single-cell cytokine combinations secreted by each sample. Each column represents a specific combination of cytokines, while the orange box represents the frequency with which that specific combination of cytokines was secreted by the corresponding sample (averages for target cells Mel624.38_PD-L1 and MelA375_PD-L1 are shown). Cytokine groups are ordered by polyfunctionality; only the 40 most polyfunctional groups present in a sample are shown. [Figure 4]Figure 1 shows the increased secretion of effector, stimulatory, and chemoattractant proteins by TCR-T cells co-expressing the costimulatory receptor PD1-41BB. Secretion frequencies of single proteins induced by co-culture of PD1-41BB cells and TCR-T cells with the target cell line Mel624.38_PD-L1. Proteins are labeled and categorized by their a) effector, b) stimulatory, c) chemoattractant, d) regulatory, and e) inflammatory functions. [Figure 5] Figure 1 shows serial killing of tumor cell spheroids. Tumor cell spheroids were generated starting with 1,000 NucLightRed-transduced cells in 96-well ULA plates 72 hours prior to co-culture. TCR-transduced T cells with and without PD1-41BB were added (10,000-38,000 per well), and tumor cell killing was monitored by real-time imaging of the NucLightRed signal using an IncuCyte S3. Fresh tumor cell spheroids, generated as described above, were added every 72-96 hours. DETAILED DESCRIPTION OF THE INVENTION

[0021] Before describing the present invention in detail with respect to some of its preferred embodiments, the following general definitions are provided.

[0022] The invention illustratively described below may suitably be practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein.

[0023] The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims.

[0024] Where the terms "comprising" are used in the present description and claims, they do not exclude other elements. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising of". Hereinafter, when a group is defined as comprising at least a certain number of embodiments, this is also understood to disclose a group that preferably consists only of these embodiments.

[0025] For the purposes of the present invention, the term "obtained" is considered to be a preferred embodiment of the term "obtainable". In the following, if, for example, an antibody is defined as obtainable from a particular source, this is understood to also disclose an antibody obtained from this source.

[0026] Where an indefinite or definite article is used when referring to a singular noun, such as "a", "an", or "the", this includes the plural of that noun, unless specifically stated otherwise. The term "about" or "approximately" in the context of the present invention refers to an interval of accuracy that a person skilled in the art would understand as still ensuring the technical effect of the feature in question. The term typically indicates a deviation of ±10%, preferably ±5%, from the indicated numerical value.

[0027] Technical terms are used in the sense or meaning common to those skilled in the art. Where a specific meaning is to be assigned to a particular term, the definition of the term will be given according to the context in which the term is used.

[0028] TCR Background TCRs are composed of two distinct and separate protein chains: the TCR alpha (α) and the TCR beta (β) chain. The TCR α chain contains a variable (V), joining (J), and constant (C) region. The TCR β chain contains a variable (V), diversity (D), joining (J), and constant (C) region. The rearranged V(D)J region of both the TCR α and TCR β chains contains hypervariable regions (CDRs, complementarity-determining regions), and the CDR3 region, in particular, determines specific epitope recognition. At the C-terminal region, both the TCR α and TCR β chains contain a hydrophobic transmembrane domain and terminate in a short cytoplasmic tail.

[0029] Typically, TCRs are heterodimers of one α-chain and one β-chain that can bind to peptide-presenting MHC molecules.

[0030] The terms "variable TCR alpha region", "TCR alpha variable chain" or "variable domain" in the context of the present invention refer to the variable region of the TCR alpha chain. The terms "variable TCR beta region" or "TCR beta variable chain" in the context of the present invention refer to the variable region of the TCR beta chain.

[0031] TCR loci and genes are named using the International Molecular Immunogenetics (IMGT) TCR nomenclature (IMGT database, www.IMGT.org; Giudicelli, V., et al. IMGT / LIGM-DB, the IMGT® comprehensive database of immunoglobulin and T cell receptor nucleotide sequences, Nucl. Acids Res., 34, D781-D784 (2006). PMID: 16381979; T cell Receptor Factsbook, LeFranc and LeFranc, Academic Press ISBN 0-12-441352-8).

[0032] target The term "epitope" generally refers to the site on an antigen, typically a (poly)peptide, recognized by a binding domain. In its broadest sense, the term "binding domain" refers to an "antigen-binding site," i.e., characterizes the domain of a molecule that binds to / interacts with a specific epitope on an antigenic target. An antigenic target may contain a single epitope, but typically contains at least two epitopes, and may contain any number of epitopes depending on the size, conformation, and type of antigen. The term "epitope" generally encompasses linear epitopes and conformational epitopes. A linear epitope is a contiguous epitope contained in a primary sequence of amino acids, typically containing at least two or more amino acids. A conformational epitope is formed by non-contiguous amino acids juxtaposed by folding of a target antigen, particularly a target (poly)peptide.

[0033] Other embodiments include: (A) NY-ESO1-specific T cell receptor (TCR), including: - a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 35, a CDR2 having the amino acid sequence of SEQ ID NO: 36 and a CDR3 having the amino acid sequence of SEQ ID NO: 37, and - a TCR beta chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 38, a CDR2 having the amino acid sequence of SEQ ID NO: 39 and a CDR3 having the amino acid sequence of SEQ ID NO: 40, and (B) A chimeric costimulatory receptor comprising: - an extracellular domain containing polypeptide derived from PD-1 - a transmembrane domain, and - an intracellular domain containing a polypeptide derived from 4-1BB; A cell comprising: It relates to cells that secrete three or more proteins.

[0034] TCR-specific sequences As used herein, "at least 80% identical," and particularly "having an amino acid sequence that is at least 80% identical," includes amino acid sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the presented amino acid sequence.

[0035] Determining percent identity between multiple sequences is preferably accomplished using the AlignX application of the Vector NTI Advance™ 10 program (Invitrogen Corporation, Carlsbad, CA, USA), which utilizes a modified Clustal W algorithm (Thompson et al., 1994. Nucl Acids Res. 22: pp. 4673-4680; Invitrogen Corporation; Vector NTI Advance TM 10 DNA and protein sequence analysis software. User's Manual, 2004, pp. 389-662). Percent identity determination is performed using standard parameters of the AlignX application.

[0036] The TCRs described herein are specific for NY-ESO-1, in particular the NY-ESO-1 epitope SLLMWITQC (SEQ ID NO: 34), and exhibit very low cross-reactivity to other epitopes or antigens.

[0037] Thus, exemplary TCRs for use in the combinations of the present invention include: - a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 35, a CDR2 having the amino acid sequence of SEQ ID NO: 36 and a CDR3 having the amino acid sequence of SEQ ID NO: 37, and - a TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 38, a CDR2 having the amino acid sequence of SEQ ID NO: 39, and a CDR3 having the amino acid sequence of SEQ ID NO: 40 Includes.

[0038] In some embodiments, the TCR comprises a variable TCR alpha region having an amino acid sequence that is at least 80% identical to SEQ ID NO:41 and a variable TCR beta region having an amino acid sequence that is at least 80% identical to SEQ ID NO:42.

[0039] In a specific embodiment, the TCR comprises a variable TCR alpha region having the amino acid sequence of SEQ ID NO:41 and a variable TCR beta region having the amino acid sequence of SEQ ID NO:42.

[0040] In some embodiments, the TCR may comprise a TCR alpha chain having an amino acid sequence identical or at least 80% identical to SEQ ID NO: 43 and a TCR beta chain having an amino acid sequence identical or at least 80% identical to SEQ ID NO: 44.

[0041] In some embodiments, the TCR may comprise a TCR alpha chain having the amino acid sequence of SEQ ID NO:43 and a TCR beta chain having the amino acid sequence of SEQ ID NO:44.

[0042] Modification In some embodiments, the amino acid sequence of the TCR and / or chimeric costimulatory receptor may contain one or more phenotypically silent substitutions.

[0043] "Phenotypically silent substitutions" are also called "conservative amino acid substitutions." The concept of "conservative amino acid substitution" is understood by those skilled in the art and preferably means that codons encoding positively charged residues (H, K, and R) are replaced with codons encoding positively charged residues, codons encoding negatively charged residues (D and E) are replaced with codons encoding negatively charged residues, codons encoding polar-neutral residues (C, G, N, Q, S, T, and Y) are replaced with codons encoding polar-neutral residues, and codons encoding non-polar neutral residues (A, F, I, L, M, P, V, and W) are replaced with codons encoding non-polar neutral residues. These variations can occur naturally, can be introduced by random mutagenesis, or can be introduced by directed mutagenesis. These changes can be made without destroying the essential properties of these polypeptides. One skilled in the art can easily and routinely screen variant amino acids and / or the nucleic acids encoding them to determine whether these variations substantially reduce or abolish the binding ability of the ligand by methods known in the art.

[0044] Those skilled in the art will understand that the nucleic acid encoding the TCR and / or chimeric costimulatory receptor can also be modified. Useful modifications in the entire nucleic acid sequence include codon optimization of the sequence. Modifications that result in conservative substitutions in the expressed amino acid sequence can be made. These variations can be made in the complementarity-determining region and non-complementarity-determining region of the amino acid sequence of the TCR chain, which do not affect function. Usually, additions and deletions should not be made in the CDR3 region.

