阅读说明：本技术 Magea1特异性t细胞受体及其用途 (MAGEA 1-specific T cell receptor and uses thereof ) 是由 卡里娜·韦纳 马诺·韦斯 西尔克·拉菲格斯特 安雅·莫施 于 2020-04-01 设计创作，主要内容包括：本发明涉及对MAGEA1衍生肽特异的分离的T细胞受体(TCR)和包含TCR的功能部分的多肽。进一步涉及多价TCR复合物、编码TCR的核酸、表达TCR的细胞和包含TCR的药物组合物。本发明还涉及用作药物的TCR,特别是用于治疗癌症的TCR。(The present invention relates to isolated T Cell Receptors (TCRs) specific for MAGEA 1-derived peptides and polypeptides comprising a functional portion of the TCR. Further relates to multivalent TCR complexes, nucleic acids encoding TCRs, cells expressing TCRs, and pharmaceutical compositions comprising TCRs. The invention also relates to TCRs for use as medicaments, in particular TCRs for use in the treatment of cancer.)

1. An isolated T Cell Receptor (TCR) specific for MAGEA 1.

2. The isolated TCR of embodiment 1, wherein the TCR specifically recognizes the amino acid sequence SEQ ID NO 1 or a fragment thereof.

3. The isolated TCR of embodiments 1 and 2, wherein the TCR specifically recognizes the HLA-A2-bound form of the amino acid sequence of SEQ ID NO 1.

4. The isolated TCR according to any one of the preceding embodiments, wherein the TCR specifically recognizes the amino acid sequence of SEQ ID No.1 presented by a molecule encoded by a gene selected from the group consisting of HLA-a 02:01, HLA-a 02:04, HLA-a 02:16 and HLA-a 02:17, preferably wherein the TCR specifically recognizes the amino acid sequence of SEQ ID No.1 presented by a molecule encoded by HLA-a 02: 01.

5. The isolated TCR of any one of the preceding embodiments, wherein the TCR comprises:

a TCR alpha chain comprising the CDR1 having the amino acid sequence of SEQ ID NO.2, the CDR2 having the amino acid sequence of SEQ ID NO. 3 and the CDR3 having the amino acid sequence of SEQ ID NO. 4,

a TCR β chain comprising the CDR1 having the amino acid sequence of SEQ ID NO. 5, the CDR2 having the amino acid sequence of SEQ ID NO. 6 and the CDR3 having the amino acid sequence of SEQ ID NO. 7.

6. The isolated TCR of any one of the preceding embodiments, wherein the TCR comprises a variable TCR a region having an amino acid sequence at least 80% identical to SEQ ID No. 8 and a variable TCR β region having an amino acid sequence at least 80% identical to SEQ ID No. 9.

7. An isolated polypeptide comprising a functional portion of a TCR of any one of embodiments 1 to 21, wherein the functional portion comprises at least one of the amino acid sequences of SEQ ID NOs 4 and 7, preferably wherein the functional portion comprises the amino acid sequences of SEQ ID NOs 2, 3, 4, 5, 6 and 7.

8. A multivalent TCR complex comprising at least two TCRs as claimed in any one of claims 1 to 6.

9. The isolated TCR of claims 1-6, the polypeptide of claim 7, the multivalent TCR complex of claim 8, wherein IFN- γ secretion is induced by binding to the amino acid sequence of SEQ ID No.1 presented by a molecule encoded by HLA-a 02: 01.

10. A nucleic acid encoding a TCR according to any one of claims 1 to 6 or encoding an isolated polypeptide according to claim 7.

11. A vector comprising the nucleic acid of claim 10, wherein the vector is preferably an expression vector, more preferably a retroviral vector or a lentiviral vector.

12. A cell expressing a TCR according to claims 1 to 6.

13. An antibody or antigen-binding fragment thereof that specifically binds to the portion of the TCR that mediates MAGEA1 specificity according to claims 1 to 6, preferably wherein the portion of the TCR that mediates MAGEA1 specificity comprises the alpha chain CDR3 of SEQ ID No. 4 and/or the beta chain CDR3 of SEQ ID No. 7.

14. A TCR according to claims 1 to 6, a polypeptide according to claim 7, a multivalent TCR complex according to claim 8, a nucleic acid according to claim 10, a vector according to claim 11, a cell according to claim 12 or an antibody according to claim 13, for use as a medicament.

15. The TCR of claims 1-6, the polypeptide of claim 7, the multivalent TCR complex of claim 8, the nucleic acid of claim 10, the vector of claim 11, the cell of claim 12, or the antibody of claim 13, for use in treating a cancer, wherein the cancer is selected from the group consisting of: prostate cancer, uterine cancer, thyroid cancer, testicular cancer, renal cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma, non-hodgkin's lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, cervical cancer, colorectal cancer, gastric adenocarcinoma, cholangiocarcinoma, breast cancer, bladder cancer, myeloid leukemia, and acute lymphoblastic leukemia, sarcoma, or osteosarcoma.

Technical Field

The present invention relates to isolated T Cell Receptors (TCRs) specific for MAGEA 1-derived peptides and polypeptides comprising a functional portion of the TCR. Further relates to multivalent TCR complexes, nucleic acids encoding TCRs, cells expressing TCRs, and pharmaceutical compositions comprising TCRs. The invention also relates to TCRs for use as medicaments, in particular TCRs for use in the treatment of cancer.

Background

MAGEA1, also known as melanoma-associated antigen 1 (M: MAGEA gea: MAGEA1, and both as ac, and both as MAGEA1, and both as MAGEA1, and both as well as macia 1, and both asMelanoma-Associated Antigen 1), is a member of the MAGEA gene family that encodes many proteins with high homology to each other. The MAGEA antigen belongs to the family of cancer/testis antigens (CTA) and is the first human tumor-associated antigen identified at the molecular level (Science 1991.254: 1643-1647/reissue in J.Immunol.2007; 178: 2617-2621). The MAGEA gene family includes 12 highly homologous genes located on chromosome Xq 28. Their expression is consistently detected in cancers of different histological origin, such as non-small cell lung cancer, bladder cancer, esophageal cancer, head and neck cancer and sarcoma, as well as myeloma, certain types of breast cancer, and also in germ cells (Front Med (Lausanne). MAGEA1 is a cytosolic/cytoplasmic protein, and peptides derived from MAGEA1 protein are presented in the MHC class I context (i.e. on HLA molecules). More specifically, the presentation of the MAGEA 1-derived epitope on the HLA-a2 molecule suggests antigen immunogenicity and this suitability as a target for cancer immunotherapy. A number of clinical trials (clinicaltralals. gov) have been conducted targeting MAGEA proteins in the field of immunotherapy. The combination of MAGEA1 expression in a variety of tumor entities, such as melanoma and lung cancer (Lancet Oncol 2003; 4: 104-09) and large numbers of patients, with the described immunogenicity of the antigen, makes MAGEA1 a promising target for T-cell mediated immunotherapy. Cancer using Adoptive Cell Transfer (ACT)The principle of symptomatic immunotherapy enables the use of the patient's own immune system for highly tumour-specific cancer treatment. ACT uses ex vivo expanded autologous (patient-derived) T cells genetically engineered to express a T Cell Receptor (TCR) specific for a defined epitope derived from an intracellular protein, such as MAGEA 1. Thus, there remains a need for a highly efficient TCR which targets only the tumor specific/restricted antigen MAGEA1 and thus has particular potential for use in cancer immunotherapy. Thus, a specific TCR for this target MAGEA1 is needed.

Disclosure of Invention

It is an object of the present invention to provide T Cell Receptors (TCRs) specific for MAGEA 1.

In one embodiment, an isolated TCR comprises: a TCR alpha chain comprising CDR1 having the amino acid sequence of SEQ ID NO.2, CDR2 having the amino acid sequence of SEQ ID NO. 3 and CDR3 having the amino acid sequence of SEQ ID NO. 4, a TCR beta chain comprising CDR1 having the amino acid sequence of SEQ ID NO. 5, CDR2 having the amino acid sequence of SEQ ID NO. 6 and CDR3 having the amino acid sequence of SEQ ID NO. 7.

The TCR specifically recognizes the amino acid sequence SEQ ID NO 1 or a fragment thereof.

In particular embodiments, the TCR specifically recognizes the HLA-A2 and/or HLA-A26 bound form of the amino acid sequence of SEQ ID NO.1, preferably HLA-A2 bound form. More specifically, the TCR specifically recognizes the amino acid sequence of SEQ ID No.1 presented by the molecule encoded by a gene selected from the group consisting of HLA-a 02:01, HLA-a 02:02, HLA-a 02:04, HLA-a 02:16, HLA-a 02:17 and HLA-a 26:01, more preferably the TCR specifically recognizes the amino acid sequence of SEQ ID No.1 presented by the molecule encoded by a gene selected from the group consisting of HLA-a 02:01, HLA-a 02:04, HLA-a 02:16 and HLA-a 02:17, even more preferably the TCR specifically recognizes the amino acid sequence of SEQ ID No.1 presented by the molecule encoded by HLA-a 02: 01.

The inventive TCRs are particularly useful because they exhibit high tumor cell recognition, high tumor cell killing, and high functional affinity. In addition, the TCR did not exhibit substantial secretion of the Th2 cytokines IL-4, IL-5 and IL-13, which is beneficial for effective tumor regression.

Preferably, the TCR does not have cross-reactivity with other MAGEA family members.

It is another object of the invention to provide T cells expressing a functional TCR specific for MAGEA 1.

TCRs according to the invention are isolated and/or purified and may be soluble or membrane-bound.

In some embodiments, the amino acid sequence of the TCR can comprise one or more phenotypically silent substitutions. In addition, the TCRs of the invention may be labeled. Useful labels are known in the art and can be coupled to a TCR or TCR variant, optionally through linkers of various lengths, using conventional methods. The term "label" or "labeling group" refers to any detectable label. Additionally or alternatively, the amino acid sequence can be modified to include a therapeutic agent or a pharmacokinetic modifying moiety. The therapeutic agent may be selected from the group consisting of immune effector molecules, cytotoxic agents, and radionuclides. The immune effector molecule may be, for example, a cytokine. The pharmacokinetic modifying moiety may be at least one polyethylene glycol repeat unit, at least one glycol group, at least one sialic acid group, or a combination thereof.

The TCR according to the invention, in particular the soluble form of the TCR, may be modified by attachment of additional functional moieties, e.g. for reducing immunogenicity, increasing hydrodynamic size (size in solution), solubility and/or stability (e.g. by enhancing protection against proteolytic degradation) and/or increasing serum half-life. Other useful functional moieties and modifications include "suicide" or "safety switches" which can be used to turn off or on effector host cells carrying the TCRs of the invention in a patient, or to turn off or on the transgenic TCRs themselves. TCRs having altered glycosylation patterns are also contemplated herein.

It is also contemplated to add a drug or therapeutic entity (e.g., a small molecule compound) to the TCR, particularly to the soluble form of the inventive TCR.

TCRs, particularly soluble forms of the TCRs of the invention, may be additionally modified to introduce additional domains (tags) that facilitate identification, tracking, purification and/or isolation of the corresponding molecules.

In some embodiments, the TCR is of the single chain type, wherein the TCR α chain and the TCR β chain are linked by a linker sequence.

Another aspect of the invention relates to a polypeptide comprising a functional portion of a TCR as described herein, wherein the functional portion comprises one of the amino acid sequences of SEQ ID NOs 4 and 7, preferably wherein the functional portion comprises the amino acid sequences of SEQ ID NOs 2, 3, 4 and 5, 6, 7.

In particular embodiments, the functional moiety comprises a TCR α variable chain and/or a TCR β variable chain.

Particular embodiments relate to multivalent TCR complexes comprising at least two TCRs as described herein. In a more specific embodiment, at least one of the TCRs is associated with a therapeutic agent.

Some embodiments relate to a TCR of the invention expressed as a functional or functional multivalent polypeptide on an effector cell, particularly an immune effector cell, wherein upon binding to the HLA-a 2-bound form of amino acid sequence SEQ ID NO:2, IFN- γ secretion is induced in the aforementioned effector cell expressing the TCR.

Another aspect of the invention relates to a nucleic acid encoding a TCR as described herein or encoding a polypeptide as described above.

Another aspect of the invention relates to a plasmid or vector comprising a nucleic acid of the present application as described above. Preferably, the vector is an expression vector, or a vector suitable for transduction or transfection of a cell, especially a eukaryotic cell. The vector may be, for example, a retroviral vector, such as a gamma-retroviral or lentiviral vector.

Another aspect of the invention relates to a cell expressing a TCR as described herein. The cells may be isolated primary cells or non-naturally occurring.

Another aspect of the invention relates to a cell comprising a nucleic acid as described above or a plasmid or vector as described above. More specifically, the cell may comprise:

a) an expression vector comprising at least one nucleic acid as described above, or

b) A first expression vector comprising a nucleic acid encoding an alpha chain of a TCR as described herein, and a second expression vector comprising a nucleic acid encoding a beta chain of a TCR as described herein.

The cells may be Peripheral Blood Lymphocytes (PBLs) or Peripheral Blood Mononuclear Cells (PBMCs). Typically, the cells are immune effector cells, particularly T cells. Other suitable cell types include γ - δ T cells and NK-like T cells and NK cells, whether modified or unmodified.

Another aspect relates to an antibody or antigen-binding fragment thereof that specifically binds a portion of a TCR as described herein that mediates specificity for MAGEA 1. In a particular embodiment, the portion of the TCR that mediates MAGEA1 specificity comprises the alpha chain CDR3 of SEQ ID NO. 4 and/or the beta chain CDR3 of SEQ ID NO. 7. In some embodiments, the portion of the TCR that mediates MAGEA1 specificity comprises the amino acid sequence of SEQ ID NOs 2, 3, 4 and 5, 6, 7.

Another aspect of the invention relates to a pharmaceutical composition comprising a TCR as described herein, a polypeptide as described herein, a multivalent TCR complex as described herein, a nucleic acid as described herein, a vector as described herein, a cell as described herein, or an antibody as described herein.

Typically, the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier.

Another aspect of the invention relates to a TCR as described herein, a polypeptide as described herein, a multivalent TCR complex as described herein, a nucleic acid as described herein, a vector as described herein, a cell as described herein, or an antibody as described herein, for use as a medicament, in particular for use in the treatment of cancer. The cancer may be a hematological cancer or a solid tumor. The cancer may be selected from the group consisting of: prostate cancer, uterine cancer, thyroid cancer, testicular cancer, renal cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma, non-hodgkin's lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, cervical cancer, colorectal cancer, gastric adenocarcinoma, cholangiocarcinoma, breast cancer, bladder cancer, myeloid leukemia, and acute lymphoblastic leukemia, sarcoma, or osteosarcoma.

