Combination of ny-ESO-1 specific t cell receptors and chimeric

Abstract

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

[0002] NY-ESO-1 and LAGE-1 are important immunotherapy target antigens belonging to the cancer / testis antigen family. Cancer / testis antigens are expressed in a variety of malignancies and testicular germ cells, but not in other adult tissues.

[0003] The success of immunotherapy using TCR-modified T cells depends not only on the choice of target antigen, but also on the selection of TCRs with high antigen specificity and sensitivity. Another challenge, especially in the treatment of solid tumors, is the immunosuppressive tumor microenvironment (TME), which negatively affects the efficacy, adaptability, and persistence of TCR-modified T cells. In addition to inhibitory cytokines and lack of essential metabolic factors, T cells also face the inhibitory checkpoint PD-1 / PD-L1 axis in the TME, which reduces T cell infiltration and leads to its exhaustion. Therefore, new strategies are needed to give TCR-modified T cells the properties to overcome the inhibitory immunosuppressive TME. More specifically, TCR-modified T cells with high specificity and enhanced proliferation, cytokine release, and cytotoxicity that target specific antigens (e.g., NY-ESO-1) are needed. Summary of the invention

[0004] To meet these needs, the present invention provides a combination of high-affinity TCR and chimeric co-stimulatory receptors that allow the generation of highly specific T cells with enhanced cytokine release, proliferation and cytotoxicity targeting antigens (e.g., NY-ESO-1), particularly multifunctional immune cells that secrete two or more cytokines.

[0005] The present invention relates to a target-specific immune cell, which expresses

[0006] (A) antigen-specific TCR, and

[0007] (B) Chimeric co-stimulatory receptors,

[0008] Wherein the target-specific immune cells secrete at least two proteins.

[0009] Therefore, one embodiment of the present invention provides a cell comprising

[0010] (A) NY-ESO-1 / LAGE-1 specific TCR, comprising

[0011] - a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 35, a CDR 2 having the amino acid sequence of SEQ ID NO: 36, and a CDR 3 having the sequence of SEQ ID NO: 37; and

[0012] - A TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO:38, a CDR 2 having the amino acid sequence of SEQ ID NO:39, and a CDR 3 having the sequence of SEQ ID NO:40.

[0013] (B) a chimeric co-stimulatory receptor, the chimeric co-stimulatory receptor comprising

[0014] - containing an extracellular domain of a polypeptide derived from PD-1,

[0015] - transmembrane domain, and

[0016] - an intracellular domain comprising a polypeptide derived from 4-1BB,

[0017] The NY-ESO-1 specific TCR used is capable of binding to a NY-ESO-1 peptide having the amino acid sequence SLLMWITQC (SEQ ID NO: 34) or a portion thereof, or an HLA-A2 bound form thereof. It provides high functional affinity and favorable tumor cell recognition and killing properties. The co-stimulatory receptor reverses the inhibitory checkpoint axis PD-1 / PD-L1 to improve T cell functionality, particularly in an inhibitory TME. Thus, the combination of the TCR of the present invention and the chimeric co-stimulatory receptor allows for improved targeting of NY-ESO-1 with high specificity and enhanced proliferation, cytokine release and cytotoxicity.

[0018] The chimeric costimulatory receptor may comprise a transmembrane domain derived from PD-1. In certain embodiments, the sequence of the chimeric costimulatory receptor may comprise the sequence of SEQ ID NO:26.

[0019] Therefore, another aspect relates to a composition comprising

[0020] - a nucleic acid encoding a NY-ESO-1 specific T cell receptor (TCR), the TCR comprising

[0021] - 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

[0022] - 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

[0023] - a nucleic acid encoding a chimeric co-stimulatory receptor comprising

[0024] - containing an extracellular domain of a polypeptide derived from PD-1,

[0025] - transmembrane domain, and

[0026] - containing an intracellular domain of a polypeptide derived from 4-1BB.

[0027] Furthermore, one aspect relates to a nucleic acid comprising

[0028] - a nucleic acid encoding a NY-ESO-1 specific T cell receptor (TCR), the TCR comprising

[0029] - 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

[0030] - 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

[0031] - a nucleic acid encoding a chimeric co-stimulatory receptor comprising

[0032] - containing an extracellular domain of a polypeptide derived from PD-1,

[0033] - transmembrane domain, and

[0034] - containing an intracellular domain of a polypeptide derived from 4-1BB.

[0035] Another aspect relates to a vector comprising a nucleic acid comprising sequences of a NY-ESO-1 specific TCR and a chimeric co-stimulatory receptor, and also includes a cell comprising the nucleic acid composition and / or the vector.

[0036] Typically, the cell is a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). In certain embodiments, the cell is a T cell.

[0037] Multifunctional immune cells

[0038] One object of the present invention relates to a cell population comprising cells expressing:

[0039] (A) Antigen-specific TCR

[0040] (B) Chimeric co-stimulatory receptor

[0041] wherein the cell population comprises cells that secrete at least two proteins.

[0042] In some embodiments, the chimeric costimulatory receptor comprises

[0043] - containing an extracellular domain of a polypeptide derived from PD-1,

[0044] - A transmembrane domain derived from PD-1, and

[0045] - an intracellular domain comprising a polypeptide derived from 4-1BB,

[0046] wherein the cell population comprises cells that secrete at least two proteins.

[0047] Another aspect of the present invention relates to a target-specific immune cell expressing

[0048] (A) antigen-specific TCR, and

[0049] (B) a chimeric co-stimulatory receptor as described herein,

[0050] Among them, target-specific immune cells secrete at least two proteins.

[0051] In some embodiments, the cells secrete at least three proteins, such as at least four or at least five proteins.

[0052] Other aspects relate to a pharmaceutical composition comprising the cells, compositions, nucleic acids and vectors defined herein. Other aspects relate to the cells, compositions, nucleic acids and vectors defined herein for use in cancer treatment.

[0053] Surprisingly, the inventors found that TCR-T cells expressing chimeric co-stimulatory receptors as defined herein exhibited higher polyfunctionality than TCR-T cells lacking chimeric co-stimulatory receptors as defined herein. The higher polyfunctionality of transgenic T cells refers to higher in vivo functionality and anti-tumor activity, and is associated with clinical outcomes.

[0054] In particular, we could show that the cell population expressing both the target-specific NY-ESO-2-specific TCR and the chimeric co-stimulatory receptor defined herein comprises cells secreting at least two proteins, such as at least three proteins, at least four proteins, at least five proteins, at least six proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1: TCR-T cells targeting NY-ESO-1 expressing PD1-41BB exhibited higher polyfunctionality compared to TCR-T cells targeting NY-ESO-1 lacking PD1-41BB. The single-cell polyfunctionality (release of 2 or more cytokines) of TCR-transgenic T cells with or without PD1-41BB was analyzed using the IsoPlexis technology. Secretion of 32 T cell cytokines / proteins by single CD8+ T cells was assessed after co-culture with PD-L1-overexpressing, NY-ESO-1-positive MelA375 and Mel624.38 tumor cells and compared with non-transduced TCR-T cells.

[0056] (A) TCR-T cells expressing PD1-41BB show a higher percentage of polyfunctional T cells compared to TCR-T cells lacking PD1-41BB. Different shades of gray indicate how many cytokines are released simultaneously by a single T cell.

[0057] (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 showed a higher polyfunctional strength index (PSI) compared with TCR-T cells lacking PD1-41BB. The various cytokines / lytic proteins released were classified and it was found that 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 had a large contribution to the excellent PSI, followed by chemo-attractive (IP-10, MIP-1β, RANTES) cytokines. Regulatory (IL-4, IL-10, IL-22, sCD137, TGF-β1) and inflammatory (IL-6, IL-17F, MCP-1) cytokines were released to a lower extent.

[0058] (C) Detailed analysis of single cell multi-cytokine release using multifunctional heatmaps shows that the multi-cytokine signatures of TCR-T cells with and without PD1-41BB are different. Notably, TCR-T cells expressing PD1-41BB contain a higher proportion of single cells that simultaneously secrete 2-6 cytokines.

[0059] Figure 2: Improved effector function of NY-ESO-1TCR-T cells co-expressing a chimeric PD1-41BB co-stimulatory receptor. (A) Flow cytometric analysis of T cells that were not transduced (UT), NY-ESO-1TCR expressing (TCR), and NY-ESO-1TCR co-expressing PD1-41BB (TCR+PD1-41BB). (B) NY-ESO-1TCR 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, **p>0.01, ***p<0.001). (C) The expression levels of NY-ESO-1 were measured by qPCR and quantified according to a standard calibration curve. The PD-L1 levels summarized here were assessed by qPCR and FACS analysis. (D) NY-ESO-1 TCR transgenic CD8+ T cells with or without PD1-41BB were stained with 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.