[0045] According to some embodiments of the invention, the amino acid sequence of the TCR and / or chimeric costimulatory receptor is modified to include a detectable label, a therapeutic agent, or a pharmacokinetic-modifying moiety.

[0046] Non-limiting examples of detectable labels are radioactive labels, fluorescent labels, nucleic acid probes, enzymes, and contrast agents.Therapeutic agents that can be associated with TCR include radioactive compounds, immunomodulatory agents, enzymes, or chemotherapeutic agents.Therapeutic agents can be encapsulated by liposomes linked to TCR, so that the compound can be slowly released at target site.This will avoid damage during transport in the body, and will ensure that therapeutic agents, such as toxins, have maximum efficiency after TCR binds to relevant antigen-presenting cells.Other examples of therapeutic agents include: Peptide cytotoxins are proteins or peptides capable of killing mammalian cells, such as ricin, diphtheria toxin, Pseudomonas exotoxin A, DNase, and RNase. Small molecule cytotoxic drugs are compounds with a molecular weight of less than 700 daltons that are capable of killing mammalian cells. Such compounds may contain toxic metals that can have cytotoxic effects. Furthermore, it should be understood that these small molecule cytotoxic drugs also include prodrugs, i.e., compounds that break down or are converted under physiological conditions to release cytotoxic drugs. Such agents may include, for example, docetaxel, gemcitabine, cisplatin, maytansine derivatives, rachelmcin, calicheamicin, etoposide, ifosfamide, irinotecan, porfimer sodium photofrin II, temozolomide, topotecan, trimetrexate glucuronate, mitoxantrone, auristatin E, vincristine, and doxorubicin; radionuclides, for example, iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210 and -213, actinium-225, and astatine-213. Association of the radionuclide with the TCR or a derivative thereof may be effected, for example, by a chelator, an immune-stimulator, also known as an immunostimulant, i.e., an immune effector molecule that stimulates an immune response. Exemplary immunostimulatory agents are cytokines, such as IL-2 and IFN-γ, antibodies or fragments thereof, such as anti-T cell or NK cell determinant antibodies (e.g., anti-CD3, anti-CD28, or anti-CD16); alternative protein scaffolds with antibody-like binding properties; superantigens, i.e., antigens and variants thereof that cause non-specific activation of T cells, resulting in polyclonal T cell activation and massive cytokine release; chemokines, such as IL-8, platelet factor 4, melanoma growth stimulating protein, complement activators; heterologous protein domains, homologous protein domains, viral / bacterial protein domains, viral / bacterial peptides.

[0047] The therapeutic agent may preferably be selected from the group consisting of immune effector molecules, cytotoxic drugs, and radionuclides. Preferably, the immune effector molecule is a cytokine.

[0048] The pharmacokinetic-modifying moiety can be, for example, at least one polyethylene glycol repeat unit, at least one glycol group, at least one sialic group, or a combination thereof. The association of the at least one polyethylene glycol repeat unit, at least one glycol group, and at least one sialic group can be induced by several methods known to those skilled in the art. In a preferred embodiment, the unit is covalently linked to the TCR. The TCR according to the present invention can be modified by one or more pharmacokinetic-modifying moieties. In particular, the soluble form of the TCR is modified by one or more pharmacokinetic-modifying moieties. The pharmacokinetic-modifying moiety can achieve beneficial changes to the pharmacokinetic profile of the therapeutic agent, such as improved plasma half-life, reduced or enhanced immunogenicity, and improved solubility.

[0049] The TCR and / or chimeric costimulatory receptor can be modified by attaching additional functional moieties, for example, to reduce immunogenicity, increase hydrodynamic size (size in solution), solubility and / or stability (e.g., by enhanced protection against proteolysis), and / or extend serum half-life.

[0050] Other useful functional moieties and modifications include "suicide" or "safety switches" that can be used to shut off effector host cells bearing the TCRs of the present invention in a patient. An example is the inducible caspase 9 (iCasp9) "safety switch" described in Gargett and Brown Front Pharmacol. 2014; 5: 235. Briefly, effector host cells are modified by well-known methods to express a caspase 9 domain whose dimerization is dependent on a small molecule dimerizing agent, such as AP1903 / CIP, resulting in rapid induction of apoptosis in the modified effector cells. The system is described, for example, in EP 2173869. Other examples of "suicide" or "safety switches" are known in the art, including, for example, expression and subsequent depletion of CD20 using herpes simplex virus thymidine kinase (HSV-TK), anti-CD20 antibodies, or myc tags (Kieback et al, Proc Natl Acad Sci US A. 2008 Jan 15;105(2):623-8).

[0051] TCRs with altered glycosylation patterns are also contemplated herein. As is known in the art, glycosylation patterns may depend on the amino acid sequence (e.g., the presence or absence of specific glycosylated amino acid residues, as described below) and / or the host cell or organism producing the protein. Glycosylation of polypeptides is typically N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. Addition of N-linked glycosylation sites to binding molecules is conveniently achieved by modifying the amino acid sequence to include one or more tripeptide sequences selected from asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline). O-linked glycosylation sites can be introduced by adding or substituting one or more serine or threonine residues to the starting sequence.

[0052] Another means of glycosylation of TCRs is by chemical or enzymatic coupling of glycosides to proteins. Depending on the coupling mode used, sugar(s) can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as cysteine, (d) free hydroxyl groups, such as serine, threonine, or hydroxyproline, (e) aromatic residues, such as phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. Similarly, deglycosylation (i.e., removal of carbohydrate moieties present on the binding molecule) can be achieved chemically, for example, by exposing the TCR to trifluoromethanesulfonic acid, or enzymatically, using endoglycosidases and exoglycosidases.

[0053] It is also contemplated that drugs such as small molecule compounds can be added to TCR, particularly the solubilized form of the TCR of the present invention.Linkage can be achieved by covalent or non-covalent interaction, for example, electrostatic force.Various linkers known in the art can be used to form drug conjugates.

[0054] Furthermore, TCRs, particularly soluble forms of the TCRs of the present invention, can be modified to introduce additional domains that aid in the identification, tracking, purification, and / or isolation of the respective molecules (tags). Thus, in some embodiments, the TCR alpha or beta chains may be modified to include an epitope tag.

[0055] An epitope tag is a useful example of a tag that can be incorporated into the TCR of the present invention. An epitope tag is a short stretch of amino acids that allows binding of a specific antibody, thereby enabling identification and tracking of the binding and migration of soluble TCRs or host cells within a patient or cultured (host) cells. Detection of epitope tags, and therefore tagged TCRs, can be achieved using several different techniques. Tags can also be used to stimulate and grow host cells bearing the TCR of the present invention by culturing the cells in the presence of a binding molecule (antibody) specific for the tag.

[0056] Generally, TCRs can be modified with various mutations that, in some cases, change the affinity and dissociation rate of the TCR to the target antigen. In particular, the mutations can increase affinity and / or decrease dissociation rate. Thus, TCRs can be mutated in at least one CDR and its variable domain framework region.

[0057] However, in a preferred embodiment, the CDRs of the TCR are not modified or, as in the case of the TCR in the example, are affinity matured in vitro. This means that the CDRs have naturally occurring sequences. This can be advantageous because in vitro affinity maturation can cause the TCR molecule to become immunogenic. This can lead to the production of anti-drug antibodies that reduce or inactivate the therapeutic effect and treatment, and / or cause side effects.

[0058] The mutations can be one or more substitutions, deletions, or insertions. These mutations can be introduced by any suitable method known in the art, such as polymerase chain reaction, restriction enzyme-based cloning, ligation-independent cloning techniques (e.g., as described in Sambrook, Molecular Cloning - 4th Edition (2012) Cold Spring Harbor Laboratory Press).

[0059] Theoretically, unpredictable TCR specificity with a risk of cross-reactivity may arise due to mispairing between endogenous and exogenous TCR chains. To avoid mispairing of TCR sequences, recombinant TCR sequences can be modified to contain murine or minimally murine Cα and Cβ regions, a technique that has been shown to efficiently enhance the correct pairing of several different transduced TCR chains. TCR murinization (i.e., replacing the human Cα and Cβ regions with their murine counterparts) is a commonly applied technique to improve the cell surface expression of TCRs in host cells. Without wishing to be bound by any particular theory, murine TCRs are thought to more effectively associate with the CD3 co-receptor; and / or preferentially pair with each other, and to be less prone to forming mixed TCRs on human T cells that have been genetically modified ex vivo to express TCRs of desired antigen specificity but still retain and express the "original" TCR.

[0060] Nine amino acids responsible for improved expression of murinized TCRs have been identified (Sommermeyer and Uckert, J Immunol. 2010 Jun 1; 184(11):6223-31), and it is envisioned that one or all of the amino acid residues in the TCR α and / or β chain constant regions will be replaced with their murine counterparts. This technique, also known as "minimal murinization," offers the advantage of enhancing cell surface expression while simultaneously reducing the number of "foreign" amino acid residues in the amino acid sequence, thereby reducing the risk of immunogenicity.