Drawings

FIG. 1 shows the effector pairs expressing TCR T15.8-4.3-83 from different donors (donors A or B; grey or black columns) loading MAGEA1_KVL(SEQ ID NO.1) specific recognition of peptide (KVL) by T2 cells. TCR-mediated target recognition of the TCR version with the fully murine C region (murC) as measured by IFN- γ secretion is shown in the left panel and TCR-mediated target recognition of the TCR version with the minimally murine human C region (mmC) is shown in the middle panel. Effector-mediated recognition by the benchmark control MAGEA1-TCR transduction is shown on the right. As a control, T2 cells loaded with the irrelevant ASTN-1 peptide (SEQ ID No.28) were incubated with T cells expressing the inventive TCR or a control TCR (ctrl. magea 1-TCR).

FIG. 2 shows IFN- γ secretion following specific recognition of KVL peptide loaded Lymphoblast Cell Lines (LCLs) by TCR T15.8-4.3-83 expressed in three different effector cell preparations (shaded columns: white, light grey and dark grey). No recognition was observed when unloaded LCL were co-cultured with the corresponding effector cells (solid color columns: white, light gray, dark gray). The boxes represent the HLA alleles responsible for presentation and recognition. In the case of HLA-A heterozygous LCLs (as indicated by the asterisks), recognition of peptides on other HLA-A can be excluded (data not shown).

Figure 3 depicts the recognition of multiple tumor cell lines of different origin mediated by an effector expressing TCR T15.8-4.3-83 (upper panel) or a benchmark ctrl. magea1-TCR (lower panel). The effector cells are from three different donors (donors A, B or C; white, light grey or dark grey columns). Target recognition by MAGEA1-TCR expressing effector cells (solid color columns) or untransduced control cells (shaded columns) was detected by measuring IFN- γ secretion. T2 cells loaded with either the KVL peptide or the irrelevant ASTN1 peptide were used as target controls and are shown on the right side of each figure (T2+ KVL, T2+ ASTN 1).

FIGS. 4A to D show TCR-mediated lysis of tumor cell lines UACC-62(A), NCI-H1703(B), Saos-2(C) and 647-V (D). Lysis of the cells was measured by the disappearance of fluorescently labeled target cells. Testing unmodified (black) or loaded with KVL peptide (grey) as positive pairIrradiated tumor cells. The target was co-cultured for up to 144 hours with effector cells from three different donors (donor A, B or C, filled circles) expressing TCR T15.8-4.3-83 (top panel), baseline Ctrl MAGEA1-TCR (middle panel) or untransduced (bottom panel). Individually cultured target cells are shown in each figure as a reference (open circles). IncuCyte was used every 2 hours^TMThe Zoom System takes and analyzes images depicting the target cell count/well over 144 hours.

Figure 5 shows the recognition of T2 cells loaded with peptides for threonine substitution scans after co-culture with either a baseline ctrl. magea1-TCR transduced (solid color bars) or untransduced (shaded bars) effectors from three different donors (donors A, B or C; white, light gray, or dark gray bars). Recognition of the original MAGEA 1-derived epitope KVL of the TCR is shown on the left. The epitope sequence shown on the x-axis represents the position of the original AA exchange to threonine. The recognition eliminated by the exchange of the original AA is highlighted with a black box. Target recognition was detected by measuring T cell mediated IFN- γ secretion.

FIG. 6 shows the recognition of T2 cells loaded with peptides for threonine substitution scans after co-culture with TCR T15.8-4.3-83 transduced (solid color bars) or untransduced (shaded bars) effectors from three different donors (donors A, B or C; white, light grey or dark grey bars). Recognition of the original MAGEA 1-derived epitope KVL of the TCR is shown on the left. The epitope sequence shown on the x-axis represents the position of the original AA exchange to threonine. The recognition eliminated by the exchange of the original AA is highlighted with a black box. Target recognition was detected by measuring T cell mediated IFN- γ secretion.

FIG. 7 shows TCR T15.8-4.3-83 mediated recognition of HEK 293T cells (A), K562 cells (B) and LCL cells (C). The recognition of target cells is detected by measuring T cell mediated IFN- γ secretion. Cell lines and T2 cells were loaded with MAGEA1 derived KVL peptide (+ KVL) or ASTN1 derived KLY peptide (+ ASTN1) as controls, or transfected with ivtRNA encoding MAGEA1, ASTN1 or 13 MAGE family members, as indicated on the x-axis. The target was co-cultured with TCR transduced (solid color column) or untransduced (shaded column) effector cells from two donors (light grey or dark grey). The background mediated by target cells cultured alone (target cells only) is given as a white column. The dotted line represents the target-independent background secretion of IFN-. gamma.s.

Figure 8 shows TCR T15.8-4.3-83 mediated recognition of unmodified (unloaded) or KVL-loaded target cells derived from healthy tissue or from induced pluripotent stem cells (iPS) representing healthy human tissue. The recognition of target cells was detected by measuring IFN- γ secretion from T cells from three different donors transduced with TCR T15.8-4.3-83 (donors A, B or C; white, light grey or dark grey columns). Background IFN- γ secretion of individual targets (target only) for each cell type is shown as a control, and recognition of T2 cells loaded with epitope KVL recognized by the TCR (T2+ KVL) and background by individual effectors (effector only) are also shown on the right side of the figure.

Fig. 9 shows representative pictures taken using a phase contrast microscope of normal cells co-cultured with effector cells. While all target cells loaded with KVL had significant TCR-mediated lysis (complete disruption of the cell layer), the TCR-expressing effector cells did not lyse unmodified normal cells.

Figure 10 shows the ability of the TCR transgenic effector T cell population to produce IFN- γ in response to MAGEA1 and HLA-a 02:01 double positive tumor cell lines. FH1, FH2, FH3 and FH4 refer to the TCRs disclosed in WO2018/170338, FH1: v α ═ WO '338 SEQ ID No. 7, V β ═ WO' 338 SEQ ID No. 5; FH 2: v α ═ WO '338 SEQ ID No.:11, V β ═ WO' 338 SEQ ID No.:9, FH3 ═ WO '338 SEQ ID No.:15, V β ═ WO' 338 SEQ ID No.: 13; 19, 17, FH1, va, WO' 338, SEQ ID No. 19; R37P1C9 refers to MAGA1-TCR as disclosed in WO 2018/104438.

Figure 11 shows the ability of the TCR transgenic effector T cell population to lyse MAGEA1 and HLA-a 02:01 double positive tumor cell lines. FH1, FH2, FH3 and FH4 refer to the TCRs disclosed in WO2018/170338 (see fig. 10). R37P1C9 refers to MAGA1-TCR as disclosed in WO 2018/104438. UTD: not transduced.

Figure 12 shows the functional affinity of different KVL peptide-specific TCRs. Functional avidity refers to the cumulative strength of multiple affinities of each non-covalent binding interaction between the transgenic TCR and pMHC complexes. FH1, FH2, FH3 and FH4 refer to the TCRs disclosed in WO2018/170338 (see fig. 10). R37P1C9 refers to MAGA1-TCR as disclosed in WO 2018/104438.

FIG. 13 serine residue for systematic replacement of MAGEA1_KVLIndividual amino acids in the peptide (serine scans) were used to determine the key amino acids in the epitope sequence.

FIG. 14 shows the secretion pattern of TH 2-specific cytokines IL-4, IL-5 and IL-13 from T cells transduced with different TCRs. UT: not transfected. T1367 represents a TCR as disclosed in WO 2014/118236. Target only: tumor cells were cultured without TCR.

Detailed Description

Before describing the invention in detail with respect to certain preferred embodiments thereof, the following general definitions are provided.

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

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.

When the term "comprising" is used in the present description and claims, other elements are not excluded. For the purposes of the present invention, the term "consisting of … …" is considered to be a preferred embodiment of the term "comprising". If in the following a group is defined comprising at least a certain number of embodiments, this should also be understood as disclosing a group preferably consisting of only these embodiments.

For the purposes of the present invention, the term "obtained" is considered to be a preferred embodiment of the term "obtainable". If in the following e.g. an antibody is defined as obtainable from a particular source, this is also to be understood as disclosing the antibody obtained from that source.

Unless specifically stated otherwise, a noun that is not defined by a quantitative term includes both the singular and the plural of that noun. The term "about" or "approximately" in the context of the present invention denotes an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term generally denotes a deviation of ± 10%, preferably ± 5%, from the indicated value.

The technical terms are used in accordance with their common sense or meaning to those skilled in the art. If a specific meaning is conveyed to certain terms, the definition of the terms will be given below in the context in which they are used.

TCR background

The TCR is composed of two distinct and independent protein chains, namely the TCR alpha (α) and TCR beta (β) chains. The TCR alpha chain comprises a variable region (V), a connecting region (J) and a constant region (C). The TCR β chain comprises a variable region (V), a diversity region (D), a joining region (J) and a constant region (C). The rearranged v (d) J regions of both TCR α and TCR β chains comprise hypervariable regions (CDRs, complementarity determining regions), of which the CDR3 region determines specific epitope recognition. In the C-terminal region, both TCR α and TCR β chains comprise a hydrophobic transmembrane domain and end with a short cytoplasmic tail.

Generally, TCRs are heterodimers of one α chain and one β chain. Such heterodimers can bind to MHC molecules presenting peptides.

The term "variable TCR α region" or "TCR α variable chain" or "variable domain" in the context of the present invention refers to the variable region of the TCR α chain. The term "variable TCR β region" or "TCR β variable chain" in the context of the present invention refers to the variable region of the TCR β chain.

TCR loci and genes use the International Immunogenetics (IMGT) TCR nomenclature (IMGT database, www.IMGT.org; Giudicelli, V., et al. IMGT/LIGM-DB, the(iii) complex database of immunoglobulin and T cell receptor nucleotide sequences, Nucl. acids Res.,34, D781-D784(2006). PMID: 16381979; t cell Receptor facebook, LeFranc and LeFranc, Academic Press ISBN 0-12-441352-8).

Target

The target of the TCRs described herein is MAGEA1(NCBI reference: NM-004988.4) derived peptide KVL (SEQ ID NO: 1).

TCR specific sequences

Some embodiments relate to isolated TCRs comprising a TCR α chain and a TCR β chain, wherein

The TCR alpha chain comprises a complementarity determining region 3(CDR3) having the sequence of SEQ ID NO. 4,

the TCR beta chain comprises a CDR3 having the amino acid sequence of SEQ ID NO 7.

Particular embodiments relate to an isolated TCR comprising:

-a TCR α chain comprising CDR1 having the amino acid sequence of SEQ ID No.2, CDR2 having the amino acid sequence of SEQ ID No. 3 and CDR3 having the sequence of SEQ ID No. 4;

-a TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID No. 5, CDR2 having the amino acid sequence of SEQ ID No. 6 and CDR3 having the sequence of SEQ ID No. 7.

In some embodiments, the TCR comprises a variable TCR α region having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID No. 8, and a variable TCR β region having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID No. 9.

One preferred embodiment relates to a TCR comprising a variable TCR alpha region having the amino acid sequence of SEQ ID NO. 8 and a variable TCR beta region having the amino acid sequence of SEQ ID NO. 9.

The TCR derived from the T cell clone T15.8-4.3-83 (which was used in the examples in transgenic form) comprised: a TCR alpha chain comprising the complementarity determining region 3(CDR3) having the sequence of SEQ ID NO. 4 and a TCR beta chain comprising the CDR3 having the amino acid sequence of SEQ ID NO. 7. In particular, the inventive TCR comprises a variable TCR α region having the amino acid sequence of SEQ ID No. 8 and a variable TCR β region having the amino acid sequence of SEQ ID No. 9.

As can be seen from the examples, the TCR according to the invention is specific for MAGEA1 and exhibits only very low cross-reactivity to other epitopes or antigens.

Other embodiments relate to isolated TCRs comprising a TCR alpha chain having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID No. 10, and a TCR beta chain having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID No. 11.

Particular embodiments relate to TCRs comprising a TCR alpha chain having the amino acid sequence of SEQ ID No. 10 and a TCR beta chain having the amino acid sequence of SEQ ID No. 11. Thus, the TCRs described herein, which are specific for the complex of HLA-a 02:01 and MAGEA1 peptide of SEQ ID NO 1, comprise a V α chain encoded by the TRAV19 x 01 gene and a V β gene encoded by the TRAV30 x 01 gene.

Other embodiments relate to isolated TCRs comprising a TCR α chain and a TCR β chain, wherein

-the variable TCR alpha region has an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID No. 8 and comprises the CDR3 region shown in SEQ ID No. 3;

the variable TCR β region has an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID No. 9 and comprises the CDR3 region shown in SEQ ID No. 7.

Preference is given to using Vector NTI Advance^TM10 programs (Invitrogen Corporation, Carlsbad CA, USA) to determine percent identity between sequences. The program used a modified Clustal W algorithm (Thompson et al, 1994.Nucl Acids Res.22: pp. 4673-4680; Invitrogen Corporation; Vector NTI Advance^TMUser's Manual,2004, pp 389-662). Percent identity was determined using the standard parameters of the AlignX application.

The TCR according to the invention is isolated or purified. By "isolated" in the context of the present invention is meant that the TCR is not present in the environment in which it originally occurs in nature. In the context of the present invention, "purified" means, for example, that the TCR is free or substantially free of other proteins and non-protein portions of the cell from which it is derived.

In some embodiments, the amino acid sequence of the TCR can comprise one or more phenotypically silent substitutions.

"phenotypically silent substitutions" are also referred to as "conservative amino acid substitutions". The concept of "conservative amino acid substitutions" is understood by the skilled person and preferably means 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 may occur spontaneously, be introduced by random mutagenesis, or may be introduced by site-directed mutagenesis. These changes can be made without destroying the essential characteristics of these polypeptides. One of ordinary skill can readily and routinely screen variant amino acids and/or nucleic acids encoding them to determine whether these variants significantly reduce or disrupt ligand binding capability by methods known in the art.

The skilled artisan understands that nucleic acids encoding TCRs can also be modified. Useful modifications in the overall nucleic acid sequence include codon optimization of the sequence. Changes can be made that result in conservative substitutions within the expressed amino acid sequence. These changes can be made in the complementarity determining regions and non-complementarity determining regions of the amino acid sequences of the TCR chain which do not affect function. Generally, additions and deletions should not be made in the CDR3 region.

According to some embodiments of the invention, the amino acid sequence of the TCR is modified to comprise a detectable label, a therapeutic agent, or a pharmacokinetic modifying moiety.

