T-cell receptors (TCRs), their uses and pharmaceutical composition.

Abstract

The present invention relates to T cell receptors (TCRs) that bind to the restricted peptide hla-a*0201 gvydgrehtv (seq id no: 1) derived from the mage-a4 protein. The TCRs of the invention demonstrate excellent specificity profiles for this mage epitope. Nucleic acids encoding the TCRs, cells engineered to present the TCRs, cells harboring the expression vectors encoding the TCRs, and pharmaceutical compositions comprising the TCRs, nucleic acids, or cells of the invention are also provided.

Description

[001] The present invention relates to T cell receptors (TCRs) that bind to the restricted decapeptide HLA-A*0201 GVYDGREHTV derived from melanoma-associated antigen (MAGE) protein A2 (amino acids 230-239). The TCRs of the invention demonstrate excellent specificity profiles for this MAGE epitope. Background of the Invention

[002] Testicular cancer antigens (TCAs) are a subclass of tumor-associated antigens (TAAs) encoded by approximately 140 genes. The expression of these antigens is restricted to immune-privileged sites such as the testes, placenta, and fetal ovary; they are not normally expressed in other tissues. The expression of these genes has been observed in malignant tumors. The immunogenicity of TCAs has led to the widespread development of cancer vaccines targeting these antigens in many solid tumors. Within this vast class of TAAs, melanoma-associated antigens (MAGEs) have emerged as promising candidates for cancer immunotherapy.

[003] More than 30 testicular cancer (TC) genes have been reported as members of multigene families that are organized into gene clusters on the X chromosome (TC-X antigens). The gene clusters are located between TC Xq24 and Xq28 and include gene families such as MAGE and NY-ESO-1. Type I MAGE gene clusters are the most extensively characterized and include the MAGE-A, MAGE-B, and MAGE-C families. MAGE-A proteins are encoded by 12 different members of the MAGE-A family (MAGEA1 to MAGE-A12 genes) and are defined by a conserved amino acid base 165-171 called the MAGE homology domain (MHD). The MHD corresponds to the single amino acid region shared by all members of the MAGE-A family. Petition 870260068719, dated 10 / 07 / 2026, p. 7 / 117 2 / 44

[004] T cells recognize and interact with complexes of cell surface molecules, referred to as human leukocyte antigens (HLA) or major histocompatibility complexes (MHCs), and peptides. Peptides are derived from larger molecules that are processed by cells that also present the HLA / MHC molecule. The interaction of T cells and HLA / peptide complexes is restricted, requiring a specific T cell for a particular combination of an HLA molecule and a peptide. If a specific T cell is not present, there is no T cell response even if the partner complex is present. Similarly, there is no response if the specific complex is absent but the T cell is present. The mechanism is involved in the immune system's response to infection, in autoimmune diseases, and in responses to abnormalities such as tumors.

[005] Some proteins in the MAGE gene family are expressed only in germ cells and cancer (MAGE-A to MAGE-C families). Others are widely expressed in normal tissues (MAGE-D to MAGE-H). All these MAGE protein families have a homologous region that is closely matched to the sequence of other MAGE proteins and comprises peptides presented as HLA / peptide complexes upon immunological recognition. Therefore, it is important to select clinical TCR candidates that are highly specific for the desired MAGE peptide / HLA-A2 antigen.

[006] MAGE A4 is a CTA member of the MAGE A gene family. Its function is unknown, although it is believed that it may play a role in embryonic development. In tumor pathogenesis, it appears to be involved in tumor transformation or aspects of tumor progression. MAGE A4 has been implicated in a large number of tumors, including seminoma and dyskeratosis. Petition 870260068719, dated 10 / 07 / 2026, page 8 / 117 3 / 44 congenital, melanoma, hepatocellular carcinoma, renal cell carcinoma, pancreatic cancer, lung cancer, colorectal cancer, and breast cancer. The GVYDGREHTV peptide (SEQ ID NO: 1) corresponds to amino acid residue numbers 230-239 of the known MAGE-A4 protein.

[007] MAGE B2 is a CTA from the MAGE B gene family. MAGE B2 is expressed in the testes and placenta, and in a significant fraction of tumors of different histological types. The GVYDGEEHSV peptide (SEQ ID NO: 2) shows cross-reactivity with MAGE A4, such that certain TCRs are able to bind to HLA molecules that present both peptides. Summary of the Invention

[008] A TCR was developed that binds to HLA molecules that present the MAG A4 GVYDGREHTV peptide, in preference to MAGE B2. In a first aspect, the present invention provides a T cell receptor (TCR) that has the property of binding to GVYDGREHTV (SEQ ID NO: 1) in complex with HLA-A*0201 with a dissociation constant of about 0.05 μM to about 20.0 μM when measured with surface plasmon resonance at 25 °C and a pH between 7.1 and 7.5 using a soluble form of the TCR, wherein the TCR comprises an alpha chain variable domain of TCR and a beta chain variable domain of TCR, and wherein the variable domains of TCR form contacts with at least residues V2, Y3 and D4 of GVYDGREHTV (SEQ ID NO: 1).