[0060] Figure 3: Multifunctionality of NY-ESO-1 TCR-T cells enhanced by co-expression of chimeric co-stimulatory receptor PD1-41BB. After 24 hours of co-culture with tumor target cell lines Mel624.38_PD-L1 and MelA375_PD-L1, single-cell proteomic analysis of a panel of 32 secreted cytokines, chemokines, and cytotoxic molecules (IsoLight technology, IsoPlexis) was used to analyze NY-ESO-1-specific TCR-T cells with (TCR+PD1-41BB) and without (TCR) co-expression of chimeric co-stimulatory receptor PD1-41BB. Co-culture with untransduced T cells (UT) from the same donor served as a control. Cells that secrete 2 or more cytokines are considered multifunctional. (A) The multifunctionality of single TCR T cells is shown as a percentage of the sample and is classified by the percentage of cells that simultaneously express 2, 3, 4, and 5+ analytes (orange shading). (B) The polyfunctional intensity index (PSI) of the samples shown is defined as the number of T cells (polyfunctional T cells in a) that secrete more than 2 effector molecules per cell, multiplied by the mean fluorescence intensity (MFI) of the proteins secreted by the respective cells, and categorized by proteins associated with effector, stimulatory, chemoattractive, regulatory, and inflammatory properties (grey shading). (C) Single cell polyfunctionality heat map shows the single cell cytokine combination secreted by each sample. Each column represents a specific cytokine combination, and the orange squares describe the frequency of secretion of a specific cytokine combination by the corresponding sample (average values ​​of target cells Mel624.38_PD-L1 and MelA375_PD-L1 are shown). Cytokine groups are sorted by polyfunctionality, and only the 40 most functional groups present in the sample are shown.

[0061] Figure 4 : TCR-T cells co-expressing the co-stimulatory receptor PD1-41BB secrete an increased frequency of effector, irritant and chemoattractant proteins. The secretion frequency of single proteins induced by co-culture of TCR T cells equipped with PD1-41BB cells and the target cell line Mel624.38_PD-L1. The proteins are shown and classified by their a) effector, b) irritant, c) chemoattractant, d) regulatory and e) inflammatory properties.

[0062] Figure 5: Serial killing of tumor cell spheroids. Tumor cell spheroids were generated starting from 1000 NucLightRed-transduced cells in 96-well ULA plates 72 hours before 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 NucLightRed signal using IncuCyte S3. Fresh tumor cell spheroids generated as described above were added every 72–96 hours. DETAILED DESCRIPTION

[0063] Before describing the present invention in detail with respect to some preferred embodiments thereof, the following general definitions are provided.

[0064] The present invention as illustratively described hereinafter suitably may be practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein.

[0065] 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.

[0066] When the term "comprising" is used in the present description and claims, it does not exclude other elements. For the purpose of the present invention, the term "consisting of" is considered a preferred embodiment of the term "comprising..." If hereinafter a group is defined as comprising at least a certain number of embodiments, this is also to be understood as disclosing a group that preferably consists of only these embodiments.

[0067] For the purposes of the present invention, the term “obtained” is considered to be a preferred embodiment of the term “obtainable.” If, for example, an antibody is defined below as being obtainable from a particular source, this is also to be understood as disclosing the antibody obtained from this source.

[0068] When an indefinite or definite article is used when referring to a singular noun, for example "a", "an" or "the", this includes the plural of the noun unless expressly stated otherwise. In the context of the present invention, the term "about" or "approximately" indicates an interval of accuracy that a person skilled in the art will understand still ensures the technical effect of the feature in question. The term generally indicates a deviation of ±10%, preferably ±5%, from the indicated numerical value.

[0069] Technical terms are used with their common sense or meaning to those skilled in the art. If certain terms are given specific meanings, the definitions of the terms will be given below based on the context in which they are used.

[0070] TCR Background

[0071] TCR is composed of two different and independent protein chains, namely TCR alpha (α) and TCR beta (β) chains. The TCR α chain contains variable (V), joining (J) and constant (C) regions. The TCR β chain contains variable (V), diversity (D), joining (J) and constant (C) regions. The rearranged V (D) J regions of both TCR α and TCR β chains contain hypervariable regions (CDR, complementary determining regions), of which the CDR3 region determines specific epitope recognition. Both TCR α and TCR β chains contain a hydrophobic transmembrane domain in the C-terminal region and end with a short cytoplasmic tail.

[0072] Typically, TCR is a heterodimer of one α chain and one β chain that can bind to MHC molecules presenting peptides.

[0073] The term "variable TCR alpha region" or "TCR alpha variable chain" or "variable domain" in the context of the present invention refers to the variable region of the TCR alpha chain. The term "variable TCR beta region" or "TCR beta variable chain" in the context of the present invention refers to the variable region of the TCR beta chain.

[0074] TCR loci and genes are assigned using the International Immunogenetics (IMGT) TCR nomenclature (IMGT Database, www.IMGT.org; Giudicelli, V., et al. IMGT / LIGM-DB, the 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) naming.

[0075] Target

[0076] The term "epitope" generally refers to a site on an antigen (usually a (poly)peptide) that is recognized by a binding domain. The term "binding domain" refers to an "antigen binding site" in its broadest sense, i.e. a domain that characterizes a molecule that binds / interacts with a specific epitope on an antigen target. An antigen target may contain a single epitope, but generally contains at least two epitopes, and may contain any number of epitopes depending on the size, conformation, and type of the antigen. The term "epitope" generally includes linear epitopes and conformational epitopes. A linear epitope is a continuous epitope contained in the primary sequence of amino acids, generally containing at least 2 or more amino acids. A conformational epitope is formed by non-continuous amino acids juxtaposed by the folding of a target antigen (particularly a target (poly)peptide).

[0077] Other embodiments relate to a cell comprising

[0078] (A) NY-ESO1-specific T cell receptor (TCR), comprising

[0079] - 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

[0080] 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

[0081] and

[0082] (B) a chimeric co-stimulatory receptor, the chimeric co-stimulatory receptor comprising

[0083] - Contains the extracellular domain of a polypeptide derived from PD-1

[0084] - transmembrane domain, and

[0085] - an intracellular domain comprising a polypeptide derived from 4-1BB,

[0086] In which cells secrete more than two proteins.

[0087] TCR-specific sequences

[0088] As used herein, "at least 80% identical", particularly "having an amino acid sequence at least 80% identical to" 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 listed amino acid sequences.

[0089] The percent identity between multiple sequences is preferably determined using Vector NTI Advance TM 10 program (Invitrogen Corporation, Carlsbad CA, USA). This program uses a modified Clustal W algorithm (Thompson et al., 1994. Nucl Acids Res. 22: pp. 4673-4680; Invitrogen Corporation; Vector NTI Advance TM 10DNA and protein sequence analysis software. User's Manual, 2004, pp. 389-662). The percent identity was determined using the standard parameters of the AlignX application.

[0090] The TCRs described herein are specific for NY-ESO-1, in particular the NY-ESO-1 epitope SLLMWITQC (SEQ ID NO: 34), and exhibit only very low cross-reactivity with other epitopes or antigens.

[0091] Thus, exemplary TCRs for use in the combinations of the invention include

[0092] - 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

[0093] - 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.

[0094] In some embodiments, the TCR comprises a variable TCR alpha region having an amino acid sequence at least 80% identical to SEQ ID NO:41 and a variable TCR beta region having an amino acid sequence at least 80% identical to SEQ ID NO:42.

[0095] 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.

[0096] In some embodiments, the TCR may comprise a TCR alpha chain having an amino acid sequence that is identical or at least 80% identical to SEQ ID NO:43 and a TCR beta chain having an amino acid sequence that is identical or at least 80% identical to SEQ ID NO:44.

[0097] 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.

[0098] Modification

[0099] In some embodiments, the amino acid sequence of the TCR and / or chimeric co-stimulatory receptor may contain one or more phenotypically silent substitutions.

[0100] "Phenotypic silent substitutions" are also referred to as "conservative amino acid substitutions". Those skilled in the art understand the concept of "conservative amino acid substitutions", preferably meaning that codons encoding positively charged residues (H, K and R) are replaced by codons encoding positively charged residues, codons encoding negatively charged residues (D and E) are replaced by codons encoding negatively charged residues, codons encoding neutral polar residues (C, G, N, Q, S, T and Y) are replaced by codons encoding neutral polar residues, and codons encoding neutral non-polar residues (A, F, I, L, M, P, V and W) are replaced by codons encoding neutral non-polar residues. These variations can occur spontaneously, introduced by random mutagenesis, or can be introduced by directed mutagenesis. These changes can be made without destroying the basic characteristics of these polypeptides. Those of ordinary skill can easily and routinely screen variant amino acids and / or nucleic acids encoding them to determine whether these variations significantly reduce or destroy ligand binding ability by methods known in the art.

[0101] The skilled person understands that the nucleic acid encoding TCR and / or chimeric costimulatory receptor can also be modified. Useful modifications in the entire nucleic acid sequence include codon optimization of the sequence. Conservative substitution changes in the amino acid sequence that lead to its expression can be made. These changes can be made in the complementary determining regions and non-complementary determining regions of the amino acid sequence of the TCR chain that do not affect function. Generally, additions and deletions should not be made in the CDR3 region.

[0102] According to some embodiments of the invention, the amino acid sequence of the TCR and / or chimeric co-stimulatory receptor is modified to include a detectable label, a therapeutic agent, or a pharmacokinetic modifying moiety.