[0061] Some embodiments relate to an isolated TCR as described herein, wherein the TCR is of the single chain type and the TCR alpha and beta chains are linked by a linker sequence.

[0062] A preferred single-chain TCR format comprises a first segment composed of an amino acid sequence corresponding to the variable TCR α region, a second segment composed of an amino acid sequence corresponding to the variable TCR β region fused to the N-terminus of an amino acid sequence corresponding to the TCR β chain constant region extracellular sequence, and a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment. Alternatively, the first segment may be composed of an amino acid sequence corresponding to the TCR β chain variable region, and the second segment may be composed of an amino acid sequence corresponding to the TCR α chain variable region sequence fused to the N-terminus of the amino acid sequence corresponding to the TCR α chain constant region extracellular sequence. The single-chain TCR may further comprise a disulfide bond between the first and second chains, and the length of the linker sequence and the position of the disulfide bond are such that the variable domain sequences of the first and second segments are oriented relative to each other substantially as in a natural T cell receptor. More specifically, the first segment may be composed of an amino acid sequence corresponding to a TCR α chain variable region sequence fused to the N-terminus of an amino acid sequence corresponding to a TCR α chain constant region extracellular sequence, the second segment may be composed of an amino acid sequence corresponding to a TCR β chain variable region fused to the N-terminus of an amino acid sequence corresponding to a TCR β chain constant region extracellular sequence, and a disulfide bond may be provided between the first and second chains. The linker sequence may be any sequence that does not impair TCR function.

[0063] In the context of the present invention, a "functional" TCR α and / or β chain fusion protein would mean a TCR or TCR variant that has been modified, for example by addition, deletion, or substitution of amino acids, which maintains at least substantial biological activity. In the case of a TCR α and / or β chain, this would mean that both chains remain capable of forming a TCR (with unmodified α and / or β chains, or with another inventive fusion protein α and / or β chain) that exerts its biological function, in particular binding of said TCR to a specific peptide-MHC complex and / or functional signaling upon specific peptide:MHC interaction.

[0064] In certain embodiments, TCRs may be engineered to be functional TCR alpha and / or beta chain fusion proteins, where the epitope tag has a length of 6 to 15 amino acids, preferably 9 to 11 amino acids. In another embodiment, TCRs may be engineered to be functional T cell receptor (TCR) alpha and / or beta chain fusion proteins, where the TCR alpha and / or beta chain fusion proteins comprise two or more epitope tags, spaced apart or directly adjacent in tandem. Fusion protein embodiments can contain two, three, four, five, or even more epitope tags, so long as the fusion protein maintains its biological activity(ies) ("functionality").

[0065] Preferred are functional TCR α and / or β chain fusion proteins according to the present invention, in which the epitope tag is selected from, but not limited to, CD20 or Her2 / neu tags, or other conventional tags, such as myc, T7, GST, or GFP tags. Myc, T7, GST, and GFP tags are epitopes derived from existing molecules. In contrast, FLAG is a synthetic epitope tag designed for high antigenicity (see, for example, U.S. Pat. Nos. 4,703,004 and 4,851,341). The myc tag is preferably used because of the availability of high-quality reagents for its detection. Naturally, epitope tags can have one or more additional functions in addition to antibody recognition. The sequences of these tags are described in the literature and are well known to those skilled in the art.

[0066] Chimeric costimulatory receptors The chimeric costimulatory receptor used in combination with the antigen-specific TCR (NY-ESO-specific TCR) - an extracellular domain containing polypeptide derived from PD-1; - a transmembrane domain, and - an intracellular domain containing a polypeptide derived from 4-1BB Includes.

[0067] As used herein, a chimeric costimulatory receptor used in combination with an antigen-specific TCR, e.g., a NY-ESO-1-specific TCR, may particularly comprise an extracellular domain containing an extracellular domain derived from PD-1 (e.g., human PD-1). In this context, the term "derived from" particularly means that each polypeptide contained in the extracellular domain comprises at least a portion of PD-1 (e.g., human PD-1), preferably the extracellular domain of PD-1. A chimeric costimulatory receptor comprising an extracellular domain derived from PD-1 has binding activity to PD-L1, PD-L2, or other inhibitory ligands of PD-1. As used herein, the term "derived from" PD-1 also allows for up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions, deletions, and / or insertions compared to the native sequence of PD-1 (e.g., human PD-1) or a portion thereof (e.g., the extracellular domain).

[0068] In one embodiment, the extracellular domain containing polypeptide derived from PD-1 comprises the sequence set forth in SEQ ID NO: 28, or an amino acid sequence at least 80% identical to SEQ ID NO: 28. In a specific embodiment, the extracellular domain containing polypeptide derived from PD-1 comprises the sequence set forth in SEQ ID NO: 28.

[0069] In one embodiment of the present invention, the chimeric costimulatory receptor comprises an extracellular domain containing a polypeptide derived from human or mouse PD-1, e.g., PD-1, comprising an amino acid sequence having up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions (preferably conservative or highly conservative substitutions), deletions, and / or insertions compared to the amino acid sequence of the extracellular domain of human PD-1, e.g., as set forth in SEQ ID NO:28.

[0070] The chimeric costimulatory receptor used herein in combination with an antigen-specific TCR, for example, a NY-ESO-1-specific TCR, further comprises a transmembrane domain operably linked between the extracellular domain and the intracellular domain. Generally, the transmembrane domain is not limited to a specific transmembrane domain. Preferably, the transmembrane domain enables stable anchoring of the fusion protein in the membrane of a cell (e.g., a T cell) expressing the fusion protein, and further enables the extracellular domain to bind to PD-L1 and, upon binding to PD-L1, transmits a signal to the intracellular domain containing a polypeptide derived from 4-1BB, respectively.

[0071] In a preferred embodiment, the transmembrane domain of the chimeric costimulatory receptor is derived from PD-1. In one embodiment, the transmembrane domain comprises the sequence set forth in SEQ ID NO: 30 or an amino acid sequence that is at least 80% identical to SEQ ID NO: 30. In a specific embodiment, the transmembrane domain containing polypeptide derived from PD-1 comprises the sequence set forth in SEQ ID NO: 30.

[0072] A chimeric costimulatory receptor used in combination with an antigen-specific TCR, e.g., a NY-ESO-1-specific TCR, herein may particularly comprise an intracellular domain containing a polypeptide derived from 4-1BB (also referred to as "41BB"), preferably the intracellular domain of 4-1BB (e.g., human 4-1BB). In this context, the term "derived from" particularly means that the polypeptide contained in the intracellular domain comprises at least a portion of 4-1BB (e.g., human 4-1BB), preferably the intracellular domain of 4-1BB. A chimeric costimulatory receptor comprising an intracellular domain derived from 4-1BB can increase the proliferation rate of T cells expressing the chimeric costimulatory receptor upon stimulation with PD-L1, PD-L2, or another inhibitory ligand of PD-1, and / or can increase the effector function (e.g., increased IFN-γ release and / or increased cytotoxicity) of T cells expressing the chimeric costimulatory receptor compared to corresponding T cells that do not express the chimeric costimulatory receptor. As used herein, the term "derived from" 4-1BB also allows for up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions, deletions, and / or insertions compared to the native sequence of 4-1BB (human or mouse, preferably human 4-1BB) or a portion thereof (e.g., the intracellular domain). In one embodiment, the intracellular domain containing the polypeptide derived from 4-1BB comprises the sequence set forth in SEQ ID NO: 32 or an amino acid sequence at least 80% identical to SEQ ID NO: 32. In a specific embodiment, the intracellular domain containing the polypeptide derived from 4-1BB comprises the sequence set forth in SEQ ID NO: 32.

[0073] "At least 80% identical," and particularly "having an amino acid sequence that is at least 80% identical," as used herein, includes amino acid sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the presented amino acid sequence.

[0074] Determining percent identity between multiple sequences is preferably accomplished using the AlignX application of the Vector NTI Advance™ 10 program (Invitrogen Corporation, Carlsbad, California, USA), which uses a modified Clustal W algorithm (Thompson et al., 1994. Nucl Acids Res. 22: pp. 4673-4680; Invitrogen Corporation; Vector NTI Advance TM 10 DNA and protein sequence analysis software. User's Manual, 2004, pp. 389-662). Percent identity determination is performed using standard parameters of the AlignX application.

[0075] Nucleic acids, nucleic acid compositions, and vectors The present invention encompasses nucleic acids encoding antigen-specific TCRs, particularly NY-ESO-1 specific TCRs as described herein, as well as corresponding nucleic acid compositions and vectors comprising said nucleic acids.

[0076] The nucleotide sequences encoding the relevant regions and domains of the NY-ESO-specific TCR are presented in Table 1.

[0077] [Table 1]

[0078] The nucleotide sequences encoding the relevant regions and domains of the chimeric costimulatory receptors are presented in Table 2.