Non-limiting examples of detectable labels are radioactive labels, fluorescent labels, nucleic acid probes, enzymes, and contrast agents. Therapeutic agents that may be associated with the TCR include radioactive compounds, immunomodulators, enzymes or chemotherapeutic agents. The therapeutic agent may be encapsulated by liposomes associated with the TCR, so that the compound may be slowly released at the target site. This will avoid damage during in vivo transport and ensure that the therapeutic agent (e.g. toxin) has the greatest effect after binding of the TCR to the relevant antigen presenting cell. Other examples of therapeutic agents are:

peptide cytotoxins, i.e., proteins or peptides having the ability to kill mammalian cells, such as ricin, diphtheria toxin, pseudomonas exotoxin A, DNase, and RNase. Small molecule cytotoxic agents, i.e., compounds having the ability to kill mammalian cells with a molecular weight of less than 700 daltons. Such compounds may contain toxic metals capable of having a cytotoxic effect. Furthermore, it is understood that these small molecule cytotoxic agents also include prodrugs, i.e., compounds that decompose or convert under physiological conditions to release the cytotoxic agent. Such agents may include, for example, docetaxel, gemcitabine, cisplatin, maytansine derivatives, lacrimycin (rachelmycin), calicheamicin (calicheamicin), etoposide, ifosfamide, irinotecan, porfilum sodium photosensitizer ii (porfimer sodium dibenzofrin ii), temozolomide, topotecan, trimetrexate gluconate, mitoxantrone, auristatin e (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 the radionuclide with the TCR or derivative thereof may be performed, for example, by a chelator; immunostimulants, also known as immunostimulants, are immune effector molecules that stimulate an immune response. 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-CD 3, anti-CD 28, or anti-CD 16); a surrogate protein scaffold with antibody-like binding properties; superantigens, i.e., antigens and mutants thereof that cause nonspecific activation of T cells, resulting in polyclonal T cell activation and release of a number of cytokines; chemokines, such as complement activators like IL-8, platelet factor 4, melanoma growth stimulating protein, etc.; heterologous protein domains, allogeneic protein domains, viral/bacterial peptides.

Antigen receptor molecules on human T lymphocytes (T cell receptor)A somatic molecule) to a complex of CD3(T3) molecules on the surface of a cell. Interference of this complex with an anti-CD 3 monoclonal antibody induces T cell activation. Thus, some embodiments relate to a TCR as described herein associated (typically by fusion to the N-or C-terminus of the α or β chain) with an anti-CD 3 antibody or a functional fragment or variant of said anti-CD 3 antibody. Antibody fragments and variants/analogs suitable for use in the compositions and methods described herein include minibodies, Fab fragments, F (ab')₂Fragments, dsFv and scFv fragments, Nanobodies^TM(ablynx (belgium), molecules comprising synthetic single immunoglobulin variable heavy domain derived from camelid (e.g. camel or llama) antibodies) and domain antibodies (molecules comprising affinity matured single immunoglobulin variable heavy domain or immunoglobulin variable light domain (domanis (belgium)) or alternative protein scaffolds exhibiting antibody-like binding properties, such as Affibodies (comprising an engineered protein a scaffold (affibody)) or anticollics (comprising an engineered Anticalins pins (German)).

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

The pharmacokinetic modifying moiety may be, for example, at least one polyethylene glycol repeat unit, at least one diol group, at least one sialic acid group, or a combination thereof. The association of the at least one polyethylene glycol repeat unit, the at least one diol group, the at least one sialic acid group can be caused in a variety of ways known to those skilled in the art. In a preferred embodiment, the unit is covalently linked to the TCR. TCRs according to the invention may 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 may effect beneficial changes to the pharmacokinetic profile of the therapeutic agent, such as improved plasma half-life, reduced or enhanced immunogenicity, and improved solubility.

TCRs according to the invention may be soluble or membrane bound. The term "soluble" refers to a TCR in soluble form (i.e., without a transmembrane or cytoplasmic domain), e.g., for use as a targeting agent for delivery of a therapeutic agent to an antigen presenting cell. For stability, the soluble α β heterodimeric TCR preferably has an introduced disulfide bond between residues of the respective constant domains, as described in, for example, WO 03/020763. One or both constant domains present in the α β heterodimers of the invention may be truncated at one or both C-termini, e.g., up to 15, or up to 10, or up to 8 or fewer amino acids. For use in adoptive therapy, α β heterodimeric TCRs can, for example, be transfected as full-length chains with both cytoplasmic and transmembrane domains. The TCR may comprise a disulfide bond corresponding to that found in nature between the respective α and β constant domains, in addition or alternatively, a non-native disulfide bond may be present.

Thus, TCRs according to the invention, in particular soluble forms of TCRs, may be modified by the attachment of additional functional moieties, e.g. for reducing immunogenicity, increasing hydrodynamic size (size in solution), solubility and/or stability (e.g. by enhancing protection against proteolytic degradation) and/or increasing serum half-life.

Other useful functional moieties and modifications include "suicide" or "safety switches" which can be used to turn off effector host cells carrying the TCRs of the invention in a patient. One example is that described by Gargett and Brown Front pharmacol.2014; 5:235, inducible caspase 9(iCasp9) "safety switch". Briefly, effector host cells are modified by well-known methods to express caspase 9 domains, whose dimerization is dependent on small molecule dimerization drugs such as AP1903/CIP, and results in rapid induction of apoptosis in the modified effector cells. This system is described, for example, in EP2173869(a 2). 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 using anti-CD 20 antibodies or a myc tag (Kieback et al Proc Natl Acad Sci U S A.2008 Jan 15; 105(2): 623-8).

TCRs having altered glycosylation patterns are also contemplated herein. As known in the art, the glycosylation pattern can depend on the amino acid sequence (e.g., the presence or absence of particular glycosylated amino acid residues as discussed below) and/or the host cell or organism producing the protein. Glycosylation of polypeptides is typically N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The N-linked glycosylation site is conveniently added to the binding molecule by altering the amino acid sequence to comprise one or more tripeptide sequences selected from asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline). O-linked glycosylation sites can be introduced by adding or replacing one or more serine or threonine residues to the starting sequence.

Another method of TCR glycosylation is by chemical or enzymatic coupling of glycosides to proteins. Depending on the coupling mode used, the sugar may 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 the carbohydrate moiety present on the binding molecule) can be accomplished chemically, for example by exposing the TCR to trifluoromethanesulfonic acid, or enzymatically by using endo-and exo-glycosidases.

It is also contemplated to add drugs such as small molecule compounds to the TCR, particularly the soluble form of the inventive TCR. Attachment may be achieved by covalent bonds or non-covalent interactions, such as by electrostatic forces. Various linkers known in the art can be used to form the drug conjugate.

TCRs, particularly soluble forms of the TCRs of the invention, may be additionally modified to introduce additional domains (tags) that facilitate identification, tracking, purification and/or isolation of the corresponding molecules. Thus, in some embodiments, the TCR α chain or TCR β chain may be modified to comprise an epitope tag.

Epitope tags are useful examples of tags that can be incorporated into the TCRs of the invention. An epitope tag is a short stretch of amino acids that allows binding of specific antibodies, thus enabling identification and tracking of binding and movement of soluble TCRs or host cells or cultured (host) cells in a patient. Detection of the epitope tag, and thus the tagged TCR, can be achieved using a variety of different techniques.

The tag can further 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.

Generally, in some cases, the TCR may be modified with various mutations that alter the affinity and off-rate (off-rate) of the TCR with the target antigen. In particular, the mutation may increase affinity and/or decrease off-rate. Thus, the TCR may be mutated in at least one CDR and its variable domain framework region.

However, in a preferred embodiment, the CDR regions of the TCR are not modified or affinity matured in vitro, for example for the TCR receptors in the examples. This means that the CDR regions have naturally occurring sequences. This may be advantageous because in vitro affinity maturation may lead to immunogenicity on the TCR molecule. This may lead to a reduction or inactivation of the therapeutic effect and the production of therapeutic anti-drug antibodies and/or cause side effects.

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

Theoretically, unpredictable TCR specificities with the risk of cross-reactivity may arise due to mismatches between endogenous and exogenous TCR chains. To avoid mismatches in TCR sequences, recombinant TCR sequences can be modified to include the most mouse-ized C α and C β regions, a technique that has been shown to be effective in enhancing correct pairing of a variety of differently transduced TCR chains. Murine humanization of TCRs (i.e., the exchange of human constant regions in the α and β chains by their murine counterparts) is a technique commonly used to improve the cell surface expression of TCRs in host cells. Without wishing to be bound by a particular theory, it is believed that the murine TCR associates more efficiently with the CD3 co-receptor; and/or preferentially pair with each other and are less likely to form mixed TCRs on human T cells that have been genetically modified ex vivo to express TCRs of the desired antigen specificity, but still retain and express their "original" TCRs.

Nine amino acids responsible for increasing the expression of the murine TCR have been identified (Sommermeyer and Uckert, J Immunol.2010 Jun 1; 184(11):6223-31) and substitution of one or all amino acid residues in the TCR alpha and/or beta chain constant regions with their murine counterpart residues is contemplated. This technique is also referred to as "minimurization" and has 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.

Some embodiments relate to an isolated TCR as described herein, wherein the TCR is of the single chain type, wherein the TCR a chain and the TCR β chain are linked by a linker sequence.

Suitable single chain TCR formats comprise a first segment consisting of an amino acid sequence corresponding to the variable TCR α region, a second segment 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 TCR β chain constant region extracellular sequence, and a linker sequence linking the C-terminus of the first segment to the N-terminus of the second segment. Alternatively, the first segment may be comprised of an amino acid sequence corresponding to the TCR β chain variable region and the second segment may be comprised of an amino acid sequence corresponding to the TCR α chain variable region sequence fused to the N-terminus of an amino acid sequence corresponding to the TCR α chain constant region extracellular sequence. The single chain TCR described above may further comprise a disulfide bond between the first and second chains, and wherein the length of the linker sequence and the position of the disulfide bond are such that the variable domain sequences of the first and second segments are mutually oriented substantially as in the native T cell receptor. More specifically, the first segment may be composed of an amino acid sequence corresponding to the TCR α chain variable region sequence fused to the N-terminus of an amino acid sequence corresponding to the TCR α chain constant region extracellular sequence, the second segment may be composed of an amino acid sequence corresponding to the TCR β chain variable region fused to the N-terminus of an amino acid sequence corresponding to the TCR β chain constant region extracellular sequence, and a disulfide bond may be provided between the first and second chains. The linker sequence may be any sequence which does not impair TCR function.

In the context of the present invention, a "functional" TCR α and/or β chain fusion protein shall mean a TCR or TCR variant which retains at least essential biological activity, e.g., is modified by addition, deletion or substitution of amino acids. In the case of the α and/or β chains of the TCR, this would mean that both chains are still capable of forming a T cell receptor (either with unmodified α and/or β chains or with another fusion protein of the invention α and/or β chains), which exerts its biological function, in particular binding to the specific peptide-MHC complex of the TCR, and/or functional signal transduction following specific peptide: MHC interaction.

In particular embodiments, the TCR may be modified to a functional T Cell Receptor (TCR) alpha and/or beta chain fusion protein, wherein the epitope-tag is 6 to 15 amino acids, preferably 9 to 11 amino acids in length. In another embodiment, the TCR may be modified to a functional T Cell Receptor (TCR) a and/or β chain fusion protein, wherein the T Cell Receptor (TCR) a and/or β chain fusion protein comprises two or more epitope-tags, either spaced apart or directly in tandem. Embodiments of the fusion protein may comprise 2, 3, 4, 5, or even more epitope-tags, as long as the fusion protein retains its biological activity/activities ("functionality").

Preferred are functional T Cell Receptor (TCR) alpha and/or beta chain fusion proteins according to the invention, wherein the epitope-tag is selected from, but not limited to, CD20 or Her 2/neu-tag, or other conventional tags such as myc-tag, FLAG-tag, T7-tag, HA (hemagglutinin) -tag, His-tag, S-tag, GST-tag or GFP-tag. myc, T7, GST, GFP tags are epitopes derived from existing molecules. In contrast, FLAG is a synthetic epitope tag designed for high antigenicity (see, e.g., U.S. patent nos. 4,703,004 and 4,851,341). The use of myc tags may be preferred because high quality reagents are available for their detection. The epitope tag may of course have one or more additional functions beyond antibody recognition. The sequences of these tags are described in the literature and are well known to those skilled in the art.

TCR variants

Another aspect of the invention relates to a polypeptide comprising a functional portion of a TCR as described herein, wherein the functional portion comprises at least one of the amino acid sequences of SEQ ID NOs 4 and 7.

The functional moiety may mediate binding of the TCR to an antigen, particularly to an antigen-MHC complex.

In one embodiment, the functional moiety comprises a TCR α variable chain and/or a TCR β variable chain as described herein.

The TCR variant molecule may have the binding properties of a TCR receptor, but may be combined with the signalling domain of effector cells (other than T cells), in particular NK cells. Thus, some embodiments relate to proteins comprising a functional portion of a TCR as described herein in combination with a signaling domain of an effector cell (e.g., NK cell).

Another aspect of the invention relates to a multivalent TCR complex comprising at least two TCRs as described herein. In one embodiment of this aspect, at least two TCR molecules are linked by a linker moiety to form a multivalent complex. Preferably, the complex is water soluble and therefore the linker moiety should be chosen accordingly. Preferably, the linker moiety is capable of attaching to a defined position on the TCR molecule, thereby minimizing the structural diversity of the complex formed. One embodiment of this aspect is provided by a TCR complex of the invention, wherein the polymer chain or peptide linker sequence extends between amino acid residues of each TCR which are not located in the TCR variable region sequence. Since the complexes of the invention may be used in medicine, the linker moiety should be selected with due regard to their pharmaceutical suitability, e.g. their immunogenicity. Examples of linker moieties that meet the above-described desired criteria are known in the art, e.g., in the art of linking antibody fragments.

Examples of linkers are hydrophilic polymers and peptide linkers. An example of a hydrophilic polymer is a polyalkylene glycol. The most common of these are based on polyethylene glycol or PEG. However, others are based on other suitable, optionally substituted, polyalkylene glycols, including polypropylene glycol, and copolymers of ethylene glycol and propylene glycol. The peptide linker comprises a chain of amino acids whose function is to create a simple linker or multimerisation domain to which the TCR molecule can be linked.

One embodiment relates to a multivalent TCR complex, wherein at least one of the TCRs is associated with a therapeutic agent.

Cytokine and chemokine release

Some embodiments relate to an isolated TCR as described herein, a polypeptide as described herein, a multivalent TCR complex as described herein, wherein IFN- γ secretion is induced by binding of a TCR of the invention expressed on an effector cell to an HLA-a 02-binding form of an amino acid sequence selected from the group consisting of SEQ ID NO: 1.

IFN- γ secretion induced by binding of a TCR of the invention expressed on effector cells to an HLA-A02-binding form of an amino acid sequence selected from the group consisting of SEQ ID NO:1 may be in excess of 500pg/ml, such as in excess of 1000pg/ml, in excess of 2000pg/ml, more preferably in excess of 4000pg/ml, most preferably even in excess of 6000 pg/ml. IFN- γ secretion may be at least 5 fold higher when bound to the HLA-A02-binding form of the amino acid sequence of SEQ ID NO:1 compared to the binding to the HLA-A02-binding form of an unrelated peptide (e.g., SEQ ID NO: 28).

The "effector cells" may be Peripheral Blood Lymphocytes (PBLs) or Peripheral Blood Mononuclear Cells (PBMCs). Typically, the effector cell is an immune effector cell, particularly a T cell. Other suitable cell types include gamma-delta T cells and NK-like T cells.