[0103] 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, immunomodulators, enzymes or chemotherapeutic agents. The therapeutic agent can be encapsulated by liposomes connected to TCR so that the compound can be slowly released at the target site. This will avoid damage during transport in the body and ensure that the therapeutic agent (e.g., toxin) plays the greatest role after TCR binds to the relevant antigen presenting cells. Other examples of therapeutic agents are:

[0104] Peptide cytotoxins, i.e., proteins or peptides that have the ability to kill mammalian cells, such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNase and RNase. Small molecule cytotoxic agents, i.e., compounds that have the ability to kill mammalian cells and have a molecular weight of less than 700 Daltons. Such compounds may contain toxic metals that can have cytotoxic effects. In addition, it should be understood that these small molecule cytotoxic agents also include prodrugs, i.e., compounds that decay or transform under physiological conditions to release cytotoxic agents. Such agents may include, for example, docetaxel, gemcitabine, cisplatin, maytansine derivatives, rachelmycin, calicheamicin, etoposide, ifosfamide, irinotecan, porfimer sodium photofrin II, temozolomide, topotecan, trimetrexate glucoronate, mitoxantrone, auristatin E, vincristine, and doxorubicin; radionuclides, such as iodine 131, rhenium 186, indium 111, yttrium 90, bismuth 210 and 213, actinium 225, and astatine 213. The association of radionuclides with TCR or its derivatives may be carried out, for example, by a chelating agent; immunostimulants, also known as immunostimulants, are immune effector molecules that stimulate immune responses. Exemplary immunostimulants are cytokines, such as IL-2 and IFN-γ, antibodies or fragments thereof, including 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 their mutants that cause non-specific activation of T cells, leading to polyclonal T cell activation and massive cytokine release; chemokines, such as IL-8, platelet factor 4, melanoma growth stimulating protein, etc.; complement activators; heterologous protein domains, allogeneic protein domains, viral / bacterial protein domains, viral / bacterial peptides.

[0105] The therapeutic agent may preferably be selected from the group consisting of an immune effector molecule, a cytotoxic agent and a radionuclide. Preferably, the immune effector molecule is a cytokine.

[0106] The pharmacokinetic modifying moiety can be, for example, at least one polyethylene glycol repeating unit, at least one diol group, at least one sialic acid group or a combination thereof. The association of at least one polyethylene glycol repeating unit, at least one diol group, at least one sialic acid group can be achieved in a variety of ways known to those skilled in the art. In a preferred embodiment, these units are 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 characteristics of the therapeutic agent, such as improving plasma half-life, reducing or enhancing immunogenicity and improving solubility.

[0107] TCRs and / or chimeric co-stimulatory receptors can be modified by attaching additional functional moieties, e.g., to reduce immunogenicity, increase hydrodynamic size (size in solution) solubility and / or stability (e.g., by enhancing protection from proteolytic degradation) and / or extend serum half-life.

[0108] Other useful functional parts and modifications include "suicide" or "safety switches", which can be used to turn off effector host cells carrying the TCR of the present invention in patients. An example is the inducible caspase 9 (iCasp9) "safety switch" described by Gargett and Brown Front Pharmacol.2014; 5:235. In short, the effector host cells are modified by well-known methods to express the caspase 9 domain, the dimerization of which depends on small molecule dimerization drugs (e.g., AP1903 / CIP), and lead to the rapid induction of apoptosis in modified effector cells. The system is described, for example, in EP2173869 (A2). Other examples of "suicide" "safety switches" are known in the art, such as herpes simplex virus thymidine kinase (HSV-TK), expression of CD20 and subsequent consumption of anti-CD20 antibodies or myc tags (Kieback et al., Proc Natl Acad Sci US A.2008 Jan 15; 105 (2): 623-8).

[0109] 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. The glycosylation of a polypeptide is typically N-connected or O-connected. N-connection refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. By changing the amino acid sequence so that it comprises one or more tripeptide sequences selected from asparagine-X-serine and asparagine-X-threonine (wherein any amino acid except proline), it is possible to conveniently achieve the addition of N-connected glycosylation sites to a binding molecule. O-connected glycosylation sites may be introduced by adding or replacing one or more serine or threonine residues in the starting sequence.

[0110] Another method for TCR glycosylation is to chemically or enzymatically couple glycosides to the protein. Depending on the coupling mode used, sugars can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as those of cysteine, (d) free hydroxyl groups, such as those of serine, threonine or hydroxyproline, (e) aromatic residues, such as those of 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 accomplished chemically (e.g., by exposing the TCR to trifluoromethanesulfonic acid) or enzymatically by using endo- and exo-glycosidases.

[0111] It is also conceivable to add drugs (e.g., small molecule compounds) to TCRs, particularly soluble forms of the TCRs of the present invention. Connection can be achieved by covalent bonds or non-covalent interactions (e.g., by electrostatic forces). Various linkers known in the art can be used to form drug conjugates.

[0112] TCR, in particular the soluble form of the TCR of the present invention, may be additionally modified to introduce additional domains that facilitate the identification, tracking, purification and / or isolation of the corresponding molecule (tag). Thus, in some embodiments, the TCR alpha chain or TCR beta chain may be modified to include an epitope tag.

[0113] Epitope tags are useful examples of tags that can be incorporated into the TCR of the present invention. Epitope tags are short stretches of amino acids that allow binding to specific antibodies, thereby enabling identification and tracking of binding and movement of soluble TCRs or host cells in patients or cultured (host) cells. Detection of epitope tags and thus labeled TCRs can be achieved using a variety of different techniques.

[0114] The tag can also be used to stimulate and expand host cells carrying the TCR of the invention by culturing the cells in the presence of a binding molecule (antibody) specific for the tag.

[0115] In general, in some examples, a TCR can be modified by various mutations that change the affinity and dissociation rate of the TCR to the target antigen. In particular, the mutation can increase the affinity and / or reduce the dissociation rate. Thus, a TCR can be mutated in at least one CDR and its variable domain framework region.

[0116] However, in preferred embodiments, the CDRs of the TCR are not modified or affinity matured in vitro, such as for the TCRs in the examples. This means that the CDRs have naturally occurring sequences. This may be advantageous because affinity maturation in vitro may lead to immunogenicity of the TCR molecule. This may lead to the production of anti-drug antibodies, thereby reducing or inhibiting the therapeutic effect and treatment and / or causing adverse reactions.

[0117] The mutations may be one or more substitutions, deletions or insertions. These mutations may be introduced by any suitable method known in the art, such as polymerase chain reaction, restriction enzyme-based cloning, ligation-independent cloning procedures, such as described in Sambrook, Molecular Cloning—4 th Edition (2012) Cold Spring Harbor Laboratory Press.

[0118] In theory, unpredictable TCR specificity with a risk of cross-reactivity may occur due to mispairing between endogenous and exogenous TCR chains. In order to avoid TCR sequence mispairing, the recombinant TCR sequence can be modified to include murinized or minimally murinized Cα and Cβ regions, a technique that has been shown to effectively enhance the correct pairing of multiple different transduced TCR chains. Murinization of TCRs (i.e., exchanging human Cα and Cβ regions with their murine counterparts) is a technique commonly used to improve cell surface expression of TCRs in host cells. Without wishing to be bound by a particular theory, it is believed that murinized TCRs associate more effectively with CD3 co-receptors; and / or preferentially pair with each other, and are less likely to form hybrid TCRs on human T cells that have been genetically modified in vitro to express TCRs with the desired antigen specificity, but still retain and express their "original" TCRs.

[0119] Nine amino acids responsible for improving the 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 amino acid residues in the constant region of the TCR α and / or β chain are substituted with their mouse counterpart residues. This technology is also referred to as "ultimate mouse humanization" and provides the advantage of enhancing cell surface expression while reducing the number of "foreign" amino acid residues in the amino acid sequence, thereby reducing the risk of immunogenicity.

[0120] Some embodiments relate to the isolated TCR described herein, wherein the TCR is of a single chain type, wherein the TCR alpha chain and the TCR beta chain are connected by a linker sequence.

[0121] Suitable single-chain TCR formats include a first fragment consisting of an amino acid sequence corresponding to the variable TCR α region, a second fragment consisting 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 extracellular sequence of the constant region of the TCR β chain, and a linker sequence connecting the C-terminus of the first fragment to the N-terminus of the second fragment. Alternatively, the first fragment may be composed of an amino acid sequence corresponding to the variable region of the TCR β chain, and the second fragment may be composed of an amino acid sequence corresponding to the variable region sequence of the TCR α chain fused to the N-terminus of an amino acid sequence corresponding to the extracellular sequence of the constant region of the TCR α chain. The above-mentioned single-chain TCR may also include a disulfide bond between the first chain and the second chain, wherein the length of the linker sequence and the position of the disulfide bond make the variable domain sequences of the first and second fragments substantially consistent with the mutual orientation in the natural T cell receptor. More specifically, the first fragment 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 an extracellular sequence of a TCR α chain constant region, the second fragment 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 an extracellular sequence of a TCR β chain constant region, and a disulfide bond may be provided between the first chain and the second chain. The linker sequence may be any sequence that does not impair the function of the TCR.

[0122] In the context of the present invention, a "functional" TCR alpha and / or beta chain fusion protein shall refer to a TCR or a TCR variant (modified, for example, by the addition, deletion or substitution of amino acids) that retains at least substantial biological activity. In the case of the alpha and / or beta chains of a TCR, this shall mean that the two chains are still able to form a TCR (with unmodified alpha and / or beta chains or with another fusion protein alpha and / or beta chain of the present invention) that exerts its biological function, in particular binding to a specific peptide-MHC complex of the TCR, and / or functional signal transduction after a specific peptide:MHC interaction.