[0079] [Table 2]

[0080] Furthermore, a further aspect is - a nucleic acid encoding a NY-ESO-1-specific T cell receptor (TCR), comprising: - a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 35, a CDR2 having the amino acid sequence of SEQ ID NO: 36 and a CDR3 having the amino acid sequence of SEQ ID NO: 37, and - a TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 38, a CDR2 having the amino acid sequence of SEQ ID NO: 39, and a CDR3 having the amino acid sequence of SEQ ID NO: 40; and - a nucleic acid encoding a chimeric costimulatory receptor comprising: - an extracellular domain containing polypeptide derived from PD-1; - a transmembrane domain, and - an intracellular domain containing a polypeptide derived from 4-1BB The present invention relates to a composition comprising:

[0081] Furthermore, one aspect is - a nucleic acid encoding a NY-ESO-1-specific T cell receptor (TCR), comprising: - a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 35, a CDR2 having the amino acid sequence of SEQ ID NO: 36 and a CDR3 having the amino acid sequence of SEQ ID NO: 37, and - a TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 38, a CDR2 having the amino acid sequence of SEQ ID NO: 39, and a CDR3 having the amino acid sequence of SEQ ID NO: 40; and - a nucleic acid encoding a chimeric costimulatory receptor comprising: - an extracellular domain containing polypeptide derived from PD-1; - a transmembrane domain, and - an intracellular domain containing a polypeptide derived from 4-1BB The present invention relates to a nucleic acid comprising:

[0082] A further embodiment refers to a vector comprising a nucleic acid comprising sequences for a NY-ESO-1 specific TCR and a chimeric costimulatory receptor. Cells comprising the nucleic acid composition and / or the vector are also encompassed.

[0083] A "nucleic acid molecule" generally refers to a polymer of DNA or RNA, which may be single-stranded or double-stranded, synthesized or obtained from a natural source (e.g., isolated and / or purified), and which may contain natural, non-natural, or modified nucleotides, and which may contain natural, non-natural, or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages, in place of the phosphodiesters found between nucleotides in unmodified oligonucleotides. Preferably, the nucleic acids described herein are recombinant. As used herein, the term "recombinant" refers to (i) a molecule constructed outside a living cell by linking natural or synthetic nucleic acid segments to a nucleic acid molecule capable of replicating in the living cell, or (ii) a molecule resulting from replication as described in (i) above. For purposes of this specification, replication can be in vitro or in vivo replication. Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using techniques known in the art or commercially available (e.g., from Genscript, Thermo Fisher, and similar companies). See, e.g., Sambrook et al. Nucleic acids can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides (see, e.g., Sambrook et al. 2001) (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization. Nucleic acids can include any nucleotide sequence that encodes a recombinant TCR and / or chimeric costimulatory receptor, polypeptide, or protein, or any functional portion or variant thereof.

[0084] For example, the present disclosure also provides isolated or purified variant nucleic acids, wherein the variant nucleic acids comprise a nucleotide sequence at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence encoding a TCR described herein. Such variant nucleotide sequences encode a functional TCR that specifically recognizes NY-ESO-1, particularly the NY-ESO-1 epitope of SEQ ID NO:34.

[0085] For example, the present disclosure also provides isolated or purified variants of nucleic acids, wherein the variant nucleic acids comprise a nucleotide sequence at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a chimeric costimulatory receptor described herein. Such variant nucleotide sequences encode a functional chimeric costimulatory receptor described herein.

[0086] As already described elsewhere herein, the nucleic acid encoding the TCR and / or chimeric costimulatory receptor may be modified. Useful modifications in the entire nucleic acid sequence may be codon optimization. Changes resulting in conservative substitutions in the translated amino acid sequence may also be made. For TCRs, these variations can be made in the complementarity-determining and non-complementarity-determining regions of the amino acid sequence of the TCR chain, which do not affect function. Usually, additions and deletions should not be made in the CDR3 region.

[0087] Another embodiment relates to a vector comprising nucleic acid encoding the TCR and chimeric costimulatory receptor described herein.

[0088] The vector is preferably a plasmid, shuttle vector, phagemid, cosmid, expression vector, retroviral vector, adenoviral vector or particle, and / or a vector used in gene therapy.

[0089] A "vector" is any molecule or composition capable of transporting a nucleic acid sequence into a suitable host cell where synthesis of the encoded polypeptide can occur. Typically and preferably, a vector is a nucleic acid that has been engineered using recombinant DNA techniques known in the art to incorporate a desired nucleic acid sequence (e.g., a nucleic acid of the present invention). A vector may comprise DNA or RNA, and / or a liposome and / or a viral particle. A vector may be a plasmid, a shuttle vector, a phagemid, a cosmid, an expression vector, a retroviral vector, a lentiviral vector, an adenoviral vector or particle, and / or a vector used in gene therapy. A vector may contain a nucleic acid sequence that allows replication in a host cell, such as an origin of replication. A vector may also contain one or more selectable marker genes and other genetic elements known to those skilled in the art. A vector is preferably an expression vector comprising a nucleic acid of the present invention operably linked to a sequence that allows expression of said nucleic acid.

[0090] Preferably, the vector is an expression vector, more preferably a retroviral vector, more particularly a gamma-retroviral or lentiviral vector.

[0091] Those skilled in the art will appreciate that the chimeric costimulatory receptor sequence and the TCR-α and TCR-β chain sequences can be contained in a single nucleic acid, e.g., a single vector, linked to an internal ribosome entry site (IRES) sequence or a 2A peptide sequence from other species, such as the porcine teschovirus (P2A) or the Thosea asigna virus 2A peptide (T2A) or the foot-and-mouth disease virus 2A peptide (F2A) (described in Szymczak et al.: Development of 2A peptide-based strategies in the design of multicistronic vectors), resulting in the expression of a single messenger RNA (mRNA) molecule under the control of a viral promoter in transduced cells.

[0092] In certain embodiments, the cells may contain nucleic acids encoding the TCRs and chimeric costimulatory receptors described herein, or vectors containing said nucleic acids.

[0093] The terms "transfection" and "transduction" are interchangeable and refer to the process of introducing an exogenous nucleic acid sequence into a host cell, e.g., a eukaryotic host cell. It should be noted that the introduction or transfer of a nucleic acid sequence can be achieved by various means, including, but not limited to, electroporation, microinjection, gene gun delivery, lipofection, superfection, and the aforementioned infection with retroviruses or other viruses suitable for transduction or transfection. Methods for cloning and exogenous expression of TCRs are described, for example, in Engels et al. (Relapse or eradication of cancer is predicted by peptide-major histocompatibility complex affinity. Cancer Cell, 23(4), 516-26. 2013). Transduction of primary human T cells with lentiviral vectors is described, for example, in Cribbs, "Simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells," BMC Biotechnol. 2013; 13: 98.

[0094] The cells described and provided in connection with the present invention, including the nucleic acid molecules or vectors described and provided herein, are preferably capable of stably or transiently (e.g., stably) expressing (constitutively or conditionally) an antigen-specific TCR, e.g., an NY-ESO-1-specific TCR, and a chimeric costimulatory receptor of the present invention. Host cells can generally be transduced or transformed by any method using any suitable nucleic acid molecule or vector. In one embodiment, host cells are transduced using a retroviral or lentiviral (e.g., retroviral) vector containing a nucleic acid molecule encoding a fusion protein of the present invention or a portion thereof (e.g., ECD, TMD, and / or ICD), as described above.

[0095] In some embodiments, the cells (including, for example, target-specific immune cells or cells of the populations of cells referred to herein) are peripheral blood lymphocytes (PBLs) or peripheral blood mononuclear cells (PBMCs). The cells can be natural killer cells or T cells. Preferably, the cells are T cells. The T cells can be CD4+ or CD8+ T cells. In some embodiments, the cells are stem cell-like memory T cells.

[0096] Stem cell-like memory T cells (TSCM) are a subpopulation of less differentiated CD8+ or CD4+ T cells characterized by the ability to self-renew and persist for long periods. When these cells encounter their antigen in vivo, they further differentiate into central memory T cells (TCM), effector memory T cells (TEM), and terminally differentiated effector memory T cells (TEMRA), with some TSCM remaining quiescent (Flynn et al., Clinical & Translational Immunology (2014)). These remaining TSCM cells exhibit the ability to build durable immune memory in vivo and are therefore considered to be an important T cell subpopulation for adoptive T cell therapy (Lugli et al., Nature Protocols 8, 33-42 (2013) Gattinoni et al., Nat. Med. 2011 Oct; 17(10): 1290-1297). Immunomagnetic selection can be used to restrict the T cell pool to stem cell memory T cell subtypes (see Riddell et al. 2014, Cancer Journal 20(2): 141-44).

[0097] Pharmaceutical Compositions, Medical Treatments, and Kits Another aspect of the present invention relates to cells comprising an antigen-specific TCR, e.g., an NY-ESO-1-specific TCR, and a chimeric costimulatory receptor, or cells comprising nucleic acid molecules encoding the above molecules described herein; nucleic acids encoding an antigen-specific TCR, e.g., an NY-ESO-1-specific TCR, and a chimeric costimulatory receptor; compositions comprising a nucleic acid encoding an antigen-specific TCR, e.g., an NY-ESO-1-specific TCR, and a nucleic acid encoding a chimeric costimulatory receptor; and pharmaceutical compositions comprising the corresponding vectors described herein.