The invention also relates to a method of identifying a TCR, or a fragment thereof, which binds to the target amino acid sequence SEQ ID No.1 or HLA-a 02, preferably or HLA-a 02:01 binding form thereof, wherein the method comprises contacting a candidate TCR, or a fragment thereof, with the amino acid sequence of SEQ ID No.1 or HLA-a 02, preferably or HLA-a 02:01 binding form thereof, and determining whether the candidate TCR, or fragment thereof, binds to the target and/or mediates an immune response.

Whether a candidate TCR, or fragment thereof, mediates an immune response can be determined, for example, by measuring cytokine secretion, such as IFN- γ secretion. As described above, cytokine secretion can be measured by an in vitro assay in which K562 cells (or other APCs) transfected with the ivtRNA encoding the amino acid sequence SEQ ID NO:1 are incubated with CD8+ -rich PBMC expressing a TCR or a molecule comprising a fragment of the TCR to be investigated.

In a particular embodiment, the inventive TCRs exhibit no significant secretion of the Th2 cytokines IL-4, IL-5 and IL-13. This may be advantageous because a Th2 cytokine response may increase tumor progression (Jager MJ, Desjardins L,t, Damato BE (eds): Current Concepts in Uveal Melanoma. Dev Ophthalmol. Basel, Karger,2012, Vol.49, p.137-; 41(8):369-378).

Nucleic acid, vector

Another aspect of the invention relates to a nucleic acid encoding a TCR as described herein or a polynucleotide encoding a TCR as described herein.

"nucleic acid molecule" generally refers to a polymer of DNA or RNA, which may be single-or double-stranded, synthetic or obtained (e.g., isolated and/or purified) from a natural source, which may comprise natural, non-natural or altered nucleotides, and which may comprise natural, non-natural or altered internucleotide linkages, such as phosphoramidate (phosphoroamidate) linkages or phosphorothioate linkages, rather than phosphodiesters found between nucleotides of unmodified oligonucleotides. Preferably, the nucleic acids described herein are recombinant. As used herein, the term "recombinant" refers to a molecule that is either (i) constructed outside a living cell by linking a natural or synthetic nucleic acid fragment to a nucleic acid molecule that is replicable in the living cell, or (ii) produced by the replication of those described in (i) above. For the purposes herein, replication may be in vitro or in vivo. 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 Genscript, Thermo Fisher, etc.). For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides), see, e.g., Sambrook et al. The nucleic acid may comprise any nucleotide sequence encoding any recombinant TCR, polypeptide, or protein, or a functional portion or functional variant thereof.

The present disclosure also provides variants of the isolated or purified nucleic acid, 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 the nucleotide sequence encoding a TCR described herein. Such variant nucleotide sequences encode functional TCRs that specifically recognize MAGEA 1.

The present disclosure also provides an isolated or purified nucleic acid comprising a nucleotide sequence that is complementary to a nucleotide sequence of any of the nucleic acids described herein or a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of any of the nucleic acids described herein.

Nucleotide sequences that hybridize under stringent conditions preferably hybridize under high stringency conditions. By "high stringency conditions" is meant that a nucleotide sequence specifically hybridizes to a target sequence (the nucleotide sequence of any of the nucleic acids described herein) in a detectably greater amount than non-specific hybridization. High stringency conditions include conditions that can distinguish between polynucleotides having exactly complementary sequences or polynucleotides that contain only a few discrete mismatches, and random sequences that happen to have several small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such small regions of complementarity are easier to melt than full-length complementarity of 14-17 bases or more, and high stringency hybridisation makes them easy to distinguish. Relatively high stringency conditions will include, for example, low salt and/or high temperature conditions, such as provided by about 0.02-0.1M NaCl, or equivalent, at a temperature of about 50-70 ℃. Such high stringency conditions are hardly tolerant of mismatches between the nucleotide sequence and the template or target strand, if any, and are particularly suitable for detecting expression of any of the TCRs described herein. It is generally believed that conditions can be made more stringent by adding increased amounts of formamide.

The nucleic acid encoding the TCR may be modified as already described elsewhere herein. Useful modifications in the overall nucleic acid sequence may be codon optimization. Changes can be made that result in conservative substitutions within the expressed amino acid sequence. These changes can be made in the complementarity determining regions and non-complementarity determining regions of the amino acid sequences of the TCR chain which do not affect function. In general, additions and deletions should not be made in the CDR3 region.

Another embodiment relates to a vector comprising a nucleic acid encoding a TCR as described herein.

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

A "vector" is any molecule or composition capable of carrying a nucleic acid sequence into a suitable host cell in which synthesis of the encoded polypeptide can occur. Typically and preferably, the vector is a nucleic acid that has been engineered to introduce a desired nucleic acid sequence (e.g., a nucleic acid of the invention) using recombinant DNA techniques known in the art. The carrier may comprise DNA or RNA and/or comprise liposomes. The vector may be a plasmid, shuttle vector, phagemid, cosmid, expression vector, retroviral vector, lentiviral vector, adenoviral vector or a particle and/or vector for gene therapy. A vector may include a nucleic acid sequence, such as an origin of replication, that allows it to replicate in a host cell. The vector may also include 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 invention operably linked to a sequence allowing the expression of said nucleic acid.

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

Cell, cell line

Another aspect of the invention relates to a cell expressing a TCR as described herein.

In some embodiments, the cell is isolated or non-naturally occurring.

In particular embodiments, the cell may comprise a nucleic acid encoding a TCR as described herein or a vector comprising the nucleic acid.

In a cell, the above-described vector comprising a nucleic acid sequence encoding the above-described TCR can be introduced or the ivtRNA encoding the TCR can be introduced. The cells may be peripheral blood lymphocytes, such as T cells. Cloning and exogenous expression methods for TCRs are described, for example, in Engels et al (Relay or organization of cancer is predicted by peptide-major histocompatibility complex affinity. cancer Cell,23(4), 516-26.2013). For example, transduction of primary human T cells with lentiviral vectors in "simplified production and concentration of viral vectors to achievehigh conversion in primary human T cells" BMC biotechnol.2013 of Cribbs; 13: 98.

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 noted that the introduction or transfer of nucleic acid sequences is not limited to the above-described methods, but may be accomplished by any number of means, including electroporation, microinjection, biolistic delivery, lipofection, superstaining, and infection by a retrovirus or other virus suitable for transduction or transfection as mentioned.

Some embodiments relate to a cell comprising:

a) an expression vector comprising at least one nucleic acid as described herein, or

b) A first expression vector comprising a nucleic acid encoding an alpha chain of a TCR as described herein, and a second expression vector comprising a nucleic acid encoding a beta chain of a TCR as described herein.

In some embodiments, the cell is a Peripheral Blood Lymphocyte (PBL) or a Peripheral Blood Mononuclear Cell (PBMC). The cell may be a natural killer cell or a T cell. Preferably, the cell is a T cell. The T cells may be CD4+ or CD8+ T cells. In some embodiments, the cell is a stem cell-like memory T cell.

Stem cell-like memory T cells (TSCMs) are a subset of less differentiated CD8+ or CD4+ T cells characterized by their ability to self-renew and persist for a long period of 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), some of which remain quiescent (Flynn et al, Clinical & Translational Immunology (2014.) these remaining TSCM cells exhibit the ability to establish persistent immunological memory in vivo, and thus are considered an important T cell subset for adoptive T cell therapy (Lugli et al, Nature Protocols 8, 33-42 (2013) gattoni et al, nat. med.2011 Oct; 17(10): 1290) immunomagnetic selection can be used to limit the T cell pool to stem cell memory T cell subtypes (see riddel et al 2014, Cancer Journal 20(2): 141-44).

Antibodies targeting TCR

Another aspect of the invention relates to an antibody or antigen-binding fragment thereof that specifically binds to a portion of the TCR that mediates the specificity of MAGEA1 as described herein. In one embodiment, the portion of the TCR that mediates MAGEA1 specificity comprises the alpha chain CDR3 of SEQ ID NO. 4 and/or the beta chain CDR3 of SEQ ID NO. 7.

The antibody or antigen binding fragment thereof can modulate the activity of the TCR. Which may or may not block TCR binding to MAGEA 1. Which can be used to modulate the therapeutic activity of the TCR or for diagnostic purposes.

Pharmaceutical compositions, drug treatments and kits

Another aspect of the invention relates to a pharmaceutical composition comprising a TCR as described herein, a polypeptide comprising a functional portion of the TCR, a multivalent TCR complex as described herein, a nucleic acid encoding a TCR, a vector comprising the nucleic acid, a cell comprising the TCR, or an antibody that specifically binds to a portion of the TCR as described herein.

Those active ingredients of the invention are preferably used in a pharmaceutical composition that can treat or at least alleviate a disease in a dosage amount that is mixed with an acceptable carrier or carrier material. Such compositions may contain, in addition to the active ingredient and carrier, filler materials, salts, buffers, stabilizers, solubilizers, and other materials known in the art.

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 ingredient. The choice of carrier depends on the application.

The pharmaceutical composition may comprise additional ingredients that enhance the activity of the active ingredient or supplement therapy. Such additional ingredients and/or factors may be part of the pharmaceutical composition to achieve a synergistic effect or to minimize adverse or undesirable effects.

The formulation or preparation and application/administration techniques of the active ingredients of the present invention are disclosed in "Remington's Pharmaceutical Sciences", Mack Publishing co. Suitable applications are parenteral applications, such as intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intra-nodal, intraperitoneal or intratumoral injections. Intravenous injection is the preferred treatment for patients.

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

The injectable composition is a pharmaceutically acceptable fluid composition comprising at least one active ingredient, such as an expanded population of T cells expressing a TCR (e.g., autologous or allogeneic to the patient to be treated). The active ingredient is typically dissolved or suspended in a physiologically acceptable carrier, and the composition may additionally contain minor amounts of one or more non-toxic auxiliary substances, such as emulsifiers, preservatives, pH buffers and the like. Such injectable compositions useful for use with the fusion proteins of the present disclosure are conventional; suitable formulations are well known to those of ordinary skill in the art.

Typically, the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier.

Thus, another aspect of the invention relates to a TCR as described herein, a polypeptide comprising a functional portion of said TCR, a multivalent TCR complex as described herein, a nucleic acid encoding said TCR, a vector comprising said nucleic acid, a cell comprising said TCR, or an antibody that specifically binds to a portion of a TCR as described herein, for use as a medicament.

Some embodiments relate to a TCR as described herein, a polypeptide comprising a functional portion of the TCR, a multivalent TCR complex as described herein, a nucleic acid encoding the TCR, a vector comprising the nucleic acid, a cell comprising the TCR, for use in treating cancer.

In one embodiment, the cancer is a hematologic cancer or a solid tumor.

Hematologic cancers, also known as blood cancers, do not form solid tumors and are therefore dispersed in the body. Examples of hematological cancers are leukemia, lymphoma or multiple myeloma. There are two major types of solid tumors, sarcomas and carcinomas. Sarcomas are tumors such as blood vessels, bones, adipose tissue, ligaments, lymphatic vessels, muscles, or tendons.

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

Also contemplated herein are pharmaceutical compositions and kits comprising one or more of: (i) an isolated TCR as described herein; (ii) a viral particle comprising a nucleic acid encoding a recombinant TCR; (iii) an immune cell, e.g., a T cell or NK cell, modified to express a recombinant TCR as described herein; (iv) a nucleic acid encoding a recombinant TCR as described herein. In some embodiments, the disclosure provides compositions comprising lentiviral vector particles (or T cells that have been modified to express recombinant TCRs using vector particles described herein) comprising a nucleotide sequence encoding a recombinant TCR described herein. Such compositions can be administered to a subject in the methods of the present disclosure as further described herein.

Compositions comprising modified T cells as described herein may be used in methods and compositions for adoptive immunotherapy according to known techniques, or variants thereof that will be apparent to those of skill in the art based on this disclosure.

In some embodiments, the cells are formulated by first harvesting them from their culture medium, then washing and concentrating the cells in a medium and container system ("pharmaceutically acceptable" carrier) suitable for administration in a therapeutically effective amount. Suitable infusion media may be any isotonic medium preparation, typically physiological saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but 5% aqueous glucose or ringer's lactate may also be used. The infusion medium may be supplemented with human serum albumin.

The number of cells in the composition for effective treatment is typically greater than 10 cells, up to 10⁶Up to and including 10⁸Or 10⁹A cell, and may exceed 10¹⁰And (4) cells. The number of cells will depend on the intended end use of the composition and the cell type contained therein. For the uses provided herein, the volume of cells is typically one liter or less, and may be 500ml or less, and even 250ml or 100ml or less. Thus, the desired cell density is typically greater than 10⁶Individual cells/ml and usually greater than 10⁷Individual cell/ml, usually 10⁸Individual cells/ml or greater. Clinically relevant numbers of immune cells can be assigned to multiple infusions, with a cumulative equal to or exceeding 10⁹、10¹⁰Or 10¹¹And (4) cells. The pharmaceutical compositions provided herein can 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 formulated by conventional methods and may be administered to a subject in a suitable dosage. Stabilizers such as lipids, nuclease inhibitors, polymers and chelating agents can prevent degradation of the composition in vivo. In compositions intended for administration by injection, surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, suspending agents, and the like,One or more of a stabilizer and an isotonic agent.

A recombinant TCR as described herein or a viral vector particle comprising a nucleotide sequence encoding a recombinant TCR provided herein can be packaged as a kit. Kits may optionally comprise one or more components, such as instructions for use, devices, and additional reagents, as well as components for performing the methods, such as tubes, containers, and syringes. An exemplary kit can comprise a nucleic acid encoding a recombinant TCR, a recombinant TCR polypeptide, or a virus provided herein, and can optionally include instructions for use, a device for detecting a virus in a subject, and a device for administering the composition to a subject.

Also contemplated herein are kits comprising a polynucleotide encoding a gene of interest (e.g., a recombinant TCR). Also contemplated herein are kits comprising a viral vector encoding a sequence of interest (e.g., a recombinant TCR) and optionally a polynucleotide sequence encoding an immune checkpoint inhibitor.

Kits contemplated herein also include kits for carrying out a method of detecting the presence of a polynucleotide encoding any one or more of the TCRs disclosed herein. In particular, such diagnostic kits may include components for performing deep sequencing to detect appropriate amplification and detection primers and other related reagents that encode the TCRs disclosed herein. In other embodiments, the kits herein may comprise reagents, such as antibodies or other binding molecules, for detecting the TCRs disclosed herein. The diagnostic kit can further comprise instructions for determining the presence of a polynucleotide encoding a TCR disclosed herein or for determining the presence of a TCR disclosed herein. The kit may further comprise instructions. The specification generally includes tangible expressions that describe: the components contained in the kit and methods of administration include methods for determining the appropriate state, appropriate dosage, and appropriate method of administration for a subject. The instructions may also include instructions to monitor the subject for the duration of the treatment period.

The kits provided herein can further comprise a device for administering the compositions described herein to a subject. Any of a variety of devices known in the art for administering drugs or vaccines can 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 dropper tubes. Typically, the device used to administer the virus of the kit will be compatible with the virus of the kit; for example, a needle-free injection device, such as a high pressure injection device, may be, but is typically not, contained in a kit in which the virus is not destroyed by the high pressure injection.