[0123] In a specific embodiment, TCR can be modified to a functional TCR alpha and / or beta chain fusion protein, wherein the length of the epitope tag is 6 to 15 amino acids, preferably 9 to 11 amino acids. In another embodiment, TCR can be modified to a functional T cell receptor (TCR) alpha and / or beta chain fusion protein, wherein the TCR alpha and / or beta chain fusion protein comprises two or more epitope tags spaced apart from each other or directly connected in series. The embodiment of the fusion protein may comprise 2, 3, 4, 5 or more epitope tags, as long as the fusion protein maintains its biological activity ("functionality").

[0124] Preferred is a functional TCR α and / or β chain fusion protein according to the present invention, wherein the epitope tag is selected from but not limited to CD20 or Her2 / neu tags, or other conventional tags, such as myc tags, FLAG tags, T7 tags, HA (hemagglutinin) tags, His tags, S tags, GST tags or GFP tags. Myc, T7, GST, 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. Patent Nos. 4,703,004 and 4,851,341). The myc tag can be preferably used because there are available high-quality reagents for detecting it. In addition to being recognized by antibodies, epitope tags can certainly have one or more additional functions. The sequences of these tags are described in the literature and are well known to those skilled in the art.

[0125] Chimeric co-stimulatory receptor

[0126] The chimeric co-stimulatory receptor used in combination with the antigen-specific TCR (NY-ESO-specific TCR) comprises

[0127] - containing an extracellular domain of a polypeptide derived from PD-1,

[0128] - transmembrane domain, and

[0129] - containing an intracellular domain of a polypeptide derived from 4-1BB.

[0130] The chimeric co-stimulatory receptor used in combination with an antigen-specific TCR (e.g., a NY-ESO-1-specific TCR) may in particular include 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 the polypeptides contained in the extracellular domain respectively contain at least a portion of PD-1 (e.g., human PD-1), preferably an extracellular domain of PD-1. The chimeric co-stimulatory receptor comprising an extracellular domain derived from PD-1 has binding activity to other inhibitory ligands of PD-L1, PD-L2 or PD-1. As used herein, the term "derived from" PD-1 also allows substitution, deletion and / or insertion of up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids compared to the native sequence of PD-1 (e.g., human PD-1) or a portion thereof (e.g., an extracellular domain).

[0131] In one embodiment, the extracellular domain containing a polypeptide derived from PD-1 comprises the sequence shown in SEQ ID NO: 28 or an amino acid sequence that is at least 80% identical to SEQ ID NO: 28. In a specific embodiment, the extracellular domain containing a polypeptide derived from PD-1 comprises the sequence shown in SEQ ID NO: 28.

[0132] In one embodiment of the present invention, the chimeric co-stimulatory receptor comprises an extracellular domain comprising a polypeptide derived from PD-1, which comprises an amino acid sequence having at most 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 or mouse PD-1 (e.g., the human PD-1 sequence shown in SEQ ID NO: 28).

[0133] The chimeric co-stimulatory receptor used in combination with antigen-specific TCR (e.g., NY-ESO-1 specific TCR) also includes a transmembrane domain operably connected between the extracellular domain and the intracellular domain herein. Generally, the transmembrane domain is not limited to a specific transmembrane domain. Preferably, the transmembrane domain allows the fusion protein to be stably anchored in the membrane of a cell (e.g., T cell) expressing the fusion protein, and further allows the extracellular domain to be bound to PD-L1 respectively, and after binding to PD-L1, allows signal transduction to the intracellular domain containing a polypeptide derived from 4-1BB.

[0134] In a preferred embodiment, the transmembrane domain of the chimeric co-stimulatory receptor is derived from PD-1. In one embodiment, the transmembrane domain comprises the sequence shown 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 a polypeptide derived from PD-1 comprises the sequence shown in SEQ ID NO: 30.

[0135] The chimeric costimulatory receptor used in combination with an antigen-specific TCR (eg, NY-ESO-1-specific TCR) may particularly include a polypeptide derived from 4-1BB (also referred to as "41BB"), preferably an intracellular domain of an intracellular domain of 4-1BB (eg, human 4-1BB). In this context, the term "derived from" refers in particular to a polypeptide contained in an intracellular domain that includes at least a portion of 4-1BB (eg, human 4-1BB), preferably an intracellular domain of 4-1BB. The chimeric costimulatory receptor comprising an intracellular domain derived from 4-1BB can increase the proliferation rate of T cells expressing the chimeric costimulatory receptor after stimulation with another inhibitory ligand of PD-L1, PD-L2 or PD-1, and / or can increase the effector function of the T cells expressing the chimeric costimulatory receptor (eg, increase IFN-γ release and / or increase cytotoxicity) compared to corresponding T cells that do not express chimeric costimulatory receptors. As used herein, the term "derived from" 4-1BB also allows for substitution, deletion and / or insertion of up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids compared to the native sequence of 4-1BB (human or mouse, preferably human 4-1BB) or a portion thereof (e.g., intracellular domain). In one embodiment, the intracellular domain of a polypeptide derived from 4-1BB comprises the sequence shown 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 of a polypeptide derived from 4-1BB comprises the sequence shown in SEQ ID NO: 32.

[0136] As used herein, "at least 80% identical", particularly "having an amino acid sequence at least 80% identical to" 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 listed amino acid sequences.

[0137] The determination of percent identity between multiple sequences is preferably performed using Vector NTI Advance TM The 10 program (Invitrogen Corporation, Carlsbad CA, USA) was used to perform the AlignX application. This program uses a modified Clustal W algorithm (Thompson et al., 1994. Nucl Acids Res. 22: pp. 4673-4680; Invitrogen Corporation; Vector NTI Advance TM10DNA and protein sequence analysis software. User's Manual, 2004, pp. 389-662). The percent identity was determined using the standard parameters of the AlignX application.

[0138] Nucleic acids, nucleic acid compositions and vectors

[0139] The present invention encompasses nucleic acids encoding the antigen-specific TCRs described herein, in particular NY-ESO-1-specific TCRs, as well as corresponding nucleic acid compositions and vectors comprising the nucleic acids.

[0140] The nucleotide sequences encoding the relevant regions and domains of NY-ESO-specific TCR are listed in Table 1:

[0141]

[0142] The nucleotide sequences encoding the chimeric co-stimulatory receptor-related regions and domains are listed in Table 2:

[0143]

[0144] Table 2

[0145] In addition, another aspect relates to a composition comprising

[0146] - a nucleic acid encoding a NY-ESO-1 specific T cell receptor (TCR), the TCR comprising

[0147] - 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

[0148] - 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

[0149] - a nucleic acid encoding a chimeric co-stimulatory receptor comprising

[0150] - containing an extracellular domain of a polypeptide derived from PD-1,

[0151] - transmembrane domain, and

[0152] - containing an intracellular domain of a polypeptide derived from 4-1BB.

[0153] Furthermore, one aspect relates to a nucleic acid comprising

[0154] - a nucleic acid encoding a NY-ESO-1 specific T cell receptor (TCR), the TCR comprising

[0155] - 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

[0156] - 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

[0157] - a nucleic acid encoding a chimeric co-stimulatory receptor comprising

[0158] - containing an extracellular domain of a polypeptide derived from PD-1,

[0159] - transmembrane domain, and

[0160] - containing an intracellular domain of a polypeptide derived from 4-1BB.

[0161] Another aspect relates to a vector comprising a nucleic acid comprising sequences of a NY-ESO-1 specific TCR and a chimeric co-stimulatory receptor, and also includes a cell comprising the nucleic acid composition and / or the vector.

[0162] "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), which may contain natural, non-natural or altered nucleotides, and may contain natural, non-natural or altered internucleotide bonds, such as phosphoramide bonds or phosphorothioate bonds, rather than phosphodiesters between nucleotides of unmodified oligonucleotides. Preferably, the nucleic acids described herein are recombinant. As used herein, the term "recombinant" refers to (i) molecules constructed outside living cells by connecting natural or synthetic nucleic acid fragments to nucleic acid molecules that can be replicated in living cells, or (ii) molecules produced by the replication of the molecules described in (i) above. For the purposes of this article, replication may be in vitro replication or in vivo replication. Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art or commercially available (e.g., from companies such as Genscript, Thermo Fisher, etc.). See, e.g., Sambrook et al., nucleic acids can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides (e.g., see Sambrook et al. 2001), which are designed to increase the biological stability of the molecule or to increase the physical stability of the double strand formed after hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides). Nucleic acids can comprise any nucleotide sequence encoding any recombinant TCR and / or chimeric costimulatory receptor, polypeptide or protein, or a functional portion or functional variant thereof.

[0163] For example, the disclosure also provides variants of isolated or purified nucleic acids, wherein the variant nucleic acid comprises a nucleotide sequence that is 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.

[0164] For example, the disclosure also provides variants of isolated or purified nucleic acids, wherein the variant nucleic acids comprise a nucleotide sequence that is 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 the chimeric costimulatory receptor described herein. Such variant nucleotide sequences encode functional chimeric costimulatory receptors as described herein.