[0098] The active ingredients of the present invention are preferably used in such pharmaceutical compositions in a dosage mixed with an acceptable carrier or carrier material that can treat or at least alleviate the disease. Such compositions can contain (in addition to the active ingredient and carrier) fillers, salts, buffers, stabilizers, solubilizers, and other materials that are well known in the state of the art.

[0099] The term "pharmaceutically acceptable" defines a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The choice of carrier will depend on the application.

[0100] The pharmaceutical composition may contain additional ingredients that enhance the activity of the active ingredient or supplement the treatment. Such additional ingredients and / or factors may be part of the pharmaceutical composition to achieve synergistic effects or to minimize adverse or unwanted effects.

[0101] Techniques for formulation or preparation and application / medication of the active ingredients of the present invention are published in "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA, latest edition. Suitable applications are parenteral applications, such as intramuscular, subcutaneous, intramedullary injection, as well as intrathecal, direct intraventricular, intravenous, intranodal, intraperitoneal, or intratumoral injection. Intravenous infusion is the preferred treatment for patients.

[0102] According to a preferred embodiment, the pharmaceutical composition is an infusion or injection.

[0103] The injectable composition is a pharmaceutically acceptable fluid composition containing at least one active ingredient, e.g., an expanded T cell population (e.g., autologous or allogeneic to the patient being treated) comprising an antigen-specific TCR, e.g., an NY-ESO-1-specific TCR, and a chimeric costimulatory receptor. The active ingredient is usually dissolved or suspended in a physiologically acceptable carrier, and the composition may additionally contain small amounts of one or more non-toxic auxiliary substances, such as emulsifiers, preservatives, and pH buffering agents. Such injectable compositions useful for use with the fusion proteins of the present disclosure are conventional, and suitable formulations are well known to those skilled in the art.

[0104] Typically, a pharmaceutical composition includes at least one pharmaceutically acceptable carrier.

[0105] Accordingly, another aspect of the present invention relates to a cell described herein, a composition described herein, a nucleic acid described herein, and / or a vector described herein for use as a medicament.

[0106] Some embodiments refer to the cells described herein, the compositions described herein, the nucleic acids described herein, and / or the vectors described herein for use in the treatment of cancer.

[0107] Thus, also contemplated are methods of treating cancer in a human or non-human animal in need thereof, comprising administering to said human or non-human animal a cell described herein, a composition described herein, a nucleic acid described herein, and / or a vector described herein.

[0108] In one embodiment, the cancer is a hematological cancer or a solid tumor.

[0109] Blood cancer, also known as blood cancer, does not form solid tumors and therefore spreads throughout the body. Examples of blood cancer are leukemia, lymphoma, or multiple myeloma. There are two main types of solid tumors: sarcoma and carcinoma. Sarcomas are, for example, tumors of blood vessels, bones, fat tissue, ligaments, lymphatic vessels, muscles, or tendons. In certain embodiments, cancer is a solid tumor.

[0110] In one embodiment, the cancer is selected from the group consisting of prostate cancer, uterine cancer, thyroid cancer, testicular cancer, renal cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma, non-Hodgkin's lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, cervical cancer, colorectal cancer, gastric adenocarcinoma, cholangiocarcinoma, breast cancer, bladder cancer, myeloid leukemia and acute lymphoblastic leukemia, carcinoma, sarcoma, or osteosarcoma.

[0111] Compositions comprising the modified T cells described herein can be utilized in methods and compositions for adoptive immunotherapy according to known techniques, or variations thereof that will be apparent to those of skill in the art based on this disclosure.

[0112] In some embodiments, cells are formulated by first harvesting them from their culture medium, then washing and concentrating the cells in a medium and a container system suitable for administration in a therapeutically effective amount (a "pharmaceutically acceptable" carrier). A suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter), although 5% dextrose in water or lactated Ringer's solution can also be used. The infusion medium can be supplemented with human serum albumin.

[0113] The number of cells in the composition for effective treatment is typically greater than 10 and up to 10 6 pieces, 10 8 pieces or 10 9 Up to 10 cells10 More than 10 cells are possible. The number of cells will depend on the intended end use of the composition and the type of cells contained therein. For the uses provided herein, the cells are generally in a volume of 1 liter or less, and may be 500 ml or less, or even 250 ml or 100 ml or less. Thus, the desired cell density is typically 10 6 cells / ml, generally above 10 7 cells / ml, typically >10 8 Clinically relevant numbers of immune cells can be distributed across multiple infusions, with cumulative cell counts exceeding 10 9 , 10 10 , or 10 11 The amount of the pharmaceutical composition provided herein is equal to or exceeds 100 cells. The pharmaceutical composition provided herein can be in various forms, for example, solid, liquid, powder, aqueous or lyophilized form. Examples of suitable pharmaceutical carriers are known in the art. Such carriers and / or additives can be formulated by conventional methods and administered to subjects in suitable doses. Stabilizing agents, such as lipids, nuclease inhibitors, polymers, and chelating agents, can protect the composition from degradation in the body. Compositions intended to be administered by injection can include one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, and isotonic agents.

[0114] The viral vector particle comprising the nucleotide sequence encoding the antigen-specific TCR provided herein, for example, NY-ESO-1-specific TCR and chimeric costimulatory receptor, can be packaged as a kit.The kit can optionally include one or more components, such as instructions for use, devices, and additional reagents, as well as components for implementing the method, such as tubes, containers, and syringes.An exemplary kit can include the nucleic acid encoding the recombinant TCR and chimeric costimulatory receptor, recombinant polypeptide, or virus provided herein, and can optionally include instructions for use, a device for detecting viruses in a subject, a device for administering the composition to a subject, and a device for administering the composition to a subject.

[0115] Also contemplated herein is a kit comprising a polynucleotide encoding an antigen-specific TCR, for example, a NY-ESO-1-specific TCR and a chimeric costimulatory receptor. Also contemplated herein is a kit comprising a viral vector encoding a sequence of interest (e.g., a recombinant TCR) and, optionally, a polynucleotide sequence encoding an immune checkpoint inhibitor.

[0116] Kits contemplated herein also include kits for performing methods for detecting the presence of polynucleotides encoding any one or more of the TCRs and / or chimeric costimulatory receptors disclosed herein. In particular, such diagnostic kits may include a set of appropriate amplification and detection primers and other related reagents for performing deep sequencing to detect polynucleotides encoding the TCRs and / or chimeric costimulatory receptors disclosed herein. In further embodiments, the kits herein may include reagents, such as antibodies or other binding molecules, for detecting the TCRs and / or chimeric costimulatory receptors disclosed herein. The diagnostic kits may also include instructions for determining the presence of polynucleotides encoding the TCRs and / or chimeric costimulatory receptors disclosed herein or for determining the presence of the TCRs and / or chimeric costimulatory receptors disclosed herein. The kits may also include instructions. The instructions typically include specific wording describing the components included in the kit, as well as the method of administration, appropriate dosages, and appropriate administration methods, including methods for determining the appropriate condition of the subject. The instructions may also include guidance for monitoring the subject over the duration of treatment.

[0117] The kit provided herein can also include a device for administering the composition described herein to a subject.Any of the various devices known in the art for administering medicines or vaccines can be included in the kit provided herein.Exemplary devices include, but are not limited to, hypodermic needles, intravenous needles, catheters, needleless injection devices, inhalers, and liquid dispensers, such as eyedroppers.Usually, the device for administering the virus in the kit is compatible with the virus in the kit; for example, a needleless injection device such as a high-pressure injection device can be included in the kit with a virus that is not damaged by high-pressure injection, but typically will not be included in the kit with a virus that is damaged by high-pressure injection.

[0118] Cytokine release Cells expressing both TCR and costimulatory molecules, such as PD1-4BB, exhibit enhanced cytokine release. Cytokines / lytic proteins secreted in large amounts by the population include IFN-γ, Gzm-B, and IP-10, MIP-1β, among others.

[0119] Surprisingly, immune cells, in particular TCR-T cells, that express a chimeric costimulatory receptor as defined herein exhibit greater polyfunctionality compared to TCR-T cells that lack a chimeric costimulatory receptor as defined herein.

[0120] Higher polyfunctionality in transgenic T cells indicates greater functionality and antitumor activity in vivo and correlates with clinical outcome.

[0121] The term "multifunctional" means that the cell secretes at least two proteins, such as at least three, at least four, at least five proteins.

[0122] Those skilled in the art will appreciate that cells secrete proteins following antigen-specific stimulation, for example, when the cells are contacted with antigen-presenting cells that present the antigen in MHC molecules.

[0123] In particular, the cell population comprises a significant percentage of cells that are multipotent cells, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 3%, 4%, 4.5%.

[0124] In particular, the cell population comprises a significant percentage of cells that express three or more proteins, at least 1%, preferably at least 1.5%, more preferably 1.75%.

[0125] In some embodiments, the multifunctional strength index is 80 or greater, preferably 90 or greater, and more preferably 100 or greater.

[0126] The polyfunctional strength index (PSI) is calculated by multiplying the strength of various secreted cytokines by the percentage of polyfunctional T cells.