[009] In embodiments, the TCR according to the invention has the property of binding to GVYDGEEHSV (SEQ ID NO: 2) in complex with HLA-A*0201 with a dissociation constant of about 20 μM to about 50 μM when measured with surface plasmon resonance at 25 °C and a pH between 7.1 and 7.5 using a soluble form of the TCR, wherein the TCR comprises a variable alpha chain domain. Petition 870260068719, dated 10 / 07 / 2026, p. 9 / 117 4 / 44 of TCR and a variable beta-chain domain of TCR. In some embodiments, the dissociation constant is above 50 μM, such as 100 μM, 200 μM, 500 μM or more.

[0010] Therefore, a TCR according to the invention is able to bind efficiently to HLA presenting GVYDGREHTV, but not to HLA presenting GVYDGEEHSV.

[0011] In some embodiments, the TCR alpha chain variable domain comprises an amino acid sequence that has at least 80% identity with the amino acid residue sequence 1-111 of SEQ ID NO: 3 (alpha chain), and / or the beta chain variable domain comprises an amino acid sequence that has at least 80% identity with the amino acid residue sequence 1-111 of SEQ ID NO: 4 (beta chain).

[0012] In a further aspect, the present invention provides a T cell receptor (TCR) having the property of binding to GVYDGREHTV (SEQ ID NO: 1) in complex with HLA-A*0201 and comprising a variable alpha chain domain of TCR and a variable beta chain domain of TCR,

[0013] the alpha chain variable domain comprising an amino acid sequence that has at least 80% identity with the amino acid residue sequence 1-111 of SEQ ID NO: 3, and / or

[0014] the beta chain variable domain comprising an amino acid sequence that has at least 80% identity with the amino acid residue sequence 1-111 of SEQ ID NO: 4.

[0015] The HLA-A2 GVYDGREHTV complex provides a cancer marker that the TCRs of the invention can target. The present invention provides such TCRs useful for the purpose of delivering cytotoxic or immune effector agents to cancer cells and / or useful for use in adoptive therapy.

[0016] TCRs are described using the TCR nomenclature of Petition 870260068719, dated 10 / 07 / 2026, page 10 / 117 5 / 44 Immunogenetics International (IMGT), and links to the public IMGT database of TCR sequences. Native alpha-beta heterodimeric TCRs have an alpha chain and a beta chain. Broadly speaking, each chain comprises variable, junction, and constant regions, and the beta chain also typically contains a short diversity region between the variable and junction regions, but this diversity region is often considered part of the junction region. Each variable region comprises three CDRs (Complementarity Determinant Regions) embedded in a structure sequence, one being the hypervariable region called CDR3. There are several types of alpha chain variable regions (Vα) and several types of beta chain variable regions (νβ) that are distinguished by their structure, the CDR1 and CDR2 sequences, and by a partially defined CDR3 sequence. The Vα types are referred to in the IMGT nomenclature by a unique TRAV number.Thus, TRAV21 defines a Vα region of TCR having a unique structure and sequences of CDR1 and CDR2, and a CDR3 sequence that is partially defined by an amino acid sequence that is conserved from TCR to TCR, but also includes an amino acid sequence that varies from TCR to TCR. Similarly, TRBV5-1 defines a Vβ region of TCR having a unique structure and sequences of CDR1 and CDR2, but with only a partially defined CDR3 sequence.

[0017] The TCR junction regions are also defined by the unique IMGT nomenclature TRAJ and TRBJ, and the constant regions by the IMGT nomenclature TRAC and TRBC.

[0018] The beta chain diversity region is referred to in IMGT nomenclature by the abbreviation TRBD, and, as mentioned, the concatenated TRBD / TRBJ regions are often considered together as the junction region. Petition 870260068719, dated 10 / 07 / 2026, p. 11 / 117 6 / 44

[0019] The α and β chains of αβ TCRs are generally considered to each have two domains, namely, variable and constant domains. The variable domain consists of a concatenation of a variable region and a junction region. In the present specification and claims, the term TCR variable domain α refers, therefore, to the concatenation of TRAV and TRAJ regions, and the term TCR constant domain α refers to the extracellular TRAC region, or to a C-terminal truncated TRAC sequence. Similarly, the term TCR variable domain β refers to the concatenation of TRBV and TRBD / TRBJ regions, and the term TCR constant domain β refers to the extracellular TRBC region, or to a C-terminal truncated TRBC sequence.

[0020] Unique sequences defined by the IMGT nomenclature are widely known and accessible to those working in the field of TCR. For example, they can be found in the public IMGT database. OT cell Receptor Factsbook, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8 also discloses sequences defined by the IMGT nomenclature, but because of its publication date and consequent time interval, the information sometimes needs to be confirmed by reference to the IMGT database.