[0165] As described elsewhere herein, nucleic acids encoding TCR and / or chimeric co-stimulatory receptors can be modified. Useful modifications in the entire nucleic acid sequence can be codon optimization. Changes resulting in conservative substitutions in the translated amino acid sequence can be made. With regard to TCR, these changes can be made in the complementary determining regions and non-complementarity determining regions of the amino acid sequence of the TCR chain that do not affect function. Generally, additions and deletions should not be made in the CDR3 region.

[0166] Another embodiment relates to a vector comprising nucleic acids encoding a TCR and a chimeric co-stimulatory receptor as described herein.

[0167] The vector is preferably a plasmid, a shuttle vector, a phagemid, a cosmid, an expression vector, a retroviral vector, an adenoviral vector or particle and / or a vector for gene therapy.

[0168] "Vector" is any molecule or composition capable of carrying a nucleic acid sequence into a suitable host cell in which the synthesis of the encoded polypeptide can be performed. Typically, and preferably, a vector is a nucleic acid engineered to integrate a desired nucleic acid sequence (e.g., nucleic acid of the present invention) using recombinant DNA techniques known in the art. The vector may comprise DNA or RNA and / or comprise liposomes and / or viral particles. The 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 for gene therapy. The vector may comprise a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. The vector may also comprise one or more selectable marker genes and other genetic elements known to those of ordinary skill in the art. The vector is preferably an expression vector comprising a nucleic acid according to the present invention that is operably linked to a sequence that allows the expression of the nucleic acid.

[0169] Preferably, the vector is an expression vector. More preferably, the vector is a retroviral vector, more particularly a gamma-retroviral or lentiviral vector.

[0170] Those skilled in the art understand that the chimeric costimulatory receptor sequence and TCR chain TCR-α and TCR-β chain sequence can be contained in one nucleic acid, such as one vector. In this case, the sequence is connected to an internal ribosome entry site (IRES) sequence or a 2A peptide sequence derived from porcine Teschen virus (P2A) or derived from other species (such as Thosea asigna virus 2A peptide (T2A) or foot-and-mouth disease virus 2A peptide (F2A)) (as 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 the transduced cell.

[0171] In certain embodiments, the cell may comprise nucleic acids encoding the TCR and chimeric co-stimulatory receptor described herein, or vectors comprising the same.

[0172] 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 is noteworthy that the introduction or transfer of nucleic acid sequences is not limited to the above methods, but can be achieved in a variety of ways, including electroporation, microinjection, gene gun delivery, lipofection, supertransfection, and the above-mentioned viral infection by retrovirus or other suitable transduction or transfection. The cloning and exogenous expression methods of TCR are described, for example, in Engels et al. (Relapse or eradication of cancer is predicted by peptide-majorhistocompatibility complex affinity. Cancer Cell, 23 (4), 516–26. 2013). 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, the use of lentiviral vectors to transduce primary human T cells is described.

[0173] Cells described and provided in the context of the present invention comprising nucleic acid molecules or vectors described and provided herein are preferably capable of stably or transiently (e.g., stably) expressing (constitutively or conditionally) antigen-specific TCRs, such as NY-ESO-1-specific TCRs, and chimeric co-stimulatory receptors of the present invention. Host cells can generally be transduced or transformed with any suitable nucleic acid molecule or vector by any method. In one embodiment, host cells are transduced with a retroviral or lentiviral (e.g., retroviral) vector comprising a nucleic acid molecule encoding a fusion protein of the present invention as described above, or a portion thereof (e.g., ECD, TMD and / or ICD).

[0174] In some embodiments, the cell (including, for example, a target-specific immune cell or a cell of a cell colony as described herein) is a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The cell can be a natural killer cell or a T cell. Preferably, the cell is a T cell. The T cell can be a CD4+ or CD8+ T cell. In some embodiments, the cell is a stem cell, such as a memory T cell.

[0175] Stem cell-like memory T cells (TSCM) are a less differentiated subset of CD8+ or CD4+ T cells, characterized by the ability to self-renew and survive for a long time. Once these cells encounter their antigens 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 in a quiescent state (Flynn et al., Clinical & Translational Immunology (2014). These remaining TSCM cells show the ability to establish persistent immune memory in vivo and are therefore considered to be an important T cell subset 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 limit the T cell pool to stem cell memory T cell subtypes, see (Riddell et al. 2014, Cancer Journal 20 (2): 141–44).

[0176] Pharmaceutical compositions, medical treatments and kits

[0177] Another aspect of the invention relates to a pharmaceutical composition comprising a cell comprising an antigen-specific TCR (e.g., a NY-ESO-1-specific TCR) and a chimeric co-stimulatory receptor; or comprising a nucleic acid molecule encoding a molecule as described herein, the nucleic acid encoding an antigen-specific TCR (e.g., a NY-ESO-1-specific TCR) and a chimeric co-stimulatory receptor; comprising a composition comprising a nucleic acid encoding an antigen-specific TCR (e.g., a NY-ESO-1-specific TCR) and a nucleic acid encoding a chimeric co-stimulatory receptor; and a corresponding vector, as described herein.

[0178] Those active ingredients of the present invention are preferably mixed with acceptable carriers or carrier materials for such pharmaceutical compositions in a dosage that allows the disease to be treated or at least alleviated. Such compositions (in addition to active ingredients and carriers) may include filler materials, salts, buffers, stabilizers, solubilizers and other materials, which are known in the art.

[0179] The term "pharmaceutically acceptable" is defined as a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The choice of carrier depends on the application.

[0180] Pharmaceutical compositions may contain additional ingredients that enhance the activity of the active ingredients or supplement the treatment. Such additional ingredients and / or factors may be part of a pharmaceutical composition to achieve a synergistic effect or to minimize adverse or undesirable effects.

[0181] Techniques for formulation or preparation and application / administration of the active ingredients of the present invention are disclosed in "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA, latest edition. Suitable applications are parenteral applications, such as intramuscular, subcutaneous, intramedullary injections, and intrathecal, direct intraventricular, intravenous, intranodal, intraperitoneal or intratumoral injections. Intravenous injection is the preferred treatment for patients.

[0182] According to a preferred embodiment, the pharmaceutical composition is an infusion or an injection.

[0183] An injectable composition is a pharmaceutically acceptable fluid composition comprising at least one active ingredient, such as an expanded T cell population (e.g., autologous or allogeneic to the patient to be treated) comprising an antigen-specific TCR (e.g., NY-ESO-1-specific TCR) and a chimeric co-stimulatory receptor. The active ingredient is typically dissolved or suspended in a physiologically acceptable carrier, and the composition may also contain a small amount of one or more non-toxic auxiliary substances, such as emulsifiers, preservatives, and pH buffers, etc. Such injectable compositions that can be used with the fusion proteins of the present disclosure are conventional; appropriate formulations are well known to those of ordinary skill in the art.

[0184] Typically, pharmaceutical compositions comprise at least one pharmaceutically acceptable carrier.

[0185] Therefore, another aspect of the present invention relates to a cell as described herein, a composition as described herein, a nucleic acid as described herein and / or a vector as described herein for use as a medicament.

[0186] Some embodiments relate to a cell described herein, a composition described herein, a nucleic acid described herein and / or a vector described herein for use in the treatment of cancer.

[0187] Thus, also contemplated are methods for treating cancer in a human or non-human animal in need thereof, comprising administering to the human or non-human animal a cell described herein, a composition described herein, a nucleic acid described herein, and / or a vector described herein.

[0188] In one embodiment, the cancer is a hematological cancer or a solid tumor.

[0189] Hematological cancers, also called blood cancers, do not form solid tumors and are therefore dispersed throughout the body. Examples of hematological cancers are leukemias, lymphomas, or multiple myeloma. There are two main types of solid tumors: sarcomas and carcinomas. Sarcomas are, for example, tumors of blood vessels, bones, fatty tissue, ligaments, lymphatic vessels, muscles, or tendons. In certain embodiments, the cancer is a solid tumor.

[0190] In one embodiment, the cancer is selected from the group consisting of prostate cancer, uterine cancer, thyroid cancer, testicular cancer, kidney cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma, non-Hodgkin lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, cervical cancer, colorectal cancer, gastric adenocarcinoma, bile duct cancer, breast cancer, bladder cancer, myeloid leukemia and acute lymphoblastic leukemia, carcinoma, sarcoma or osteosarcoma.

[0191] Compositions comprising the modified T cells described herein can be used in methods and compositions for adoptive immunotherapy according to known techniques, or variations thereof that will be apparent to those skilled in the art based on this disclosure.

[0192] In some embodiments, the cells are configured by first harvesting the cells from their culture medium, then washing and concentrating the cells in a therapeutically effective amount in a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier). Suitable infusion media can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter), but 5% dextrose in water or Ringer's lactate may also be used. The infusion medium may be supplemented with human serum albumin.