[0127] In particular, the inventors have been able to show that a cell population expressing both a target-specific TCR, e.g., a NY-ESO-1-specific TCR, and a chimeric costimulatory receptor, as defined herein, comprises cells that secrete at least two proteins, e.g., at least three proteins, at least four proteins, at least five proteins.

[0128] Accordingly, another aspect of the present invention relates to an in vitro assay that indicates the titer of a population of T cells by indicating whether the population of T cells contains a significant proportion of polyfunctional T cells as defined herein.

[0129] Thus, what is generally desired are highly potent, target-specific T cells that secrete at least two, at least three, at least four, or at least five proteins selected from the group of effector proteins, stimulatory cytokines, and chemoattractant cytokines.

[0130] In particular, the present invention comprises the steps of: - measuring the proportion of polyfunctional immune cells in the first population of cells; - assessing the titer of a first population of immune cells and assessing the titer of a second population of T cells, wherein an increased proportion of multipotent cells in the first population compared to the second population indicates that the first population has a higher titer than the second population; The present invention relates to an in vitro assay that indicates the titer of a population of immune cells, comprising:

[0131] Measurement of the proportion of polyfunctional immune cells can be performed by IsoLight® technology (Isoplexis).

[0132] Thus, a T cell population comprising target-specific T cells is desirable, the T cell population comprising T cells that secrete at least two, at least three, at least four, or at least five proteins selected from the group consisting of effector proteins, stimulatory cytokines, and chemoattractant cytokines.

[0133] The effector protein may be selected from the group consisting of Gzm-B, IFN-γ, perforin, TNF-α, TNF-β, MIP-1α.

[0134] In one embodiment, the stimulatory cytokine may be selected from the group consisting of GM-CSF, IL-2, IL-7, IL-8, IL-9, IL-12. In one embodiment, the stimulatory protein is selected from GM-CSF, IL-2, and IL-8. In one embodiment, the stimulatory protein is GM-CSF.

[0135] The chemoattractant cytokine may be selected from IP-10 and MIP-1β.

[0136] In some embodiments, each of the cells secreting at least two proteins secretes at least one of the proteins selected from the group consisting of Gzm-B, IFN-γ, perforin, TNF-α, TNF-β, MIP-1α, GM-CSF, IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IP-10, and MIP-1β. In some embodiments, each of the cells secreting at least two proteins secretes at least one of the proteins selected from the group consisting of Gzm-B, IFN-γ, perforin, TNF-α, TNF-β, GM-CSF, IL-2, IL-8, MIP-1β, and IP-10. In some embodiments, each of the cells secreting at least two proteins secretes at least one of the proteins selected from the group consisting of IFN-γ, Gzm-B, GM-CSF, MIP-1β, and IP-10. In some embodiments, each of the cells secreting at least two proteins secretes at least one of the proteins selected from the group consisting of IFN-γ, Gzm-B, and GM-CSF. In some embodiments, each of the cells secreting at least two proteins secretes at least one of the proteins selected from the group consisting of IFN-γ, Gzm-B, and IP-10. In some embodiments, each of the cells secreting at least two proteins secretes at least one of the proteins selected from the group consisting of IFN-γ and Gzm-B.

[0137] Typically, the cell population does not include cells that secrete significant amounts of IL-6 and / or IL-10.

[0138] Other embodiments include: (A) a NY-ESO-1 specific TCR as defined herein; and (B) A T cell expressing a chimeric costimulatory receptor as defined herein, wherein the T cell secretes at least two, at least three, at least four, or at least five proteins selected from the group consisting of effector proteins, stimulatory cytokines, and chemoattractant cytokines.

[0139] Accordingly, some embodiments relate to a cell population comprising T cells that express (A) an NY-ESO-1-specific TCR as defined herein and (B) a chimeric costimulatory receptor as defined herein, wherein the cell population comprises T cells that secrete at least two, at least three, at least four, or at least five proteins selected from the group consisting of effector proteins, stimulatory cytokines, and chemoattractant cytokines.

[0140] experiment [Example 1] NY-ESO-1-targeting TCR-T cells expressing PD1-41BB exhibit higher polyfunctionality compared with NY-ESO-1-targeting TCR-T cells lacking PD1-41BB. TCR-transgenic T cells with or without PD1-41BB were analyzed for their single-cell multifunctionality (release of two or more proteins) using IsoLight® technology (IsoPlexis). So-called "multifunctional" T cells secrete multiple proteins (e.g., cytokines and other effector proteins, such as Gzm-B), thereby enabling diverse effector functions and highly efficient immune responses. After 20 hours of coculture with PD-L1-overexpressing NY-ESO-1-positive MelA375 or Mel624.38 tumor cell lines, single CD8+ T cells were isolated from tumor cells using CD8 microbeads (Miltenyi Biotec) and loaded onto IsoCode chips (IsoPlexis). The cytokine / protein secretion of 32 T cells was analyzed using the IsoLight device and IsoSpeak software (IsoPlexis). TCR-T cells expressing PD1-41BB showed a higher percentage of polyfunctional T cells (meaning that T cells secrete two or more proteins) compared with TCR-T cells lacking PD1-41BB (Figure 1A). The polyfunctional strength index (PSI) was calculated by multiplying the intensities of various secreted proteins / cytokines by the percentage of polyfunctional T cells. TCR-T cells expressing PD1-41BB showed a higher PSI compared with TCR-T cells lacking PD1-41BB (Figure 1B).

[0141] Classification of the various proteins / cytokines released revealed a high contribution of effector proteins (Gzm-B, IFN-γ, MIP-1a, perforin, TNF-α, TNF-β) and stimulatory cytokines (GM-CSF, IL-12, IL-2, IL-5, IL-8, IL-9) to superior PSI, followed by chemoattractant cytokines (IP-10, MIP-1β, RANTES). Regulatory (IL-4, IL-10, IL-22, sCD137, TGF-β1) and proinflammatory (CP-1, IL-17F, IL-8) cytokines were released to a lesser extent. Detailed analysis of single-cell multiprotein / cytokine release using multifunctional heatmaps showed that TCR-T cells with and without PD1-41BB differed in their multiprotein / cytokine signatures (Figure 1C). Notably, PD1-41BB-expressing TCR-T cells contain a higher proportion of single cells secreting two to six proteins / cytokines simultaneously, indicating superior functionality compared to TCR-T cells lacking PD1-41BB. The main proteins / cytokines released by PD1-41BB-expressing TCR-T cells are Gzm-B, IFN-γ, GM-CSF, and MIP-1β, which belong to the effector protein and stimulatory cytokine families, thus indicating enhanced functionality and potential antitumor activity of TCR-T cells.

[0142] [Example 2] Improved effector function of NY-ESO-1 TCR-T cells co-expressing chimeric PD1-41BB costimulatory receptor. Flow cytometry analysis of untransduced (UT), NY-ESO-1 TCR-expressing (TCR), and NY-ESO-1 TCR co-expressing PD1-41BB (TCR+PD1-41BB) T cells was performed (Figure 2A). NY-ESO-1 TCR transgenic CD8+ T cells with or without PD1-41BB were cultured with tumor cell lines expressing NY-ESO-1 and PD-L1 at different levels, and the levels of released IFN-γ were determined by ELISA (t-test, ** indicates p>0.01, *** indicates p<0.001) (Figure 2B). NY-ESO-1 expression levels were measured by qPCR and quantified against a standard calibration curve. PD-L1 levels, summarized here, were assessed by both qPCR and FACS analysis (Figure 2C). NY-ESO-1 TCR transgenic CD8+ T cells with or without PD1-41BB were stained with a dye membrane tracker and co-cultured with NY-ESO-1+PD-L1+ tumor cell lines for 5 days. Proliferation was analyzed by flow cytometry, and data were evaluated using FlowJo (Figure 2d).

[0143] [Example 3] Enhanced NY-ESO-1 TCR-T cell polyfunctionality by coexpression of the chimeric costimulatory receptor PD1-41BB. NY-ESO-1-specific TCR-T cells with (TCR+PD1-41BB) and without (TCR) coexpression of the chimeric costimulatory receptor PD1-41BB were analyzed using single-cell proteomic analysis of a panel of 32 secreted cytokines, chemokines, and cytotoxic molecules after 24 hours of coculture with the tumor-targeting cell lines Mel624.38_PD-L1 and MelA375_PD-L1 (IsoLight technology, IsoPlexis). Coculture with untransduced T cells (UT) from the same donor served as a control. Cells secreting two or more cytokines are considered polyfunctional. The polyfunctionality of single TCR T cells is shown as the percentage of samples and categorized into the percentage of cells expressing 2, 3, 4, and 5+ cytokines (gray fill) simultaneously (Figure 3A). The polyfunctional intensity index (PSI) of the indicated samples was defined as the number of T cells secreting three or more effector molecules per cell (polyfunctional T cells in A) multiplied by the mean fluorescence intensity (MFI) of the proteins secreted by each cell, categorized into effector, stimulatory, chemoattractant, regulatory, and inflammatory proteins (gray fill) (Figure 3B). Single-cell polyfunctionality heatmaps show the single-cell cytokine combinations secreted by each sample. Each column represents a specific combination of cytokines, while orange boxes represent the frequency with which that specific combination of cytokines was secreted by the corresponding sample (averages for target cells Mel624.38_PD-L1 and MelA375_PD-L1 are shown) (Figure 3C).