The kits provided herein may further comprise a device for administering a compound, e.g., a T cell activator or stimulator, or a TLR agonist, e.g., a TLR4 agonist, to a subject. Any of a variety of devices known in the art for administering a drug to a subject can be included in the kits provided herein. Exemplary devices include hypodermic needles, intravenous needles, catheters, needle-free injection devices, but are not limited to, hypodermic needles, intravenous needles, catheters, needle-free injection devices, inhalers, and liquid dispensers, such as droppers. Generally, the device used to administer the kit compound will be compatible with the desired method of administration of the compound.

Experiment of

1. Experiment: isolation of MAGEA 1-specific TCR

To isolate T cells specific for the MAGEA 1-derived epitope and restricted to any MHC molecule of interest, an in vitro priming method was used. Priming System mature dendritic cells (mDCs) from HLA-A02: 01 negative donors were used as antigen presenting cells to initiate antigen specific T cell responses and autologous CD8⁺Expansion of T cells. In vitro transcribed RNA (ivtRNA) encoding a minigene (SEQ ID NO:27) and translated to an amino acid sequence comprising MAGEA1 derived peptide KVLEYVIKV (as referenced to SEQ ID NO:1) was used as the source of the specific antigen. At the same time, human HLA-a 02:01 encoding ivtRNA was used as a source of restriction elements transfected into mdcs to establish allogeneic priming from this dedicated HLA allele (as described in WO 2007/017201). Following electroporation into mdcs, the minigene encoding ivtRNA is translated into protein, which is subsequently processed and presented as a peptide by the co-transfected mDC expressing the transgenic HLA-a 02:01 molecule. T cells are derived from the sameIn vitro co-culture of donor ivtRNA transfected mdcs resulted in de novo induction of antigen-specific T cells that were the source of the corresponding TCR. After expansion, antigen-specific T cells can be enriched and single cell cloned by limiting dilution or FACS-based single cell sorting.

1.1 Experimental layout

Dendritic cell priming of T cells for isolation and identification of high affinity TCRs was accomplished using peptide presentation by allogeneic HLA-a 02:01 molecules according to the following protocol:

mature dendritic cells were generated within 8 days using a suitable maturation mixture of DCs according to Jonuleit et al (Jonuleit et al 1997, Eur.J.Immunol.1997,27: 3135-3142). Antigen presenting cells (8 day mdcs) were from healthy donors and cells were transfected by electroporation with 20 μ g ivtRNA encoding MG _ X1 and 20 μ g ivtRNA encoding an allogeneic HLA molecule (HLA-a × 02: 01). The prepared mdcs were then autologous CD-rich 8 with healthy donors⁺In a ratio of 1:20 in a suitable cell culture medium supplemented with IL-7 (5 ng/ml on day 0) and IL-2 (50U/ml every two to three days) at 37 ℃ (6% CO₂) The co-culture was carried out for about 14 days. Subsequently, HLA-A02: 01MAGEA1 was used_278-286Identification of the multimer (ProImmune) MAGEA1_278-286Specific cells and then isolated by single cell sorting using FACS techniques.

1.2 results

The in vitro priming method described led to the identification of T cell clones expressing candidate TCR T15.8-4.3-83. TCR sequences were identified using NGS analysis, reconstituted and transgene expressed in appropriate effector T cells to fully characterize the TCR in terms of function and safety.

2. Experiment: epitope specificity

To ensure the function of the transgenically expressed recombinant TCR, TCR-transduced effector T cells were co-cultured with peptide-loaded T2 cells (target cells). TCR-mediated recognition of the MHC complex results in the activation of TCR-expressing T cells and secretion of certain cytokines (e.g., IFN-. gamma.). To analyze secreted IFN-. gamma.in the co-culture supernatants, an ELISA was performed.

2.2 Experimental layout

T2 cells expressing HLA-A02 were loaded with saturating amounts of peptides (10)^-5M). T2 cells loaded with MAGEA 1-derived KVL peptide (SEQ ID NO:1) served as positive targets, and T2 cells loaded with the irrelevant ASTN1 peptide (SEQ ID NO:28) served as negative targets. As effector cells, populations of CD 8-rich T cells from two different donors were transduced to stably express recombinant TCR T15.8-4.3-83. Effectors were prepared to express two different versions of TCR T15.8-4.3-83, one version with murine C region (murC) and the other version with human minimal murine C region (mmC). The control MAGEA1-TCR (baseline TCR) was also expressed in T cells from two different donors and analyzed.

Effector and target cells were co-cultured in 96-well round bottom plates at 2.5: 1E: T. After approximately 20 hours of co-incubation, supernatants were harvested and analyzed by ELISA (standard sandwich ELISA, BD human IFN- γ ELISA kit).

2.3 results

TCR-expressing effector cells secreted IFN- γ only after coculture with KVL peptide-loaded T2 cells, indicating specific recognition of presented peptide MHC complexes mediated by transgenic TCR T15.8-4.3-83 and demonstrating its function. Negative targets (T2 cells loaded with irrelevant ASTN1 peptide (SEQ ID NO:28, KLYGLDWAEL)) were not recognized (FIG. 1). The transgenic TCR mediates specific target recognition and IFN- γ secretion, which is independent of the C region of the TCR.

3. Experiment: restriction analysis

Since TCRs recognize their specific epitopes only when combined with specific MHC molecules, not only the level of antigen expression but also the HLA type (in vitro and in vivo) of the target cell defines the target as a positive or negative target. In other words, depending on the HLA type, the patient may or may not be included in the TCR-based ACT treatment regimen.

To assess which HLA molecules, other than HLA-a 02:01, can be loaded with MAGEA 1-derived KVL peptide and can be recognized by T cells expressing TCR T15.8-4.3-83, a detailed restriction analysis was performed. Thus, 53 LCL cell lines (EBV-transformed B cells) covering the most common HLA allotypes in european and north american white were used as APCs (antigen presenting cells) and co-cultured with TCR-expressing effector cells not loaded or loaded with KVL peptides (fig. 2).

3.1 Experimental layout:

53 LCL cell lines were loaded with saturating amounts of MAGEA 1-derived KVL peptide (10)^-5M; SEQ ID NO:1) for about 1.5 hours, washed and then co-cultured with effectors expressing TCR T15.8-4.3-83 from different effector preparations. The unloaded LCL was used as a control. Co-cultures were set up at 1: 1E: T in 96-well round plates and after about 20 hours supernatants were collected and analyzed for IFN- γ levels by ELISA (standard sandwich ELISA, BD human IFN- γ ELISA kit).

3.2 results

Restriction analysis of TCR T15.8-4.3-83 indicated the potential of TCRs to recognize their MAGEA 1-derived epitopes, which are presented not only on HLA-a 02:01, but also to varying degrees on HLA-a 02:04, HLA-a 02:16 and HLA-a 02: 17. Fig. 2 shows only data that resulted in peptide-specific recognition of those LCLs (in 53 tests) that were above the background level detected for the unloaded LCLs.

4. Experiment: tumor cell recognition

To analyze the efficacy, specificity and applicability of T cell receptors for clinical applications, a panel of tumor cell lines were tested for TCR-mediated recognition by measuring IFN- γ secretion after co-culture with TCR-transduced effector cells.

4.1 experimental layout (FIG. 3):

effector cells from 3 different donors were transduced with TCR T15.8-4.3-83 and the baseline ctrl. magea 1-TCR. TCR transduced or untransduced effectors were co-cultured with different tumor cell lines. Cell lines U266, NCI-H1703, UACC-62, Saos-2 and Mel624.38(HLA-A2+/MAGEA1+) and HLA-A2 transfected cell lines KYO-1 and OPM-2(HLA-A2+/MAGEA1+) were used as positive targets. Cell lines NCI-H1755, 647-V and CMK (HLA-A2+/MAGEA1-), unmodified OPM-2 and KYO-1(HLA-A2-/MAGEA1+) as negative targets. HLA-a2 expression of each cell line was analyzed by FACS (data not shown), and MAGEA1 expression data was generated using qPCR or extracted from publicly available databases known to those skilled in the art. As a control, all effectors were also loaded with saturating concentrations (10)^-5M) of the MAGEA 1-derived KVL peptide (SEQ ID NO:1) or the unrelated ASTN1 peptide (SEQ ID NO: 28). For co-culture, effectors and targets were seeded at 2.5: 1E: T in 96-well round plates, supernatants were collected after about 20 hours of co-culture, and levels of IFN- γ were analyzed by ELISA.

4.2 results (FIG. 3):

the potential of the TCR to efficiently recognize tumor cells was evaluated by co-culturing T15.8-4.3-83 transduced effectors with a panel of different tumor cell lines. As shown in FIG. 3, both T15.8-4.3-83 and Ctrl MAGEA 1-TCRs mediated specific recognition of HLA-A2-positive and MAGEA 1-positive tumor cell lines U266, NCi-H1703, UACC-62, Saos-2, and Mel624.38. KYO-1 and OPM-2 cells were recognized only after transfection with the ivtRNA encoding HLA-A2.

Non-transfected KYO-1 and OPM-2(HLA-A2-/MAGEA 1-positive) as well as NCI-H1755, 647-V and CMK (HLA-A2+/MAGEA1-) served as negative controls. No negative control was recognized whenever the antigen MAGEA1 was not expressed or the required restriction element was not present. This efficient recognition of only the MAGEA1 positive and HLA-A2 positive cell line indicates the efficient and specific tumor cell recognition capability of the TCR.

4.3 Experimental layout (FIG. 10)

20000 TCR transgenic effector T cells were co-cultured with 20000 tumor cells in 96-well round bottom plates. Standard IFN-. gamma.ELISA was performed 20 hours after co-culture with the melanoma cell line UACC-257, the melanoma cell line UACC-62 or the osteosarcoma cell line SAOS-2. Values above 4000pg are extrapolated using a cubic polynomial.

4.5 results (FIG. 10):

the ability of the TCR transgenic effector T cell population to produce IFN- γ in response to MAGEA1 and HLA-a 02:01 double positive tumor cell lines was evaluated. T15.8-4.3-83 showed enhanced tumor cell recognition ability to all three cell lines UACC-257, UACC-62 or SAOS-2 compared to TCRs FH1, FH2, FH3 and FH4 disclosed in WO2018/170338 and R37P1C9 disclosed in WO 2018/104438. UACC-62 is only recognized by T15.8-4.3-83.

5. Experiment: tumor cell killing

TCR notThe ability to only recognize and effectively lyse and thereby kill tumor cells is of great importance and clinical significance. To analyze the killing ability of TCR T15.8-4.3-83, IncuCyte was used^TMThe Zoom apparatus was subjected to a 6 day long term killing test. IncuCyte^TMThe device is a microscope-based system that allows real-time imaging of cells. A panel of tumor cell lines was seeded into wells of a 96-well flat-bottom plate and co-cultured with TCR-expressing effectors from 3 different donors. The tumor cell line stably expresses nuclear-restricted red fluorescent protein mKate2, so that IncuCyte^TMThe Zoom System is able to determine the exact number of red fluorescent cells in each well. An increase in the number of cells per well over time would imply the growth of tumor cells, while a decrease in the number of cells per well would indicate TCR-mediated killing.

5.1 Experimental layout (FIGS. 4A-4D)

IncuCyte was used according to the manufacturer's instructions^TM NucLight^TMRed Lentivirus Reagent stably transduced tumor cell lines 647-V (urinary bladder urothelial carcinoma), UACC-62 (melanoma), Saos-2 (osteosarcoma) and NCI-H1703 (lung adenocarcinoma) with nuclear-restricted Red fluorescent protein mKate 2. Tumor cells were seeded in 96-well flat-bottom plates and allowed to adhere to plastic overnight. As a positive control, tumor cells were loaded with saturating concentrations of MAGEA 1-derived KVL peptide (10)^-5M) for about 1.5 hours, washed and then used for co-cultivation. KVL-loaded or unmodified tumor cells were cultured alone or co-cultured with TCR T15.8-4.3-83 transduced effectors from 3 different donors. Co-culture with untransduced effectors served as negative controls. IncuCyte was used every 2 hours^TMZoom System photographs were taken and IncuCyte was used^TMThe Zoom software (2016 version B) was used for the analysis.

5.2 results (FIGS. 4A-4D)

FIGS. 4A-4D show TCR-mediated antigen-specific cleavage of red fluorescent target cell lines 647-V (HLA-A2+/MAGEA1-), UACC-62(HLA-A2+/MAGEA1+), Saos-2(HLA-A2+/MAGEA1+), and NCI-H1703(HLA-A2+/MAGEA1+), respectively. Although untransduced effector cells had no effect on the growth of any tumor cells (cell number increased over time), the effector cells transduced with TCR T15.8-4.3-83 effectively killed tumor cells NCI-H1703, UACC-62 and Saos-2 (cell number decreased over time) expressing the antigens MAGEA1 and HLA-A2. MAGEA 1-negative 647-V cells were not killed. When artificially loaded with KVL peptide as a control, all tumor cells were killed. Tumor cells cultured alone, unloaded or loaded with KVL peptide, were also analyzed and their growth is shown in each figure as a reference.

5.3 Experimental layout (FIG. 11)

20000 TCR transgenic effector T cells and 7500T cellsNucLight Red lentivirus transduced SAOS-2 cells were co-cultured. Tumor cells were inoculated the day before the start of co-culture. After addition of effector cells, plates were transferred to IncuCyteApparatus and at 37 ℃ and 6% CO₂The red fluorescent cells were monitored for expansion for 72 hours under conditions and pictures were taken every 4 hours. Using IncuCyteThe software calculates the cell count of red fluorescent tumor cells per well per measurement point (1/mm)²). Each measurement point represents the average of 3 technical replicates.

5.4 results (FIG. 11)

SAOS-2 cells cultured in the presence of untransduced T cells showed strong proliferation. Co-culture of different TCR transgenic T cells induced a decrease in SAOS-2 cell number. Interestingly, the killing rates and extent varied between different TCR transgenic effector cells. In comparison to TCRs FH1, FH2, FH3 and FH4 disclosed in WO2018/170338 and R37P1C9 disclosed in WO2018/104438, TCR T15.8-4.3-83 showed the highest capacity to lyse MAGEA1 and HLA-a x 02:01 double positive tumor cell lines.

6. Experiment: TCR epitope recognition motifs

To analyze the specific epitope recognition motif of the TCR in detail, a threonine substitution scan and/or a serine scan was performed. By replacing the original amino acids of the epitope with threonine (or serine, respectively), the positions within the epitope that are essential for TCR-mediated recognition can be determined. This recognition depends on the correct binding of the peptide to the HLA molecule and on the interaction of the peptide MHC complex with the TCR itself, both of which are affected by a single amino acid substitution.