[0193] The number of cells in the composition for effective treatment is generally greater than 10 cells, and up to 10 6 Cells, up to and including 10 8 or 10 9 cells, and can exceed 10 10 The number of cells will depend on the end use of the composition and the type of cells contained therein. For the purposes provided herein, the volume of cells is typically 1 liter or less, can be 500 ml or less, or even 250 ml or 100 ml or less. Thus, the density of cells is typically greater than 10 6 cells / ml and usually greater than 10 7 cells / ml, usually 10 8 cells / ml or greater. Clinically relevant immune cell counts can be assigned to cumulatively equal to or greater than 10 9 , 10 10 or 10 11 The pharmaceutical compositions provided herein may be in various forms, such as solid, liquid, powder, aqueous or lyophilized forms. Examples of suitable pharmaceutical carriers are known in the art. Such carriers and / or additives may be prepared by conventional methods and may be administered to a subject at a suitable dose. Stabilizers, such as lipids, nuclease inhibitors, polymers and chelating agents, may protect the composition from degradation in vivo. In compositions intended to be administered by injection, one or more of surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers and isotonic agents may be included.

[0194] Viral vector particles comprising nucleotide sequences encoding antigen-specific TCRs (e.g., NY-ESO-1-specific TCRs) and chimeric co-stimulatory receptors provided herein can be packaged as a kit. The kit can optionally include one or more components, such as instructions for use, devices, and additional reagents, and components for practicing the method, such as tubes, containers, and syringes. Exemplary kits can include nucleic acids, recombinant polypeptides, or viruses encoding recombinant TCRs and chimeric co-stimulatory receptors provided herein, and can optionally include instructions for use, devices for detecting viruses in a subject, devices for administering the composition to a subject, and devices for administering the composition to a subject.

[0195] Also contemplated herein are kits comprising polynucleotides encoding antigen-specific TCRs (e.g., NY-ESO-1-specific TCRs) and chimeric co-stimulatory receptors. Also contemplated herein are kits comprising viral vectors encoding sequences of interest (e.g., recombinant TCRs) and optionally polynucleotide sequences encoding immune checkpoint inhibitors.

[0196] The kit considered herein also includes a kit for implementing a method for detecting the presence of any one or more polynucleotides encoding the TCR and / or chimeric costimulatory receptors disclosed herein. In particular, such diagnostic kits may include appropriate amplification and detection primer sets and other related reagents for deep sequencing to detect polynucleotides encoding the TCR and / or chimeric costimulatory receptors disclosed herein. In a further embodiment, the kit herein may include reagents for detecting TCR and / or chimeric costimulatory receptors disclosed herein, such as antibodies or other binding molecules. The diagnostic kit may also include instructions for determining the presence of polynucleotides encoding the TCR and / or chimeric costimulatory receptors disclosed herein or for determining the presence of the TCR and / or chimeric costimulatory receptors disclosed herein. The kit may also include instructions. The instructions generally include a description of the components contained in the kit and the method of administration, including a specific expression of the method for determining the appropriate state, appropriate dosage and appropriate method of administration of the subject. The instructions may also include guidance for monitoring the subject during treatment.

[0197] The kits provided herein may also include a device for administering the compositions described herein to a subject. Any of the various devices known in the art for administering drugs or vaccines may be included in the kits provided herein. Exemplary devices include, but are not limited to, hypodermic needles, intravenous needles, catheters, needleless injection devices, inhalers, and liquid dispensers, such as eye droppers. Typically, the device for administering the virus of the kit will be compatible with the virus of the kit; for example, a needleless injection device (e.g., a high-pressure injection device) may be included in a kit with a virus that is not damaged by high-pressure injection, but is generally not included in a kit with a virus that is damaged by high-pressure injection.

[0198] Cytokine release

[0199] Cells expressing TCR and co-stimulatory molecules (e.g., PD1-4BB) showed enhanced cytokine release. The population secreted large amounts of cytokines / lytic proteins, especially IFN-γ, Gzm-B, IP-10, and MIP-1β.

[0200] Surprisingly, immune cells, in particular TCR-T cells, expressing a chimeric co-stimulatory receptor as defined herein, exhibit higher polyfunctionality compared to TCR-T cells lacking a chimeric co-stimulatory receptor as defined herein.

[0201] The higher polyfunctionality of genetically modified T cells means higher in vivo functionality and anti-tumor activity and is associated with clinical outcomes.

[0202] The term "multifunctional" means that the cell secretes at least 2, such as at least 3, at least 4, at least 5 proteins.

[0203] The skilled artisan understands that cells secrete proteins following antigen-specific stimulation, for example when the cell comes into contact with an antigen-presenting cell that presents the antigen in an MHC molecule.

[0204] In particular, the cell population comprises a significant proportion of cells, e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 3%, 4%, 4.5%, of multifunctional cells.

[0205] In particular, the cell population comprises a significant proportion of cells, such as at least 1%, preferably at least 1.5%, more preferably 1.75%, of cells expressing three or more proteins.

[0206] In some embodiments, the multifunctional strength index is 80 or more, preferably 90 or more, more preferably 100 or more.

[0207] The polyfunctional intensity index (PSI) was calculated by multiplying the intensity of each secreted cytokine by the percentage of polyfunctional T cells.

[0208] In particular, we can show that a population of cells expressing a target-specific (e.g. NY-ESO-1 specific) TCR and a chimeric co-stimulatory receptor as defined herein comprises cells secreting at least two proteins (e.g. at least three proteins, at least four proteins, at least five proteins).

[0209] Therefore, another aspect of the invention relates to an in vitro assay that indicates the potency of a T cell population by indicating whether the T cell population comprises a significant proportion of multifunctional T cells as defined herein.

[0210] Thus, in general, what is needed are highly efficient target-specific T cells that secrete at least two, at least three, at least four, at least five proteins selected from effector proteins, stimulatory cytokines, and chemoattractant cytokines.

[0211] In particular, the present invention relates to an in vitro assay indicating the potency of an immune cell population, comprising the following steps

[0212] - measuring the proportion of multifunctional immune cells in the first cell population,

[0213] - assessing the potency of the first immune cell population and assessing the potency of the second T cell population, wherein an increased proportion of multifunctional cells in the first cell population compared to the second cell population indicates that the first cell population has a higher potency than the second cell population.

[0214] The proportion of multifunctional immune cells can be measured by Technology (Isoplexis) is used.

[0215] Thus, a T cell population comprising target-specific T cells is desired, wherein the cell population comprises T cells that secrete at least two, at least three, at least four, at least five proteins selected from the group consisting of effector proteins, stimulatory cytokines, and chemoattractant cytokines.

[0216] The effector protein may be selected from the group consisting of Gzm-B, IFN-γ, perforin, TNF-α, TNF-β, MIP-1α.

[0217] In one embodiment, the stimulatory cytokine can 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.

[0218] The chemoattractant cytokine may be selected from IP-10 and MIP-1β.

[0219] In some embodiments, each of the cells that secrete at least two proteins secretes at least one protein 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 that secrete at least two proteins secretes at least one protein 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 that secrete at least two proteins secretes at least one protein selected from the group consisting of IFN-γ, Gzm-B, GM-CSF, MIP-1β, and IP-10. In some embodiments, each of the cells that secrete at least two proteins secretes at least one protein selected from the group consisting of IFN-γ, Gzm-B, and GM-CSF. In some embodiments, each of the cells that secrete at least two proteins secretes at least one protein selected from the group consisting of IFN-γ, Gzm-B and IP-10. In some embodiments, each of the cells that secrete at least two proteins secretes at least one protein selected from the group consisting of IFN-γ and Gzm-B.

[0220] Typically, the cell population does not contain cells that secrete significant amounts of IL-6 and / or IL-10.

[0221] Other embodiments relate to T cells expressing (A) a NY-ESO-1-specific TCR as defined herein and (B) a chimeric co-stimulatory receptor as defined herein, wherein the T cell secretes at least two, at least three, at least four, at least five proteins selected from the group consisting of effector proteins, stimulatory cytokines, and chemoattractant cytokines.

[0222] Thus, some embodiments relate to a cell population comprising T cells expressing (A) a NY-ESO-1-specific TCR as defined herein and (B) a chimeric co-stimulatory receptor as defined herein, wherein the cell population comprises T cells that secrete at least two, at least three, at least four, at least five proteins selected from the group consisting of effector proteins, stimulatory cytokines, and chemoattractant cytokines.

[0223] experiment

[0224] Example 1: TCR-T cells targeting NY-ESO-1 expressing PD1-41BB exhibited higher polyfunctionality compared to TCR-T cells targeting NY-ESO-1 lacking PD1-41BB.

[0225] use The single-cell polyfunctionality (release of 2 or more proteins) of TCR transgenic T cells with or without PD1-41BB was analyzed using the IsoPlexis technology. So-called "polyfunctional" T cells secrete a variety of proteins (especially cytokines and other effector proteins such as Gzm-B), thereby achieving various effector functions and efficient immune responses. After co-culture with PD-L1-overexpressing, NY-ESO-1-positive MelA375 or Mel624.38 tumor cell lines, single CD8 + T cells were loaded onto the IsoCode chip (IsoPlexis). The secretion of 32 T cell cytokines / proteins was analyzed using the IsoLight device and IsoSpeak software (IsoPlexis). TCR-T cells expressing PD1-41BB showed a higher percentage of polyfunctional T cells, i.e., T cells that secrete 2 or more proteins ( Figure 1A The polyfunctional strength index (PSI) was calculated by multiplying the intensity of various secreted proteins / cytokines by the percentage of polyfunctional T cells. TCR-T cells expressing PD1-41BB showed a higher PSI ( Figure 1B ).