[0144] [Example 4] Increased frequency of secretion of effector, stimulatory, and chemoattractant proteins by TCR-T cells co-expressing the costimulatory receptor PD1-41BB. Secretion frequencies of single proteins induced by co-culture of TCR T cells with PD1-41BB cells and the target cell line Mel624.38_PD-L1. Proteins are displayed and categorized according to their a) effector, b) stimulatory, c) chemoattractant, d) regulatory, and e) inflammatory functions (Figure 4).

[0145] [Example 5] Serial killing of tumor cell spheroids. Tumor cell spheroids were generated starting with 1,000 NucLightRed-transduced cells in 96-well ULA plates 72 hours prior to coculture. TCR-transduced T cells with and without PD1-41BB were added (10,000–38,000 per well), and tumor cell killing was monitored by real-time imaging of the NucLightRed signal using an IncuCyte S3. Fresh tumor cell spheroids, generated as described above, were added every 72–96 hours (Figure 5).

[0146] The present application further includes the following items: Item 1. (A) Antigen-specific TCR, and (B) Chimeric costimulatory receptor A cell population comprising cells expressing comprising cells secreting at least two proteins, Cell population.

[0147] Item 2. Chimeric costimulatory receptors - an extracellular domain containing polypeptide derived from PD-1; - a transmembrane domain, and - an intracellular domain containing a polypeptide derived from 4-1BB; Including, the cell population comprises cells that secrete at least two proteins; The cell population described in item 2.

[0148] Item 3. Chimeric costimulatory receptors - an extracellular domain containing polypeptide derived from PD-1; - a transmembrane domain derived from PD-1, and - an intracellular domain containing a polypeptide derived from 4-1BB; Including, the cell population comprises cells that secrete at least two proteins; The cell population described in item 2.

[0149] Item 4. (A) NY-ESO-1 / LAGE-1-specific TCRs containing: - a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 35, a CDR2 having the amino acid sequence of SEQ ID NO: 36 and an amino acid sequence of SEQ ID NO: 37; and - a TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 38, a CDR2 having the amino acid sequence of SEQ ID NO: 39, and a CDR3 having the amino acid sequence of SEQ ID NO: 40; and (B) a chimeric costimulatory receptor comprising: - an extracellular domain containing polypeptide derived from PD-1; - a transmembrane domain, and - an intracellular domain containing a polypeptide derived from 4-1BB; comprising a cell expressing the cell population comprises cells that secrete at least two proteins; The cell population described in item 3.

[0150] Item 5. The cell population according to any one of Items 1 to 4, comprising cells that secrete at least three types of proteins.

[0151] Item 6. The cell population according to any one of Items 1 to 5, comprising cells that secrete at least four types of proteins.

[0152] Item 7. The cell population according to any one of Items 1 to 6, comprising cells that secrete at least five types of proteins.

[0153] Item 8. The cell population of any one of items 1 to 7, wherein the protein is selected from the group consisting of an effector protein, a stimulatory cytokine, and a chemoattractant cytokine.

[0154] Item 9. The cell population of item 8, wherein the effector protein is selected from the group consisting of Gzm-B, IFN-γ, perforin, TNF-α, TNF-β, and MIP-1α.

[0155] Item 10. The cell population of item 9, wherein the effector protein is selected from the group consisting of Gzm-B, IFN-γ, perforin, TNF-α, and TNF-β.

[0156] Item 11. The cell population according to Items 8 to 9, wherein the stimulatory cytokine is selected from the group consisting of GM-CSF, IL-2, IL-5, IL-7, IL-8, IL-9, and IL-12.

[0157] Item 12. The cell population according to items 8 to 11, wherein the stimulatory cytokine is selected from the group consisting of GM-CSF, IL-2, IL-7, IL-8, IL-9, and IL-12.

[0158] Item 13. The cell population according to Items 8 to 12, wherein the chemoattractant cytokine is selected from IP-10 and MIP-1β.

[0159] Item 14. The cell population of any one of items 1 to 13, wherein the protein is selected from the group consisting of Gzm-B, IFN-γ, perforin, TNF-α, TNF-β, MIP-1α, GM-CSF, IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IP-10, and MIP-1β.

[0160] Item 15. The cell population of any one of items 1 to 14, wherein the protein is selected from the group consisting of Gzm-B, IFN-γ, perforin, TNF-α, TNF-β GM-CSF, IL-2, IL-8, MIP-1β, and IP-10.

[0161] Item 16. The cell population according to any one of items 1 to 15, wherein the protein is selected from the group consisting of Gzm-B, IFN-γ, perforin, TNF-α, TNF-β GM-CSF, IL-2, and MIP-1β.

[0162] Item 17. The cell population of any one of items 1 to 16, wherein the protein is selected from the group consisting of IFN-γ, Gzm-B, and IP-10.

[0163] Item 18. The cell population of any one of items 1 to 17, wherein the protein is selected from the group consisting of IFN-γ and Gzm-B.

[0164] Item 19. The cell population according to any one of items 4 and 6 to 18, wherein the TCR is capable of binding to the NY-ESO peptide having the amino acid sequence set forth in SEQ ID NO: 34 or a portion thereof, preferably an HLA-A2 binding form thereof.

[0165] Item 20. The cell population according to Item 19, wherein HLA-A2 is a molecule encoded by HLA-A*02:01, HLA-A*02:02, HLA-A*02:04, or HLA-A*02:09, preferably a molecule encoded by HLA-A*2:01.

[0166] Item 21. The cell population according to any one of Items 3 to 20, wherein the extracellular domain containing the polypeptide derived from PD-1 comprises the sequence of SEQ ID NO: 28, and the intracellular domain containing the polypeptide derived from 4-1BB comprises the sequence of SEQ ID NO: 32.

[0167] Item 22. The cell population of any one of Items 3 to 21, wherein the transmembrane domain is derived from PD-1, preferably the transmembrane domain containing the polypeptide derived from PD-1 comprises the sequence of SEQ ID NO: 30, and preferably the chimeric costimulatory receptor comprises the sequence of SEQ ID NO: 26.

[0168] Item 23. The cell population of any one of items 1 to 22 for use in treating cancer.

[0169] Item 24. (A) Antigen-specific TCR, and (B) Chimeric costimulatory receptor a target-specific immune cell expressing secrete at least two proteins, Target-specific immune cells.

[0170] Item 25. Chimeric costimulatory receptors - an extracellular domain containing polypeptide derived from PD-1; - a transmembrane domain, and - an intracellular domain containing a polypeptide derived from 4-1BB; Including, Target-specific immune cells secrete at least two proteins, 25. The target-specific immune cell of item 24.

[0171] Item 26. Chimeric costimulatory receptors - an extracellular domain containing polypeptide derived from PD-1; - a transmembrane domain derived from PD-1, and - an intracellular domain containing a polypeptide derived from 4-1BB; Including, Target-specific immune cells secrete at least two proteins, 26. The target-specific immune cell of item 25.

[0172] Item 27. Target-specific immune cells (A) NY-ESO-1 / LAGE-1-specific TCR containing: - a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 35, a CDR2 having the amino acid sequence of SEQ ID NO: 36 and an amino acid sequence of SEQ ID NO: 37; and - a TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 38, a CDR2 having the amino acid sequence of SEQ ID NO: 39, and a CDR3 having the amino acid sequence of SEQ ID NO: 40; (B) a chimeric costimulatory receptor comprising: - an extracellular domain containing polypeptide derived from PD-1; - a transmembrane domain, and - an intracellular domain containing a polypeptide derived from 4-1BB; Expressing secrete at least two proteins, 27. The target-specific immune cell according to any one of items 24 to 26.

[0173] Item 28. The target-specific immune cell according to any one of Items 24 to 27, which secretes at least three types of proteins.

[0174] Item 29. The target-specific immune cell according to any one of Items 24 to 28, which secretes at least four types of proteins.

[0175] Item 30. The target-specific immune cell according to any one of Items 24 to 29, which secretes at least five types of proteins.

[0176] Item 31. The target-specific immune cell according to any one of items 24 to 30, wherein the protein is selected from the group consisting of an effector protein, a stimulatory cytokine, and a chemoattractant cytokine.

[0177] Item 32. The target-specific immune cell according to Item 31, wherein the effector protein is selected from the group consisting of Gzm-B, IFN-γ, perforin, TNF-α, TNF-β, and MIP-1α.

[0178] Item 33. The target-specific immune cell according to Item 32, wherein the effector protein is selected from the group consisting of Gzm-B, IFN-γ, perforin, TNF-α, and TNF-β.

[0179] Item 34. The target-specific immune cell according to any one of items 31 to 33, wherein the stimulatory cytokine is selected from the group consisting of GM-CSF, IL-2, IL-5, IL-7, IL-8, IL-9, and IL-12.