6.1 Experimental layout (FIGS. 5 and 6)

To specifically define the epitope recognition motif of TCR T15.8-4.3-8, effectors expressing TCR T15.8-4.3-83 and expressing the benchmark MAGEA1-TCR from 3 different donors were prepared. T cells were co-cultured with T2 cells loaded with the TCR-specific epitope KVL (SEQ ID NO:1) or with a peptide in which each individual amino acid residue was successively replaced by threonine (threonine replacement scan). T2 cells were loaded with saturating concentrations (10)^-5M), washed and co-cultured with effectors at 1: 1E: T. After co-incubation for about 20 hours, supernatants were collected and assayed for secreted IFN-. gamma.by ELISA.

6.2 results (FIGS. 5 and 6)

The results of threonine substitution scans with effectors transduced with TCR T15.8-4.3-83 (as shown in figure 6) or transduced with a benchmark ctrl. magea1-TCR (as shown in figure 5) demonstrated the different specificity of each TCR for its epitope and epitope-derived derivatives. For reference, recognition of MAGEA 1-derived KVL epitopes is shown on the left side of each figure. Mage a1-TCR failed to recognize peptides with threonine substitutions at positions 4 and 7 (E and I, fig. 5).

In contrast, TCR T15.8-4.3-83 appeared to be highly sensitive to amino acid substitutions at positions 1,3 and 5 (K, L and Y), as indicated by the lack of peptide recognition (figure 6).

The exchange of a single amino acid within the epitope significantly interfered with both TCR-mediated recognitions, demonstrating their highly selective recognition patterns, and the positions crucial for recognition were clearly different for the two analyzed TCRs.

6.3. Experimental layout (fig. 13)

20000 TCR transgenic T cells and 20000 10 load^-5T2 cells co-cultured with the M peptide. Labeling was performed after 20 hours of co-cultureQuasi IFN-. gamma.ELISA (values above 4000pg were extrapolated using a cubic polynomial). Instead of the above-mentioned threonine residues, MAGEA1 was systematically replaced with serine residues_KVLIndividual amino acids in the peptide (serine scan).

6.4 results (FIG. 13)

T cells transduced with T15.8-4.3-83TCR exhibited different recognition motifs (based on serine scanning) at different fixed positions than T cells transduced with FH1 or R37P1C9 TCR.

The combination of threonine and serine scans indicated that the first position in the epitope (lysine) and the 5 th position in the epitope (tyrosine) appeared to be particularly important for T15.8-4.3-83 TCR.

7, experiment: members of the MAGE family

Since 13 members of the MAGE family contain peptide sequences (only 2-4 mismatched amino acids) that are highly similar to MAGEA 1-derived KVL peptides, an in-depth analysis was performed to further investigate the potential TCR T15.8-4.3-83 mediated cross-recognition. The expression of members of the MAGE family is described not only in cancer and testis, but also in vital organs, making potential cross-recognition of protein-derived peptides an exclusion criterion for TCRs in clinical development.

To investigate the recognition of endogenously processed and presented peptides derived from other MAGE family members, all 13 members of the MAGE family containing peptide sequences similar to the epitope KVLEYVIKV of MAGEA1 were recombinantly expressed in 3 cell lines of different origin. Potential cross-recognition of protein-derived MAGE peptides was analysed by co-culturing cell lines expressing recombinant MAGE family members and effectors expressing TCR T15.8-4.3-83.

7.1 Experimental layout

Producing an IvtRNA encoding a member of the MAGE family: MAGEA8(NCBI reference sequence (Login): NM-001166400.1), MAGEA9 (NM-005365.4), MAGEA11 (NM-005366.4), MAGEB1 (NM-002363.4), MAGEB2 (NM-002363.4), MAGEB3 (NM-002365.4), MAGEB5 (NM-001271752.1), MAGEB16 (NM-001099921.1), MAGEB17 (NM-001277307.1), MAGEB18 (NM-173699.3), MAGEC2 (NM-016249.3), MAGED2 (NM-014599.5) and MAGEE2 (NM-138703.4). Production of peptides KLY encoding MAGEA1 (NM-004988.4) and ASTN1IvtRNA was used as positive and negative control RNA. The tumor cell lines HEK 293T (HLA-A2+/MAGE-), LCL (HLA-A2+/MAGE-) and HLA-A2 transduced K562(MAGE-) were transfected with 20. mu.g of ivtRNA encoding one of the 13 MAGE family members, MAGEA1 or ASTN1 peptide KLY, respectively. Transgene expression by MAGE family members was analyzed by FACS (data not shown). As a control, T2 cells and 3 cell lines were additionally loaded with MAGEA 1-derived KVL peptide (10)^-5M) or ASTN 1-derived KLY peptides (10)^-5M). Peptide-loaded control cells and transfected cell lines were seeded into 96-well round bottom plates 3 hours post-transfection and co-cultured with untransduced or TCR T15.8-4.3-83 transduced effectors from 2 donors at an E: T ratio of 1: 1. After co-incubation for about 20 hours, supernatants were collected and assayed for secreted IFN-. gamma.by ELISA.

7.2 results

FIG. 7 shows TCR T15.8-4.3-83 mediated recognition of recombinant MAGE family members expressed in HEK 293T cells (A), K562 cells (B) and LCL cells (C). Recognition of T2 cells and HEK 293T, K562 or LCL cells loaded externally with either KVL peptide or ASTN1 peptide served as controls. While the untransduced effector did not show any specific recognition, the TCR T15.8-4.3-83 transduced effector from 2 donors specifically recognized all 3 cell lines transfected with full-length MAGEA1 encoding ivtRNA. This suggests that RNA encoding MAGEA1 is efficiently translated into proteasomal processing of MAGEA1 protein and protein-derived KVL peptide, followed by loading and presentation of epitopes by HLA-a2 molecules on the cell surface. None of the cell lines transfected with ASTN1 peptide, nor any recombinantly expressed MAGE family members above background level were identified. Peptides derived from members of the MAGE family which can be recognised following artificial loading of target cells (as described earlier) are not processed by endogenously expressed MAGE proteins nor loaded onto MHC molecules or presented on the cell surface. Thus, these peptides do not qualify as immunogenic T cell epitopes and are not considered clinically relevant targets for TCR T15.8-4.3-83 mediated cross recognition.

8. Experiment: normal cell analysis

To investigate the safety of TCRs, TCR-mediated recognition of cells from healthy tissue of different origin must be studied and excluded. Thus, the effectors expressing the TCR were co-cultured with normal cells from kidney (renal cortical epithelial cells obtained from PromoCell), lung (pulmonary fibroblasts obtained from Lonza), and iPS-derived hepatocytes, cardiomyocytes, and endothelial cells (obtained by Cellular Dynamics). The cells studied were endogenously negative to the antigen MAGEA1, and therefore potential MAGEA 1-unrelated off-target toxicity could be identified.

8.1 Experimental layout

Normal cells were thawed and cultured for one week prior to co-culture as specified by the supplier. Cells were seeded in 96-well flat-bottom plates and co-cultured with TCR T15.8-4.3-83 transduced or untransduced effectors from 3 different donors (data not shown). HLA-A2 expression of target cells was confirmed by antibody staining and FACS (data not shown) and tested for unmodified or overloaded (10)^-5M) MAGEA 1-derived KVL (SEQ ID NO:1) peptide as a control. As a control, effector cells and target cells were seeded separately, and T2 cells were loaded with epitope KVL of TCR (10)^-5M; SEQ ID NO: 1). After approximately 20 hours of co-incubation, supernatants were collected and analyzed by ELISA. After about 48 hours of co-culture, IncuCyte was used^TMThe Zoom apparatus (Essen BioScience Inc.) takes phase contrast images to visualize potential toxic effects on lysis and detachment of adherent target cells.

8.2 results

As shown in figure 8, co-culture of TCR T15.8-4.3-83 transduced effectors from 3 donors resulted in recognition of only cells artificially loaded with KVL peptide, representing the potential of target cells to correctly present TCR epitopes on their cell surface in the context of MHC molecules. Unmodified normal cells were not recognized by effectors expressing transgenic TCR T15.8-4.3-83, indicating that the receptor studied is safe.

To visualize the potential toxic effects on target cells mediated by TCR-transduced effectors, phase-contrast images were taken of different target cells cultured alone, co-cultured with TCR T15.8-4.3-83 transduced effectors, or KVL peptide-loaded target cells co-cultured with TCR-expressing effectors. Figure 9 shows representative pictures of co-cultures with effector cells from one of three donors. While all target cells loaded with KVL had significant TCR-mediated lysis (complete disruption of the cell layer), the TCR-expressing effector cells did not lyse unmodified normal cells.

9 experiment: functional affinity

To measure the functional affinity of different KVL peptide-specific TCRs, half maximal relative IFN- γ release (EC50 value) was determined when co-cultured with T2 cells loaded with graded amounts of KVL peptide.

Functional avidity refers to the cumulative strength of multiple affinities of each non-covalent binding interaction between the transgenic TCR and pMHC complexes.

9.1 Experimental layout (FIG. 12)

Functional avidity of TCR transgenic T cell populations was determined as a gradient of loading with KVL peptide (10)^-4M to 10^-12M) of T2 cells were co-cultured for half maximal relative IFN-. gamma.release (EC50 value). Standard IFN-. gamma.ELISA was performed after 20 hours of co-cultivation (values greater than 4000pg were extrapolated using a cubic polynomial).

9.2 results (FIG. 12)

Different KVL-specific TCRs have different functional affinities for T2 cells loaded with KVL peptide. TCR T15.8-4.3-83 showed lower EC50 values, i.e. high functional avidity for 2 cells loaded with KVL peptide compared to TCRs FH1, FH2, FH3 and FH4 disclosed in WO2018/170338 and R37P1C9 disclosed in WO 2018/104438.

10 experiment: cytokine secretion

The TCR secretion patterns of TH 2-specific cytokines IL-4, IL-5 and IL-13 from T cells transduced with different TCRs were compared. Inflammation of Th2 type has been proposed to promote tumor growth (Jager MJ, Desjardins L,t, Damato BE (eds): Current Concepts in Uveal Melanoma. Dev Ophthalmol. Basel, Karger,2012, Vol.49, p.137-; 41(8):369-378). Therefore, low or undetectable secretion levels of Th2 may be beneficial for tumor regression.

10.1 Experimental layout (FIG. 14)

20000 TCR transgenic effector T cells were co-cultured with 20000 tumor cells in round bottom 96-well plates. Use ofMAP Kit basisThe protocol in the system analyzes the cytokines secreted in the cell-free supernatant.

10.2 results (FIG. 14)

T cells transduced with T1367 TCR secrete a variety of TH2 cytokines, particularly IL-4, IL-5 and IL-13. T cells transduced with 15.8-4.3-83 TCRs did not exhibit equivalent secretion of TH2 cytokines, whereas T1367 disclosed in WO2014/118236 exhibited significant secretion of these cytokines.

The present application also includes the following embodiments:

embodiment 1: an isolated T Cell Receptor (TCR) specific for MAGEA 1.

Embodiment 2: the isolated TCR according to embodiment 1, wherein the TCR specifically recognizes the amino acid sequence SEQ ID NO 1 or a fragment thereof.

Embodiment 3: the isolated TCR according to embodiments 1 and 2, wherein the TCR specifically recognizes the HLA-A2 and/or HLA-A26 binding form of the amino acid sequence of SEQ ID NO.1, preferably the HLA-A2 binding form.

Embodiment 4: the isolated TCR according to any one of the preceding embodiments, wherein the TCR specifically recognizes the amino acid sequence of SEQ ID No.1 presented by a molecule encoded by a gene selected from the group consisting of HLA-a 02:01, HLA-a 02:04, HLA-a 02:16 and HLA-a 02: 17.

Embodiment 5: the isolated TCR according to any one of the preceding embodiments, wherein the TCR specifically recognizes the amino acid sequence of SEQ ID No.1 presented by a molecule encoded by a gene selected from the group consisting of HLA-a 02:01, HLA-a 02:04, HLA-a 02:16 and HLA-a 02: 17.

Embodiment 6: the isolated TCR according to any one of the preceding embodiments, wherein the TCR specifically recognizes the amino acid sequence of SEQ ID NO:1 presented by a molecule encoded by HLA-a 02: 01.

Embodiment 7: the isolated TCR according to any one of the preceding embodiments, wherein the TCR comprises

A TCR alpha chain comprising the CDR1 having the amino acid sequence of SEQ ID NO.2, the CDR2 having the amino acid sequence of SEQ ID NO. 3 and the CDR3 having the amino acid sequence of SEQ ID NO. 4,

a TCR β chain comprising the CDR1 having the amino acid sequence of SEQ ID NO. 5, the CDR2 having the amino acid sequence of SEQ ID NO. 6 and the CDR3 having the amino acid sequence of SEQ ID NO. 7.

Embodiment 8: the isolated TCR according to any one of the preceding embodiments, wherein the TCR comprises

A variable TCR α region having an amino acid sequence at least 80% identical to SEQ ID No. 8 and a variable TCR β region having an amino acid sequence at least 80% identical to SEQ ID No. 9.

Embodiment 9: the isolated TCR according to any one of the preceding embodiments, wherein the TCR comprises

A variable TCR alpha region having the amino acid sequence of SEQ ID NO. 8 and a variable TCR beta region having the amino acid sequence of SEQ ID NO. 9.

Embodiment 10: the isolated TCR according to any one of the preceding embodiments, wherein the TCR comprises

A TCR alpha chain having an amino acid sequence at least 80% identical to SEQ ID NO. 10 and a TCR beta chain having an amino acid sequence at least 80% identical to SEQ ID NO. 11.

Embodiment 11: the isolated TCR according to any one of the preceding embodiments, wherein the TCR comprises

A TCR alpha chain having the amino acid sequence of SEQ ID NO 10 or SEQ ID NO 12 and a TCR beta chain having the amino acid sequence of SEQ ID NO 11 or SEQ ID NO 13.

Embodiment 12: an isolated TCR according to any one of the preceding embodiments, wherein the TCR comprises a TCR a chain and a TCR β chain, wherein

The variable TCR alpha region has an amino acid sequence which is at least 80% identical to SEQ ID NO. 8 and comprises a CDR3 having the amino acid sequence shown in SEQ ID NO. 4,

the variable TCR β region has an amino acid sequence at least 80% identical to SEQ ID No. 9 and comprises CDR3 having the amino acid sequence shown in SEQ ID No. 7.

Embodiment 13: the isolated TCR according to any one of the preceding embodiments, wherein the TCR is purified.

Embodiment 14: the isolated TCR according to any one of the preceding embodiments, wherein the amino acid sequence of the TCR comprises one or more phenotypically silent substitutions.

Embodiment 15: the isolated TCR according to any one of the preceding embodiments, wherein the amino acid sequence of the TCR is modified to comprise a detectable label, a therapeutic agent, or a pharmacokinetic modification moiety.

Embodiment 16: the isolated TCR according to embodiment 15, wherein the therapeutic agent is selected from the group consisting of an immune effector molecule, a cytotoxic agent, and a radionuclide.

Embodiment 17: the isolated TCR according to embodiment 16, wherein the immune effector molecule is a cytokine.

Embodiment 18: the isolated TCR according to any one of the preceding embodiments, wherein the TCR is soluble or membrane-bound.