[0226] The various proteins / cytokines released were classified and it was found that 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) had the largest contribution to the excellent PSI, followed by chemoattractant cytokines (IP-10, MIP-1β, RANTES). Regulatory (IL-4, IL-10, IL-22, sCD137, TGF-β1) and inflammatory (CP-1, IL-17F, IL8) cytokines were released to a lesser extent. A detailed analysis of single cell multi-protein / cytokine release was performed using a multifunctional heat map, which showed that the multi-protein / cytokine signatures of TCR-T cells with and without PD1-41BB were different ( Figure 1C). Notably, TCR-T cells expressing PD1-41BB contained a higher proportion of single cells that simultaneously secreted 2-6 proteins / cytokines, indicating superior functionality compared to TCR-T cells lacking PD1-41BB. The major proteins / cytokines released by TCR-T cells expressing PD1-41BB were Gzm-B, IFN-γ, GM-CSF, and MIP-1β, belonging to the families of effector proteins and stimulatory cytokines, thus indicating high TCR-T cell functionality and potential anti-tumor activity.

[0227] Example 2: Improved effector function of NY-ESO-1 TCR-T cells co-expressing chimeric PD1-41BB co-stimulatory receptors. Flow cytometric analysis of untransduced (UT), NY-ESO-1 TCR expressing (TCR), and NY-ESO-1 TCR co-expressing PD1-41BB (TCR+PD1-41BB) T cells ( Figure 2A ). NY-ESO-1TCR 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 level of released IFN-γ was measured by ELISA (t test, **p>0.01, ***p<0.001) ( Figure 2B ). NY-ESO-1 expression levels were measured by qPCR and quantified based on a standard calibration curve. PD-L1 levels summarized here were assessed by qPCR and FACS analysis ( Figure 2C ). NY-ESO-1TCR transgenic CD8+ T cells with or without PD1-41BB were stained with a dye membrane tracer 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).

[0228] Example 3: Multifunctionality of NY-ESO-1 TCR-T cells enhanced by co-expression of chimeric co-stimulatory receptor PD1-41BB. After 24 hours of co-culture with tumor target cell lines Mel624.38_PD-L1 and MelA375_PD-L1, NY-ESO-1-specific TCR-T cells with (TCR+PD1-41BB) and without co-expression of (TCR) chimeric co-stimulatory receptor PD1-41BB were analyzed using single-cell proteomic analysis (IsoLight technology, IsoPlexis) of a panel of 32 secreted cytokines, chemokines and cytotoxic molecules. Co-culture with untransduced T cells (UT) from the same donor served as a control. Cells that secrete 2 or more cytokines are considered multifunctional. The multifunctionality of single TCR T cells is shown as a percentage of the sample and is classified by the percentage of cells (grey shading) that simultaneously express 2, 3, 4 and 5+ analytes ( Figure 3A The polyfunctional intensity index (PSI) of the samples shown is defined as the number of T cells secreting more than 2 effector molecules per cell (polyfunctional T cells in A), multiplied by the mean fluorescence intensity (MFI) of the proteins secreted by the respective cells and categorized by proteins associated with effector, stimulatory, chemoattractant, regulatory, and inflammatory properties (grey shading) ( Figure 3B ). The single cell multifunctionality heat map shows the single cell cytokine combination secreted by each sample. Each column represents a specific cytokine combination, and the orange squares describe the frequency of the corresponding sample secreting a specific cytokine combination (the average values ​​of target cells Mel624.38_PD-L1 and MelA375_PD-L1 are shown) ( Figure 3C ).

[0229] Example 4: TCR-T cells co-expressing the co-stimulatory receptor PD1-41BB secrete increased frequencies of effector, stimulatory, and chemoattractant proteins. Secretion frequencies of single proteins induced by co-culture of TCR T cells equipped with PD1-41BB cells and the target cell line Mel624.38_PD-L1. Proteins are shown and classified by their functions of a) effect, b) stimulation, c) chemoattraction, d) regulation, and e) inflammatory properties ( Figure 4 ).

[0230] Example 5: Serial killing of tumor cell spheroids. Tumor cell spheroids were generated starting with 1000 NucLightRed transduced cells in a 96-well ULA plate 72 hours before 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 the IncuCyte S3. Fresh tumor cell spheroids generated as described above were added every 72–96 hours ( Figure 5 ).

[0231] This application also includes the following items:

[0232] Item 1. A cell population, comprising cells expressing:

[0233] (A) antigen-specific TCR, and

[0234] (B) Chimeric co-stimulatory receptors,

[0235] wherein the cell population comprises cells that secrete at least two proteins.

[0236] Item 2. A cell population according to Item 2, wherein the chimeric co-stimulatory receptor comprises

[0237] - containing an extracellular domain of a polypeptide derived from PD-1,

[0238] - transmembrane domain, and

[0239] - an intracellular domain comprising a polypeptide derived from 4-1BB,

[0240] wherein the cell population comprises cells that secrete at least two proteins.

[0241] Item 3. A cell population according to Item 2, wherein the chimeric co-stimulatory receptor comprises

[0242] - containing an extracellular domain of a polypeptide derived from PD-1,

[0243] - A transmembrane domain derived from PD-1, and

[0244] - an intracellular domain comprising a polypeptide derived from 4-1BB,

[0245] wherein the cell population comprises cells that secrete at least two proteins.

[0246] Item 4. The cell population according to Item 3, wherein the cell population comprises cells expressing:

[0247] (A) NY-ESO-1 / LAGE-1 specific TCR, comprising

[0248] - a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 35, a CDR 2 having the amino acid sequence of SEQ ID NO: 36, and a CDR 3 having the sequence of SEQ ID NO: 37; and

[0249] - a TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 38, a CDR 2 having the amino acid sequence of SEQ ID NO: 39, and a CDR 3 having the sequence of SEQ ID NO: 40; and

[0250] (B) a chimeric co-stimulatory receptor, the chimeric co-stimulatory receptor comprising

[0251] - containing an extracellular domain of a polypeptide derived from PD-1,

[0252] - transmembrane domain, and

[0253] - an intracellular domain comprising a polypeptide derived from 4-1BB,

[0254] wherein the cell population comprises cells that secrete at least two proteins.

[0255] Item 5. A cell population according to any of the preceding items, wherein the cell population comprises cells that secrete at least three proteins.

[0256] Item 6. A cell population according to any of the preceding items, wherein the cell population comprises cells that secrete at least four proteins.

[0257] Item 7. A cell population according to any of the preceding items, wherein the cell population comprises cells that secrete at least five proteins.

[0258] Item 8. A cell population according to any of the preceding items, wherein the protein is selected from the group consisting of effector proteins, stimulatory cytokines and chemoattractant cytokines.

[0259] Item 9. A cell population according to 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.

[0260] Item 10. A cell population according to Item 9, wherein the effector protein is selected from the group consisting of Gzm-B, IFN-γ, perforin, TNF-α, and TNF-β.

[0261] Item 11. A 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.

[0262] Item 12. A 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.

[0263] Item 13. A cell population according to Items 8 to 12, wherein the chemoattractant cytokine is selected from IP-10 and MIP-1β.

[0264] Item 14. A cell population according to any of the preceding items, 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.

[0265] Item 15. A cell population according to any one of the preceding items, 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.

[0266] Item 16. The cell population according to any one of the preceding items, wherein the protein is selected from the group consisting of Gzm-B, IFN-γ, perforin, TNF-α, TNF-βGM-CSF, IL-2, and MIP-1β.

[0267] Item 17. The cell population according to any one of the preceding items, wherein the protein is selected from the group consisting of IFN-γ, Gzm-B and IP-10.

[0268] Item 18. The cell population according to any one of the preceding items, wherein the protein is selected from the group consisting of IFN-γ and Gzm-B.

[0269] Item 19. A cell population according to any one of Items 4 and 6 to 18, wherein the TCR is capable of binding to a NY-ESO peptide having an amino acid sequence as shown in SEQ ID NO: 34 or a portion thereof, preferably an HLA-A2 binding form thereof.

[0270] Item 20. A cell population according to Item 19, wherein the HLA-A2 is an HLA-A*02:01, HLA-A*02:02, HLA-A*02:04 or HLA-A*02:09 encoded molecule, preferably an HLA-A*2:01 encoded molecule.

[0271] Item 21. A cell population according to any one of Items 3 to 20, wherein the extracellular domain containing a polypeptide derived from PD-1 comprises the sequence of SEQ ID NO: 28, and

[0272] Wherein the intracellular domain containing the polypeptide derived from 4-1BB comprises the sequence of SEQ ID NO: 32.

[0273] Item 22. A cell population according to any one of Items 3 to 21, wherein the transmembrane domain is derived from PD-1, wherein preferably the transmembrane domain containing a polypeptide derived from PD-1 comprises the sequence of SEQ ID NO:30, preferably wherein the chimeric co-stimulatory receptor comprises the sequence of SEQ ID NO:26.

[0274] Item 23. A cell population according to any one of the preceding items, for use in treating cancer.

[0275] Item 24. A target-specific immune cell expressing

[0276] (A) antigen-specific TCR, and

[0277] (B) Chimeric co-stimulatory receptors,

[0278] Wherein the target-specific immune cells secrete at least two proteins.