[0180] Item 35. The target-specific immune cell according to any one of items 31 to 34, wherein the stimulatory cytokine is selected from the group consisting of GM-CSF, IL-2, IL-7, IL-8, IL-9, and IL-12.

[0181] Item 36. The target-specific immune cell according to any one of Items 31 to 35, wherein the chemoattractant cytokine is selected from IP-10 and MIP-1β.

[0182] Item 37. The target-specific immune cell of any one of items 24 to 36, wherein the protein is selected from the group consisting of Gzm-B, IFN-γ, perforin, TNF-α, TNF-β, MIP-1α, GM-CSF, IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IP-10, and MIP-1β.

[0183] Item 38. The target-specific immune cell according to any one of items 24 to 37, wherein the protein is selected from the group consisting of Gzm-B, IFN-γ, perforin, TNF-α, TNF-β GM-CSF, IL-2, IL-8, MIP-1β, and IP-10.

[0184] Item 39. The cell population according to any one of Items 24 to 38, wherein the protein is selected from the group consisting of Gzm-B, IFN-γ, perforin, TNF-α, TNF-β, GM-CSF, IL-2, and MIP-1β.

[0185] Item 40. The target-specific immune cell according to any one of items 24 to 39, wherein the protein is selected from the group consisting of IFN-γ, Gzm-B, and IP-10.

[0186] Item 41. The target-specific immune cell according to any one of items 24 to 40, wherein the protein is selected from the group consisting of IFN-γ and Gzm-B.

[0187] Item 42. The target-specific immune cell according to any one of items 27 and 28 to 41, wherein the TCR is capable of binding to the NY-ESO peptide having the amino acid sequence set forth in SEQ ID NO: 34 or a portion thereof, preferably an HLA-A2-binding form thereof.

[0188] Item 43. The target-specific immune cell according to Item 42, wherein HLA-A2 is a molecule encoded by HLA-A*02:01, HLA-A*02:02, HLA-A*02:04, or HLA-A*02:09.

[0189] Item 44. The target-specific immune cell according to Item 43, wherein HLA-A2 is a molecule encoded by HLA-A*2:01.

[0190] Item 45. The target-specific immune cell according to any one of Items 25 to 43, wherein the extracellular domain containing the polypeptide derived from PD-1 comprises the sequence of SEQ ID NO: 28, and the intracellular domain containing the polypeptide derived from 4-1BB comprises the sequence of SEQ ID NO: 32.

[0191] Item 46. The target-specific immune cell according to any one of Items 26 to 45, wherein the transmembrane domain is derived from PD-1, preferably the transmembrane domain containing the polypeptide derived from PD-1 comprises the sequence of SEQ ID NO: 30, and preferably the chimeric costimulatory receptor comprises the sequence of SEQ ID NO: 26.

[0192] Item 47. The target-specific immune cell according to any one of items 24 to 46, which is a lymphocyte.

[0193] Item 48. The target-specific immune cell according to any one of items 24 to 47 for use in treating cancer.

Claims

1. (A) NY-ESO-1 / LAGE-1 specific TCR including the following - A TCRα chain comprising CDR1 having the amino acid sequence of SEQ ID NO: 35, CDR2 having the amino acid sequence of SEQ ID NO: 36, and CDR3 having the amino acid sequence of SEQ ID NO: 37; and - A TCRβ chain comprising CDR1 having the amino acid sequence of SEQ ID NO: 38, CDR2 having the amino acid sequence of SEQ ID NO: 39, and CDR3 having the amino acid sequence of SEQ ID NO: 40; (B) Chimeric costimulatory receptors including the following - Extracellular domain containing polypeptide derived from PD-1, - Transmembrane domain, and - Intracellular domain containing polypeptide derived from 4-1BB Target-specific immune cells that express [specific expression].

2. The aforementioned chimeric costimulatory receptor is - Extracellular domain containing an extracellular domain derived from PD-1, - Transmembrane domains derived from PD-1, and - Intracellular domain containing an intracellular domain derived from 4-1BB A target-specific immune cell according to claim 1, comprising:

3. The target-specific immune cell according to claim 2, wherein the extracellular domain derived from PD-1 comprises an amino acid sequence having up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to the extracellular domain derived from PD-1 presented in SEQ ID NO:

28.

4. The target-specific immune cell according to claim 1, wherein the intracellular domain derived from 4-1BB includes an amino acid sequence having up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to the intracellular domain derived from 4-1BB presented in SEQ ID NO:

32.

5. The target-specific immune cell according to claim 1, wherein the extracellular domain derived from PD-1 comprises the sequence of SEQ ID NO: 28, and the intracellular domain derived from 4-1BB comprises the sequence of SEQ ID NO:

32.

6. The target-specific immune cell according to claim 2, wherein the transmembrane domain derived from PD-1 comprises an amino acid sequence having up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to the transmembrane sequence derived from PD-1 presented in SEQ ID NO:

30.

7. The target-specific immune cell according to claim 6, wherein the transmembrane domain is derived from PD-1, and preferably contains a polypeptide derived from PD-1, and the transmembrane domain includes the amino acid sequence of SEQ ID NO:

30.

8. Preferably, the target-specific immune cell according to claim 1, wherein the chimeric costimulatory receptor comprises the amino acid sequence of SEQ ID NO:

26.

9. A target-specific immune cell according to claim 1, which is a lymphocyte.

10. A target-specific immune cell according to claim 1, which is a T cell.

11. The target-specific immune cell according to claim 1, wherein the TCR comprises a variable TCRα region having an amino acid sequence that is at least 80% identical to SEQ ID NO: 41, and a variable TCRβ region having an amino acid sequence that is at least 80% identical to SEQ ID NO:

42.

12. The target-specific immune cell according to claim 1, wherein the TCR comprises a variable TCRα region having the amino acid sequence of SEQ ID NO: 41 and a variable TCRβ region having the amino acid sequence of SEQ ID NO:

42.

13. The target-specific immune cell according to claim 1, wherein the TCR can bind to an NY-ESO peptide having the amino acid sequence presented in SEQ ID NO: 34 or a portion thereof, preferably in its HLA-A2 binding form.

14. The target-specific immune cell according to claim 1, wherein the HLA-A2 is a molecule encoded by HLA-A*02:01, HLA-A*02:02, HLA-A*02:04, or HLA-A*02:09, preferably a molecule encoded by HLA-A*2:

01.

15. A cell population comprising target-specific immune cells according to claims 1 to 14.

16. The cell population according to claim 15, comprising cells that secrete at least two types of proteins.

17. The cell population according to claim 16, comprising cells that secrete at least three types of proteins.

18. The cell population according to claim 16, comprising cells that secrete at least six types of proteins.

19. The cell population according to claim 16, wherein the protein is selected from the group consisting of effector proteins, stimulating cytokines, and chemoattracting cytokines.

20. The cell population according to claim 16, wherein at least 5% of the population secretes two or more effector proteins.

21. The cell population according to claim 16, wherein at least 4% of the population secretes two or more selected from effector proteins, stimulating cytokines, and chemoattracting cytokines.

22. The cell population according to claim 16, wherein the cells secrete at least one of the proteins selected from RANTES, MIP-1 alpha, perforin, TNF-alpha, and TNF-beta.

23. The cell population according to claim 19, wherein the effector protein is selected from the group consisting of Gzm-B, IFN-γ, perforin, TNF-α, TNF-β, and MIP-1α.

24. The cell population according to claim 19, wherein the effector protein is selected from the group consisting of Gzm-B, IFN-γ, perforin, TNF-α, and TNF-β.

25. The cell population according to claim 19, wherein the stimulating cytokine is selected from the group consisting of GM-CSF, IL-2, IL-5, IL-7, IL-8, IL-9, and IL-12.

26. The cell population according to claim 19, wherein the stimulating cytokine is selected from the group consisting of GM-CSF, IL-2, IL-7, IL-8, IL-9, and IL-12.

27. The cell population according to claim 19, wherein the chemoattractant cytokine is selected from IP-10 and MIP-1β.

28. The cell population according to claim 16, wherein the protein is selected from the group consisting of Gzm-B, IFN-γ, perforin, TNF-α, TNF-β, MIP-1α, GM-CSF, IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IP-10, and MIP-1β.

29. The cell population according to claim 16, wherein the protein is selected from the group consisting of Gzm-B, IFN-γ, perforin, TNF-α, TNF-β, GM-CSF, IL-2, IL-8, MIP-1β, and IP-10.

30. The cell population according to claim 16, wherein the protein is selected from the group consisting of Gzm-B, IFN-γ, perforin, TNF-α, TNF-β, GM-CSF, IL-2, and MIP-1β.

31. The cell population according to claim 16, wherein the protein is selected from the group consisting of IFN-γ, Gzm-B, and IP-10.

32. The cell population according to claim 16, wherein the protein is selected from the group consisting of IFN-γ and Gzm-B.

33. A pharmaceutical composition comprising the target-specific immune cells as defined in claims 1 to 14.

34. The pharmaceutical composition according to claim 33 for treating cancer in humans or non-human animals.

35. The pharmaceutical composition according to claim 34, wherein the cancer is a solid tumor.

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