Embodiment 19: the isolated TCR according to embodiment 15, wherein the pharmacokinetic modifying moiety is at least one polyethylene glycol repeat unit, at least one diol group, at least one sialic acid group, or a combination thereof.

Embodiment 20: the isolated TCR according to any one of the preceding embodiments, wherein the TCR is of the single-chain type, wherein the TCR a chain and the TCR β chain are linked by a linker sequence.

Embodiment 21: the isolated TCR according to embodiments 1-20, wherein the TCR a chain or the TCR β chain is modified to comprise an epitope tag.

Embodiment 22: an isolated polypeptide comprising a functional portion of a TCR according to any one of embodiments 1 to 21, wherein the functional portion comprises at least one of the amino acid sequences of SEQ ID NOs 4 and 7.

Embodiment 23: an isolated polypeptide comprising a functional portion of a TCR according to any one of embodiments 1 to 21, wherein the functional portion comprises the amino acid sequences of SEQ ID NOs 2, 3, 4, 5, 6 and 7.

Embodiment 24: the isolated polypeptide according to embodiment 21, wherein the functional moiety comprises a TCR a variable chain and/or a TCR β variable chain.

Embodiment 25: a multivalent TCR complex comprising a TCR as embodied in any one of embodiments 1 to 21.

Embodiment 26: an isolated TCR according to embodiments 1 to 21, a polypeptide according to embodiments 22 to 24, a multivalent TCR complex according to embodiment 25, wherein IFN- γ secretion is induced by binding to the amino acid sequence of SEQ ID NO 1 presented by a molecule encoded by HLA-a 02: 01.

Embodiment 27: a nucleic acid encoding a TCR according to any one of embodiments 1 to 21 or encoding a polypeptide according to embodiments 22 to 24.

Embodiment 28: a vector comprising the nucleic acid of embodiment 27.

Embodiment 29: the vector according to embodiment 28, wherein said vector is an expression vector.

Embodiment 30: the vector according to embodiment 28 or 29, wherein said vector is a retroviral vector.

Embodiment 31: the vector according to embodiment 28 or 29, wherein said vector is a lentiviral vector.

Embodiment 32: a cell expressing a TCR according to embodiments 1 to 21.

Embodiment 33: the cell according to embodiment 32, wherein the cell is isolated or non-naturally occurring.

Embodiment 34: the cell according to embodiments 32 and 33, wherein the cell comprises a nucleic acid according to embodiment 27 or a vector according to embodiments 28 to 31.

Embodiment 35: the cell of embodiments 32-34, wherein the cell comprises:

a) an expression vector comprising at least one nucleic acid as embodied in embodiment 27, or

b) A first expression vector comprising a nucleic acid encoding an alpha chain of a TCR as embodied in any one of embodiments 1 to 21, and a second expression vector comprising a nucleic acid encoding a beta chain of a TCR as embodied in any one of embodiments 1 to 21.

Embodiment 36: the cell according to any one of embodiments 32 to 35, wherein the cell is a Peripheral Blood Lymphocyte (PBL) or a Peripheral Blood Mononuclear Cell (PBMC).

Embodiment 37: the cell according to any one of embodiments 32-36, wherein the cell is a T cell.

Embodiment 38: an antibody, or antigen-binding fragment thereof, that specifically binds to a portion of a TCR according to embodiments 1 to 21 that mediates specificity for MAGEA 1.

Embodiment 39: the antibody according to embodiment 38, wherein the portion of the TCR that mediates MAGEA1 specificity comprises at least one of the CDRs shown in SEQ ID NOs 2, 3, 4, 5, 6 and 7, preferably the alpha chain CDR3 of SEQ ID No. 4 and/or the beta chain CDR3 of SEQ ID No. 7, more preferably the alpha chain CDRs shown in SEQ ID NOs 2, 3 and 4 and the beta chain CDRs of SEQ ID NOs 5, 6 and 7.

Embodiment 40: a pharmaceutical composition comprising a TCR according to embodiments 1 to 21, a polypeptide according to embodiments 22 to 24, a multivalent TCR complex according to embodiment 25, a nucleic acid according to embodiment 27, a vector according to embodiments 28 to 31, a cell according to any one of embodiments 32 to 37, or an antibody according to embodiments 38 to 39.

Embodiment 41: the pharmaceutical composition according to embodiment 40, wherein the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier.

Embodiment 42: a TCR according to embodiments 1 to 21, a polypeptide according to embodiments 22 to 24, a multivalent TCR complex according to embodiment 25, a nucleic acid according to embodiment 27, a vector according to embodiments 28 to 31, a cell according to any one of embodiments 32 to 37, or an antibody according to embodiments 38 to 39, for use as a medicament.

Embodiment 43: a TCR according to embodiments 1 to 21, a polypeptide according to embodiments 22 to 24, a multivalent TCR complex according to embodiment 25, a nucleic acid according to embodiment 27, a vector according to claims 28 to 31 or a cell according to any one of embodiments 32 to 37, for use in treating cancer.

Embodiment 44: a TCR, polypeptide, multivalent TCR complex, nucleic acid, vector, or cell according to embodiment 43, wherein the cancer is a hematologic cancer or a solid tumor.

Embodiment 45: a TCR, polypeptide, multivalent TCR complex, nucleic acid, vector, or cell according to embodiments 43 and 44, wherein the cancer is selected from the group consisting of: prostate cancer, uterine cancer, thyroid cancer, testicular cancer, renal cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma, non-hodgkin's lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, cervical cancer, colorectal cancer, gastric adenocarcinoma, cholangiocarcinoma, breast cancer, bladder cancer, myeloid leukemia, and acute lymphoblastic leukemia, sarcoma, or osteosarcoma.

Embodiment 46: a TCR, polypeptide, multivalent TCR complex, nucleic acid, vector or cell according to embodiments 43 and 44, wherein the cancer is preferably selected from the group consisting of a sarcoma or osteosarcoma.

Sequence listing

<110> GeneMedical immunotherapy finite responsible Co. (Medigene immunology GmbH)