[0279] Item 25. A target-specific immune cell according to Item 24, wherein the chimeric co-stimulatory receptor comprises

[0280] - containing an extracellular domain of a polypeptide derived from PD-1,

[0281] - transmembrane domain, and

[0282] - an intracellular domain comprising a polypeptide derived from 4-1BB,

[0283] Wherein the target-specific immune cells secrete at least two proteins.

[0284] Item 26. A target-specific immune cell according to Item 25, wherein the chimeric co-stimulatory receptor comprises

[0285] - containing an extracellular domain of a polypeptide derived from PD-1,

[0286] - A transmembrane domain derived from PD-1, and

[0287] - an intracellular domain comprising a polypeptide derived from 4-1BB,

[0288] Wherein the target-specific immune cells secrete at least two proteins.

[0289] Item 27. A target-specific immune cell according to any one of Items 24 to 26, wherein the target-specific immune cell expresses

[0290] (A) NY-ESO-1 / LAGE-1 specific TCR, comprising

[0291] - a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 35, a CDR 2 having the amino acid sequence of SEQ ID NO: 36, and a CDR 3 having the sequence of SEQ ID NO: 37; and

[0292] a TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 38, a CDR 2 having the amino acid sequence of SEQ ID NO: 39, and a CDR 3 having the sequence of SEQ ID NO: 40;

[0293] (B) a chimeric co-stimulatory receptor, the chimeric co-stimulatory receptor comprising

[0294] - containing an extracellular domain of a polypeptide derived from PD-1,

[0295] - transmembrane domain, and

[0296] - an intracellular domain comprising a polypeptide derived from 4-1BB,

[0297] Wherein the target-specific immune cells secrete at least two proteins.

[0298] Item 28. The target-specific immune cell according to any one of Items 24 to 27, wherein the target-specific immune cell secretes at least three proteins.

[0299] Item 29. A target-specific immune cell according to any one of Items 24 to 28, wherein the target-specific immune cell secretes at least four proteins.

[0300] Item 30. The target-specific immune cell according to any one of Items 24 to 29, wherein the target-specific immune cell secretes at least five proteins.

[0301] Item 31. A target-specific immune cell according to any one of Items 24 to 30, wherein the protein is selected from the group consisting of effector proteins, stimulatory cytokines and chemoattractant cytokines.

[0302] Item 32. A 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.

[0303] Item 33. A 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.

[0304] Item 34. A 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.

[0305] Item 35. A 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.

[0306] Item 36. A 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.

[0307] Item 37. A target-specific immune cell according to 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.

[0308] Item 38. A 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.

[0309] Item 39. A 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.

[0310] 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.

[0311] Item 41. A 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.

[0312] Item 42. A target-specific immune cell according to any one of Items 27 and 28 to 41, wherein the TCR is capable of binding to a NY-ESO peptide having the amino acid sequence shown in SEQ ID NO: 34 or a portion thereof, preferably an HLA-A2-bound form thereof.

[0313] Item 43. A target-specific immune cell according to Item 42, 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.

[0314] Item 44. A target-specific immune cell according to Item 43, wherein the HLA-A2 is an HLA-A*2:01 encoded molecule.

[0315] Item 45. A target-specific immune cell according to any one of Items 25 to 43, wherein the extracellular domain containing a polypeptide derived from PD-1 comprises the sequence of SEQ ID NO: 28, and

[0316] Wherein the intracellular domain containing the polypeptide derived from 4-1BB comprises the sequence of SEQ ID NO: 32.

[0317] Item 46. A target-specific immune cell according to any one of Items 26 to 45, wherein the transmembrane domain is derived from PD-1, wherein preferably the transmembrane domain containing a polypeptide derived from PD-1 comprises the sequence of SEQ ID NO:30, preferably wherein the chimeric co-stimulatory receptor comprises the sequence of SEQ ID NO:26.

[0318] Item 47. A target-specific immune cell according to any one of Items 24 to 46, wherein the target-specific immune cell is a lymphocyte.

[0319] Item 48. A target-specific immune cell according to any one of Items 24 to 47, for use in treating cancer.

Claims

1. A target-specific immune cell, wherein the target-specific immune cell expresses (A) NY-ESO-1 / LAGE-1 specific TCR, comprising - a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 35, a CDR 2 having the amino acid sequence of SEQ ID NO: 36, and a CDR 3 having the 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 CDR 2 having the amino acid sequence of SEQ ID NO: 39, and a CDR 3 having the sequence of SEQ ID NO: 40; (B) a chimeric co-stimulatory receptor, the chimeric co-stimulatory receptor comprising - containing an extracellular domain of a polypeptide derived from PD-1, - transmembrane domain, and - containing an intracellular domain of a polypeptide derived from 4-1BB.

2. The target-specific immune cell according to claim 2, wherein the chimeric co-stimulatory receptor comprises - contains an extracellular domain derived from the extracellular domain of PD-1, - A transmembrane domain derived from PD-1, and - contains an intracellular domain derived from the intracellular domain of 4-1BB.

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

28.

4. The target-specific immune cell of any one of the preceding claims, wherein the intracellular domain derived from 4-1BB comprises an amino acid sequence having at most 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions compared to the intercellular domain derived from 4-1BB as shown in SEQ ID NO:

32.

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

32.

6. The target-specific immune cell according to any one of claims 2 to 5, wherein the transmembrane domain derived from PD-1 comprises an amino acid sequence having at most 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions compared to the transmembrane sequence derived from PD-1 as shown in SEQ ID NO:

30.

7. The target-specific immune cell according to claim 6, wherein the transmembrane domain is derived from PD-1, wherein preferably the transmembrane domain containing the polypeptide derived from PD-1 comprises the sequence of SEQ ID NO:

30.

8. The target-specific immune cell according to any one of the preceding claims, preferably wherein the chimeric co-stimulatory receptor comprises the sequence of SEQ ID NO:

26.

9. The target-specific immune cell according to any one of the preceding claims, wherein the target-specific immune cell is a lymphocyte.

10. The target-specific immune cell according to any one of the preceding claims, wherein the target-specific immune cell is a T cell.

11. A target-specific immune cell according to any one of the preceding claims, wherein the TCR comprises a variable TCR alpha region having an amino acid sequence at least 80% identical to SEQ ID NO:41 and a variable TCR beta region having an amino acid sequence 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 TCR α region having the amino acid sequence of SEQ ID NO: 41 and a variable TCR β region having the amino acid sequence of SEQ ID NO:

42.

13. The target-specific immune cell according to any one of the preceding claims, wherein the TCR is capable of binding to the NY-ESO peptide having the amino acid sequence as shown in SEQ ID NO: 34 or a portion thereof, preferably an HLA-A2-bound form thereof.

14. The target-specific immune cell according to any one of the preceding claims, wherein the HLA-A2 is an HLA-A*02:01, HLA-A*02:02, HLA-A*02:04 or HLA-A*02:09 encoded molecule, preferably an HLA-A*2:01 encoded molecule.

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

16. The cell population according to claim 15, wherein the cell population comprises cells that secrete at least two proteins.

17. The cell population of claim 16, wherein the cell population comprises cells that secrete at least three proteins.

18. The cell population of claim 16 or 17, wherein the cell population comprises cells that secrete at least six proteins.

19. The cell population according to any one of claims 16 to 18, wherein the protein is selected from the group of effector proteins, stimulatory cytokines and chemoattractant cytokines.

20. The cell population of any one of claims 16 to 19, wherein at least 5% of the population secretes more than one effector protein.

21. The cell population of any one of claims 16 to 20, wherein at least 4% of the population secretes more than one selected from effector proteins, stimulatory cytokines, and chemoattractant cytokines.

22. The cell population according to any one of claims 16 to 21, wherein the cells secrete at least one protein selected from the group consisting of RANTES, MIP-1α, perforin, TNF-α and TNF-β.

23. The cell population according to claims 19 to 22, wherein the effector protein is selected from the group consisting of Gzm-B, IFN-γ, perforin, TNF-α, TNF-β, MIP-1α.

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

25. The cell population according to claims 19 to 24, wherein the stimulatory cytokine is selected from the group consisting of GM-CSF, IL-2, IL-5, IL-7, IL-8, IL-9, IL-12.

26. The cell population according to claims 19 to 25, wherein the stimulatory cytokine is selected from the group consisting of GM-CSF, IL-2, IL-7, IL-8, IL-9, IL-12.

27. The cell population of claims 19 to 26, wherein the chemoattractant cytokine is selected from IP-10 and MIP-1β.

28. The cell population of claims 16 to 27, 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 of claims 16 to 28, 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 any one of claims 16 to 29, wherein the protein is selected from the group consisting of Gzm-B, IFN-γ, perforin, TNF-α, TNF-β, GM-CSF, IL-2, MIP-1β.

31. The cell population according to any one of claims 16 to 30, wherein the protein is selected from the group consisting of IFN-γ, Gzm-B and IP-10.

32. The cell population of any one of claims 16 to 31, wherein the protein is selected from the group consisting of IFN-γ and Gzm-B.

33. A pharmaceutical composition comprising a target-specific immune cell as defined in claims 1 to 14 or a cell population as defined in claims 15 to 32.

34. A method of treating cancer in a human or non-human animal in need thereof, comprising administering to said human or non-human animal a target-specific immune cell according to claims 1 to 14 or a cell population according to claims 15 to 33.

35. The method of claim 34, wherein the cancer is a solid tumor.

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