<120> MAGEA1 specific T cell receptor and uses thereof

<130> M11421

<150> EP19167440

<151> 2019-04-04

<160> 28

<170> PatentIn version 3.5

<210> 1

<211> 9

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 1

Lys Val Leu Glu Tyr Val Ile Lys Val

1 5

<210> 2

<211> 7

<212> PRT

<213> Intelligent people

<400> 2

Thr Arg Asp Thr Thr Tyr Tyr

1 5

<210> 3

<211> 8

<212> PRT

<213> Intelligent people

<400> 3

Arg Asn Ser Phe Asp Glu Gln Asn

1 5

<210> 4

<211> 15

<212> PRT

<213> Intelligent people

<400> 4

Cys Ala Leu Ser Glu Val Ala Ser Gly Gly Ser Tyr Ile Pro Thr

1 5 10 15

<210> 5

<211> 6

<212> PRT

<213> Intelligent people

<400> 5

Gly Thr Ser Asn Pro Asn

1 5

<210> 6

<211> 5

<212> PRT

<213> Intelligent people

<400> 6

Ser Val Gly Ile Gly

1 5

<210> 7

<211> 14

<212> PRT

<213> Intelligent people

<400> 7

Cys Ala Trp Ser Gly Ser Gly Gly Asn Gln Pro Gln His Phe

1 5 10

<210> 8

<211> 138

<212> PRT

<213> Intelligent people

<400> 8

Met Leu Thr Ala Ser Leu Leu Arg Ala Val Ile Ala Ser Ile Cys Val

1 5 10 15

Val Ser Ser Met Ala Gln Lys Val Thr Gln Ala Gln Thr Glu Ile Ser

20 25 30

Val Val Glu Lys Glu Asp Val Thr Leu Asp Cys Val Tyr Glu Thr Arg

35 40 45

Asp Thr Thr Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Gly Glu

50 55 60

Leu Val Phe Leu Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile

65 70 75 80

Ser Gly Arg Tyr Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn

85 90 95

Phe Thr Ile Thr Ala Ser Gln Val Val Asp Ser Ala Val Tyr Phe Cys

100 105 110

Ala Leu Ser Glu Val Ala Ser Gly Gly Ser Tyr Ile Pro Thr Phe Gly

115 120 125

Arg Gly Thr Ser Leu Ile Val His Pro Tyr

130 135

<210> 9

<211> 130

<212> PRT

<213> Intelligent people

<400> 9

Met Leu Cys Ser Leu Leu Ala Leu Leu Leu Gly Thr Phe Phe Gly Val

1 5 10 15

Arg Ser Gln Thr Ile His Gln Trp Pro Ala Thr Leu Val Gln Pro Val

20 25 30

Gly Ser Pro Leu Ser Leu Glu Cys Thr Val Glu Gly Thr Ser Asn Pro

35 40 45

Asn Leu Tyr Trp Tyr Arg Gln Ala Ala Gly Arg Gly Leu Gln Leu Leu

50 55 60

Phe Tyr Ser Val Gly Ile Gly Gln Ile Ser Ser Glu Val Pro Gln Asn

65 70 75 80

Leu Ser Ala Ser Arg Pro Gln Asp Arg Gln Phe Ile Leu Ser Ser Lys

85 90 95

Lys Leu Leu Leu Ser Asp Ser Gly Phe Tyr Leu Cys Ala Trp Ser Gly

100 105 110

Ser Gly Gly Asn Gln Pro Gln His Phe Gly Asp Gly Thr Arg Leu Ser

115 120 125

Ile Leu

130

<210> 10

<211> 278

<212> PRT

<213> Artificial sequence

<220>

<223> mouse-derived constant region

<400> 10

Met Leu Thr Ala Ser Leu Leu Arg Ala Val Ile Ala Ser Ile Cys Val

1 5 10 15

Val Ser Ser Met Ala Gln Lys Val Thr Gln Ala Gln Thr Glu Ile Ser

20 25 30

Val Val Glu Lys Glu Asp Val Thr Leu Asp Cys Val Tyr Glu Thr Arg

35 40 45

Asp Thr Thr Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Gly Glu

50 55 60

Leu Val Phe Leu Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile

65 70 75 80

Ser Gly Arg Tyr Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn

85 90 95

Phe Thr Ile Thr Ala Ser Gln Val Val Asp Ser Ala Val Tyr Phe Cys

100 105 110

Ala Leu Ser Glu Val Ala Ser Gly Gly Ser Tyr Ile Pro Thr Phe Gly

115 120 125

Arg Gly Thr Ser Leu Ile Val His Pro Tyr Ile Gln Asn Pro Asp Pro

130 135 140

Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys

145 150 155 160

Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp

165 170 175

Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met

180 185 190

Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe

195 200 205

Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe

210 215 220

Phe Pro Ser Ser Asp Val Pro Cys Asp Val Lys Leu Val Glu Lys Ser

225 230 235 240

Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly

245 250 255

Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr

260 265 270

Leu Arg Leu Trp Ser Ser

275

<210> 11

<211> 307

<212> PRT

<213> Artificial sequence

<220>

<223> mouse-derived constant region

<400> 11

Met Leu Cys Ser Leu Leu Ala Leu Leu Leu Gly Thr Phe Phe Gly Val

1 5 10 15

Arg Ser Gln Thr Ile His Gln Trp Pro Ala Thr Leu Val Gln Pro Val

20 25 30

Gly Ser Pro Leu Ser Leu Glu Cys Thr Val Glu Gly Thr Ser Asn Pro

35 40 45

Asn Leu Tyr Trp Tyr Arg Gln Ala Ala Gly Arg Gly Leu Gln Leu Leu

50 55 60

Phe Tyr Ser Val Gly Ile Gly Gln Ile Ser Ser Glu Val Pro Gln Asn

65 70 75 80

Leu Ser Ala Ser Arg Pro Gln Asp Arg Gln Phe Ile Leu Ser Ser Lys

85 90 95

Lys Leu Leu Leu Ser Asp Ser Gly Phe Tyr Leu Cys Ala Trp Ser Gly

100 105 110

Ser Gly Gly Asn Gln Pro Gln His Phe Gly Asp Gly Thr Arg Leu Ser

115 120 125

Ile Leu Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val Phe

130 135 140

Glu Pro Ser Lys Ala Glu Ile Ala His Thr Gln Lys Ala Thr Leu Val

145 150 155 160

Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp

165 170 175

Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro

180 185 190

Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser

195 200 205

Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe

210 215 220

Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr

225 230 235 240

Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp

245 250 255

Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly Val

260 265 270

Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu

275 280 285

Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg

290 295 300

Lys Asp Phe

305

<210> 12

<211> 274

<212> PRT

<213> Artificial sequence

<220>

<223> murine constant region

<400> 12

Met Leu Thr Ala Ser Leu Leu Arg Ala Val Ile Ala Ser Ile Cys Val

1 5 10 15

Val Ser Ser Met Ala Gln Lys Val Thr Gln Ala Gln Thr Glu Ile Ser

20 25 30

Val Val Glu Lys Glu Asp Val Thr Leu Asp Cys Val Tyr Glu Thr Arg

35 40 45

Asp Thr Thr Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Gly Glu

50 55 60

Leu Val Phe Leu Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile

65 70 75 80

Ser Gly Arg Tyr Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn

85 90 95

Phe Thr Ile Thr Ala Ser Gln Val Val Asp Ser Ala Val Tyr Phe Cys

100 105 110

Ala Leu Ser Glu Val Ala Ser Gly Gly Ser Tyr Ile Pro Thr Phe Gly

115 120 125

Arg Gly Thr Ser Leu Ile Val His Pro Tyr Ile Gln Asn Pro Glu Pro

130 135 140

Ala Val Tyr Gln Leu Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys

145 150 155 160

Leu Phe Thr Asp Phe Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu

165 170 175

Ser Gly Thr Phe Ile Thr Asp Lys Thr Val Leu Asp Met Lys Ala Met

180 185 190

Asp Ser Lys Ser Asn Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe

195 200 205

Thr Cys Gln Asp Ile Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser

210 215 220

Asp Val Pro Cys Asp Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp

225 230 235 240

Met Asn Leu Asn Phe Gln Asn Leu Ser Val Met Gly Leu Arg Ile Leu

245 250 255

Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp

260 265 270

Ser Ser

<210> 13

<211> 303

<212> PRT

<213> Artificial sequence

<220>

<223> murine constant region

<400> 13

Met Leu Cys Ser Leu Leu Ala Leu Leu Leu Gly Thr Phe Phe Gly Val

1 5 10 15

Arg Ser Gln Thr Ile His Gln Trp Pro Ala Thr Leu Val Gln Pro Val

20 25 30

Gly Ser Pro Leu Ser Leu Glu Cys Thr Val Glu Gly Thr Ser Asn Pro

35 40 45

Asn Leu Tyr Trp Tyr Arg Gln Ala Ala Gly Arg Gly Leu Gln Leu Leu

50 55 60

Phe Tyr Ser Val Gly Ile Gly Gln Ile Ser Ser Glu Val Pro Gln Asn

65 70 75 80

Leu Ser Ala Ser Arg Pro Gln Asp Arg Gln Phe Ile Leu Ser Ser Lys

85 90 95

Lys Leu Leu Leu Ser Asp Ser Gly Phe Tyr Leu Cys Ala Trp Ser Gly

100 105 110

Ser Gly Gly Asn Gln Pro Gln His Phe Gly Asp Gly Thr Arg Leu Ser

115 120 125

Ile Leu Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Thr Leu Phe

130 135 140

Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val

145 150 155 160

Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp

165 170 175

Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Ala

180 185 190

Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val

195 200 205

Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln Val

210 215 220

Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro

225 230 235 240

Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala Asp

245 250 255

Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly Val Leu Ser Ala Thr

260 265 270

Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu

275 280 285

Val Ser Gly Leu Val Leu Met Ala Met Val Lys Lys Lys Asn Ser

290 295 300

<210> 14

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimization

<400> 14

acacgggaca ccacctacta c 21

<210> 15

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimization

<400> 15

cggaacagct tcgacgagca gaac 24

<210> 16

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimization

<400> 16

tgcgccctga gcgaagtggc cagcggcggc tcttacatcc ctaca 45

<210> 17

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimization

<400> 17

ggcaccagca atcccaac 18

<210> 18

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimization

<400> 18

agcgtcggca tcggc 15

<210> 19

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimization

<400> 19

tgtgcttgga gtggcagcgg cggcaatcag cctcagcact tt 42

<210> 20

<211> 414

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimization

<400> 20

atgctgacag cctctctgct gagagccgtg atcgccagca tctgtgtggt gtctagcatg 60

gcccagaaag tgacacaggc ccagaccgag atcagcgtgg tggaaaaaga agatgtgacc 120

ctggactgcg tgtacgagac acgggacacc acctactacc tgttctggta caagcagcct 180

cctagcggcg agctggtgtt cctgatcaga cggaacagct tcgacgagca gaacgagatc 240

tccggccggt acagctggaa cttccagaag tccaccagca gcttcaactt caccatcacc 300

gccagccagg tggtggatag cgccgtgtat ttttgcgccc tgagcgaagt ggccagcggc 360

ggctcttaca tccctacatt tggcagaggc accagcctga tcgtgcaccc ttat 414

<210> 21

<211> 390

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimization

<400> 21

atgctgtgtt ctctgctggc tctgctgctg ggcacctttt ttggcgtcag aagccagacc 60

atccaccagt ggcctgctac actggtgcag cctgttggaa gccctctgag cctggaatgt 120

accgtggaag gcaccagcaa tcccaacctg tactggtaca gacaggccgc tggaagagga 180

ctgcagctgc tgttttacag cgtcggcatc ggccagatca gcagcgaggt tccacagaat 240

ctgagcgcca gcagacccca ggacagacag tttatcctga gcagcaagaa gctgctgctg 300

agcgacagcg gcttctacct gtgtgcttgg agtggcagcg gcggcaatca gcctcagcac 360

tttggagatg gcacccggct gagcatcctg 390

<210> 22

<211> 837

<212> DNA

<213> Artificial sequence

<220>

<223> mouse-derived constant region, codon optimization

<400> 22

atgctgacag cctctctgct gagagccgtg atcgccagca tctgtgtggt gtctagcatg 60

gcccagaaag tgacacaggc ccagaccgag atcagcgtgg tggaaaaaga agatgtgacc 120

ctggactgcg tgtacgagac acgggacacc acctactacc tgttctggta caagcagcct 180

cctagcggcg agctggtgtt cctgatcaga cggaacagct tcgacgagca gaacgagatc 240

tccggccggt acagctggaa cttccagaag tccaccagca gcttcaactt caccatcacc 300

gccagccagg tggtggatag cgccgtgtat ttttgcgccc tgagcgaagt ggccagcggc 360

ggctcttaca tccctacatt tggcagaggc accagcctga tcgtgcaccc ttatattcag 420

aaccccgatc ctgccgtgta ccagctgaga gacagcaaga gcagcgacaa gagcgtgtgt 480

ctgttcaccg acttcgacag ccagaccaac gtgtcccaga gcaaggacag cgacgtgtac 540

atcaccgaca agaccgtgct ggacatgcgg agcatggact tcaagagcaa cagcgccgtg 600

gcctggtcca acaagagcga tttcgcctgc gccaacgcct tcaacaatag cattatcccc 660

gaggacacat tcttccccag ctccgatgtg ccctgcgacg tgaagctggt ggaaaagagc 720

ttcgagacag acaccaacct gaacttccag aacctgagcg tgatcggctt cagaatcctg 780

ctgctgaagg tggccggctt caatctgctg atgaccctga gactgtggtc cagctga 837

<210> 23

<211> 924

<212> DNA

<213> Artificial sequence

<220>

<223> mouse-derived constant region, codon optimization

<400> 23

atgctgtgtt ctctgctggc tctgctgctg ggcacctttt ttggcgtcag aagccagacc 60

atccaccagt ggcctgctac actggtgcag cctgttggaa gccctctgag cctggaatgt 120

accgtggaag gcaccagcaa tcccaacctg tactggtaca gacaggccgc tggaagagga 180

ctgcagctgc tgttttacag cgtcggcatc ggccagatca gcagcgaggt tccacagaat 240

ctgagcgcca gcagacccca ggacagacag tttatcctga gcagcaagaa gctgctgctg 300

agcgacagcg gcttctacct gtgtgcttgg agtggcagcg gcggcaatca gcctcagcac 360

tttggagatg gcacccggct gagcatcctg gaagatctga acaaggtgtt ccctccagag 420

gtggccgtgt tcgagccttc taaggccgag attgcccaca cacagaaagc cacactcgtg 480

tgcctggcta ccggcttctt tcctgaccac gtggaactgt cttggtgggt caacggcaaa 540

gaggtgcaca gcggcgtcag cacagatccc cagcctctga aagaacagcc cgctctgaac 600

gacagccggt actgtctgag cagcagactg agagtgtccg ccacattctg gcagaacccc 660

agaaaccact tcagatgcca ggtgcagttc tacggcctga gcgagaacga tgagtggacc 720

caggatagag ccaagcctgt gacacagatc gtgtctgccg aagcctgggg cagagccgat 780

tgtggaatta ccagcgccag ctaccatcag ggcgtgctgt ctgccacaat cctgtacgag 840

atcctgctgg gcaaagccac tctgtacgcc gtgctggtgt ctgccctggt gctgatggcc 900

atggtcaaga gaaaggactt ttga 924

<210> 24

<211> 825

<212> DNA

<213> Artificial sequence

<220>

<223> murine constant region, codon optimization

<400> 24

atgctgacag cctctctgct gagagccgtg atcgccagca tctgtgtggt gtctagcatg 60

gcccagaaag tgacacaggc ccagaccgag atcagcgtgg tggaaaaaga agatgtgacc 120

ctggactgcg tgtacgagac acgggacacc acctactacc tgttctggta caagcagcct 180

cctagcggcg agctggtgtt cctgatcaga cggaacagct tcgacgagca gaacgagatc 240

tccggccggt acagctggaa cttccagaag tccaccagca gcttcaactt caccatcacc 300

gccagccagg tggtggatag cgccgtgtat ttttgcgccc tgagcgaagt ggccagcggc 360

ggctcttaca tccctacatt tggcagaggc accagcctga tcgtgcaccc ttatatccag 420

aatccggagc ccgccgtata ccagctgaag gaccctagaa gccaggacag caccctgtgc 480

ctgttcaccg acttcgacag ccagatcaac gtgcccaaga ccatggaaag cggcaccttc 540

atcaccgaca agacagtgct ggacatgaag gccatggaca gcaagtccaa cggcgcaatc 600

gcctggtcca accagaccag cttcacatgc caggacatct tcaaagagac aaacgccaca 660

taccccagca gcgacgtgcc ctgtgatgcc accctgacag agaagtcctt cgagacagac 720

atgaacctga acttccagaa tctgtccgtg atgggcctga gaatcctgct gctgaaggtg 780

gccggcttca atctgctgat gaccctgcgg ctgtggtcca gctga 825

<210> 25

<211> 912

<212> DNA

<213> Artificial sequence

<220>

<223> murine constant region

<400> 25

atgctgtgtt ctctgctggc tctgctgctg ggcacctttt ttggcgtcag aagccagacc 60

atccaccagt ggcctgctac actggtgcag cctgttggaa gccctctgag cctggaatgt 120

accgtggaag gcaccagcaa tcccaacctg tactggtaca gacaggccgc tggaagagga 180

ctgcagctgc tgttttacag cgtcggcatc ggccagatca gcagcgaggt tccacagaat 240

ctgagcgcca gcagacccca ggacagacag tttatcctga gcagcaagaa gctgctgctg 300

agcgacagcg gcttctacct gtgtgcttgg agtggcagcg gcggcaatca gcctcagcac 360

tttggagatg gcacccggct gagcatcctg gaagatctcc ggaacgtgac cccccctaaa 420

gtgaccctgt tcgaacccag caaggccgag atcgccaaca agcagaaagc caccctcgtg 480

tgcctggcca gaggcttctt ccccgaccat gtggaactgt cttggtgggt caacggcaaa 540

gaggtgcaca gcggagtgtc caccgaccct caggcctaca aagagagcaa ctacagctac 600

tgcctgagca gcagactgcg ggtgtccgcc accttctggc acaacccccg gaaccacttc 660

aggtgccagg tgcagtttca cggcctgagc gaagaggaca agtggcccga aggctccccc 720

aagcccgtga cccagaatat ctctgccgag gcctggggca gagccgactg tggaattacc 780

agcgccagct accaccaggg cgtgctgtct gccaccatcc tgtacgagat cctgctgggc 840

aaggccaccc tgtacgccgt gctggtgtct ggcctggtgc tgatggccat ggtcaagaag 900

aagaacagct ga 912

<210> 26

<211> 395

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 26

Met Trp Val Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp Ile Gly

1 5 10 15

Ala Ala Pro Leu Ile Leu Ser Arg Ile Val Gly Gly Trp Glu Cys Glu

20 25 30

Lys His Ser Gln Pro Trp Gln Val Leu Val Ala Ser Arg Gly Arg Ala

35 40 45

Val Cys Gly Gly Val Leu Val His Pro Gln Trp Val Leu Thr Ala Ala

50 55 60

His Cys Ile Arg Asn Lys Ser Val Ile Leu Leu Gly Arg His Ser Leu

65 70 75 80

Phe His Pro Glu Asp Thr Gly Gln Val Phe Gln Val Ser His Ser Phe

85 90 95

Pro His Pro Leu Tyr Asp Met Ser Leu Leu Lys Asn Arg Phe Leu Arg

100 105 110

Pro Gly Asp Asp Ser Ser His Asp Leu Met Leu Leu Arg Leu Ser Glu

115 120 125

Pro Ala Glu Leu Thr Asp Ala Val Lys Val Met Asp Leu Pro Thr Gln

130 135 140

Glu Pro Ala Leu Gly Thr Thr Cys Tyr Ala Ser Gly Trp Gly Ser Ile

145 150 155 160

Glu Pro Glu Glu Phe Leu Thr Pro Lys Lys Leu Gln Cys Val Asp Leu

165 170 175

His Val Ile Ser Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val

180 185 190

Thr Lys Phe Met Leu Cys Ala Gly Arg Trp Thr Gly Gly Lys Ser Thr

195 200 205

Cys Ser Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly Val Leu Gln

210 215 220

Gly Ile Thr Ser Trp Gly Ser Glu Pro Cys Ala Leu Pro Glu Arg Pro

225 230 235 240

Ser Leu Tyr Thr Lys Val Val His Tyr Arg Lys Trp Ile Lys Asp Thr

245 250 255

Ile Val Ala Asn Pro His His His His His His Tyr Phe Ser Lys Glu

260 265 270

Glu Trp Glu Lys Met Lys Ala Ser Glu Lys Ile Phe Tyr Val Tyr Met

275 280 285

Lys Arg Lys Tyr Glu Ala Met Thr His His His His His His Asp Lys

290 295 300

Thr Gly Phe His Phe Cys Gly Gly Ser Leu Ile Ser Glu Asp Trp Val

305 310 315 320

Val Thr Ala Ala His Cys Gly Val Arg Thr Ser His His His His His

325 330 335

His Ser Ser Pro Gly Val Tyr Ala Arg Val Thr Lys Leu Ile Pro Trp

340 345 350

Val Gln Lys Ile Leu Ala Ala Asn His His His His His His Pro Arg

355 360 365

Ala Leu Ala Glu Thr Ser Tyr Val Lys Val Leu Glu Tyr Val Ile Lys

370 375 380

Val Ser Ala Arg Val Arg Phe Phe Phe Pro Ser

385 390 395

<210> 27

<211> 1188

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 27

atgtgggtcc cggttgtctt cctcaccctg tccgtgacgt ggattggtgc tgcacccctc 60

atcctgtctc ggattgtggg aggctgggag tgcgagaagc attcccaacc ctggcaggtg 120

cttgtggcct ctcgtggcag ggcagtctgc ggcggtgttc tggtgcaccc ccagtgggtc 180

ctcacagctg cccactgcat caggaacaaa agcgtgatct tgctgggtcg gcacagcctg 240

tttcatcctg aagacacagg ccaggtattt caggtcagcc acagcttccc acacccgctc 300

tacgatatga gcctcctgaa gaatcgattc ctcaggccag gtgatgactc cagccacgac 360

ctcatgctgc tccgcctgtc agagcctgcc gagctcacgg atgctgtgaa ggtcatggac 420

ctgcccaccc aggagccagc actggggacc acctgctacg cctcaggctg gggcagcatt 480

gaaccagagg agttcttgac cccaaagaaa cttcagtgtg tggacctcca cgttatttcc 540

aacgacgtgt gtgcgcaagt tcaccctcag aaggtgacca agttcatgct gtgtgctgga 600

cgctggacag ggggcaaaag cacctgctcg ggtgattctg ggggcccact tgtctgtaac 660

ggtgtgcttc aaggtatcac gtcatggggc agtgaaccgt gtgccctgcc cgaaaggcct 720

tccctgtaca ccaaggtggt gcattaccgg aagtggatca aggacaccat cgtggccaac 780

ccccatcacc atcaccacca ctacttctct aaggaagagt gggaaaagat gaaagcctcg 840

gagaaaatct tctatgtgta tatgaagaga aagtatgagg ctatgactca tcaccatcac 900

caccacgaca aaaccggctt ccacttctgc gggggctccc tcatcagcga ggactgggtg 960

gtcaccgctg cccactgcgg ggtcaggacc tcccatcacc atcaccacca ctccagccct 1020

ggcgtgtacg cccgtgtcac caagctcata ccttgggtgc agaagatcct ggctgccaac 1080

catcaccatc accaccaccc aagggccctc gctgaaacca gctatgtgaa agtccttgag 1140

tatgtgatca aggtcagtgc aagagttcgc tttttcttcc catcctga 1188

<210> 28

<211> 10

<212> PRT

<213> Intelligent people

<400> 28

Lys Leu Tyr Gly Leu Asp Trp Ala Glu Leu

1 5 10