[0021] A TCR according to the invention comprises an extracellular alpha chain domain, as shown in SEQ ID NO: 2 (TRAV10 + TRAC) and an extracellular beta chain domain, as shown in SEQ ID NO: 3 (TRBV24-1 + TRBC-2). The terms parental TCR, parental MAGE-A4 TCR, are used herein synonymously to refer to this TCR comprising the extracellular alpha and beta chains of SEQ ID Nos: 2 and 3, respectively. It is desirable to provide TCRs that are mutated or modified relative to the parental TCR that have higher affinity and / or a slower dissociation rate. Petition 870260068719, dated 10 / 07 / 2026, p. 12 / 117 7 / 44 for the peptide-HLA complex than the parental TCR.

[0022] For the purpose of providing a reference TCR against which the binding profile of such mutated or modified TCRs can be compared, it is convenient to use a soluble TCR according to the invention, which has the extracellular sequence of the alpha chain of the parental MAGE-A4 TCR given in SEQ ID NO: 3 and the extracellular sequence of the beta chain of the parental MAGE-A4 TCR given in SEQ ID NO: 4. This TCR is referred to herein as the reference TCR or the reference MAGE-A4 TCR. Note that SEQ ID NO: 5 comprises the extracellular sequence of the parental alpha chain of SEQ ID NO: 3 and that C162 has been replaced by T162 (i.e., T48 of TRAC). Similarly, SEQ ID NO: 6 is the extracellular sequence of the parental beta chain of SEQ ID NO: 4, and C169 was replaced by S169 (i.e., S57 of TRBC2), A187 was replaced by C187, and D201 was replaced by N201.These cysteine ​​substitutions relative to the parental extracellular alpha and beta chain sequences allow the formation of an interchain disulfide bond, which stabilizes the refolded soluble TCR, i.e., the TCR formed by refolding of extracellular alpha and beta chains. Using a stable disulfide-linked soluble TCR as the reference TCR allows for a more convenient assessment of binding affinity and binding half-life. The TCRs of the invention may comprise the mutations described above.

[0023] The TCRs of the invention may be of non-natural occurrence and / or purified and / or manipulated. The TCRs of the invention may have more than one mutation present in the alpha chain variable domain and / or in the beta chain variable domain relative to the parental TCR. Genetically modified TCR and mutant TCR are used herein synonymously and generally mean a TCR that has one or more mutations introduced relative to the parental TCR, in particular in its alpha chain variable domain and / or in the beta chain variable domain. Petition 870260068719, dated 10 / 07 / 2026, page 13 / 117 8 / 44 beta. This mutation(s) may improve the binding affinity for GVYDGREHTV (SEQ ID NO: 1) in complex with HLA-A*020.101. In certain embodiments, there are 1, 2, 3, 4, 5, 6, 7 or 8 mutations in the alpha chain variable domain, for example, 4 or 8 mutations, and / or 1, 2, 3, 4 or 5 mutations in the beta chain variable domain, for example, 5 mutations. In some embodiments, the variable α-chain domain of the TCR of the invention may comprise an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid residue sequence 1-111 of SEQ ID NO: 3.In some embodiments, the variable β-chain domain of the TCR of the invention may comprise an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with the amino acid residue sequence 1-111 of SEQ ID NO: 4.

[0024] The variable alpha chain domain of a TCR of the invention may have the following mutation: M4 V or L with reference to the numbering shown in SEQ ID NO: 3, and / or

[0025] The beta-chain variable domain may have at least one of the following mutations: N10 E with reference to the numbering shown in SEQ ID NO: 4.

[0026] The variable alpha chain domain of a TCR of the invention may comprise the amino acid sequence of amino acid residues 1-105 of SEQ ID NO: 3, 5 or 7 to 8.

[0027] or an amino acid sequence in which its amino acid residues 1-27, 34-47, and 54-90 have at least 90% or 95% of Petition 870260068719, dated 10 / 07 / 2026, p. 14 / 117 9 / 44 identity with the amino acid residue sequence 1-27, 34-47, and 54-90, respectively, of SEQ ID NO: 3, 5, or 7 to 8, and in which amino acid residues 28-33, 48-53, and 91-105 have at least 90% or 95% identity with the amino acid residue sequence 28-33, 48-53, and 91-105, respectively, of SEQ ID NO: 3, 5, or 7 to 8.

[0028] In the alpha-string variable domain, the sequence of

[0029] (i) amino acid residues 1-26 thereof may have (a) at least 90% identity with the amino acid residue sequence 1-26 of SEQ ID NO: 3 or (b) may have one, two or three amino acid residues inserted or deleted relative to the sequence of (a);

[0030] (ii) amino acid residues 28-33 is VSPFSN

[0031] (iii) amino acid residues 33-49 thereof may have (a) at least 90% identity with the amino acid residue sequence 34-47 of SEQ ID NO: 3 or (b) may have one, two or three amino acid residues inserted or deleted relative to the sequence of (a);

[0032] (iv) amino acid residues 48-53 may be LTIMTF or LTRMTF or LTIVTF or LTILTF

[0033] (v) amino acid residues 55-89 thereof may have at least 90% identity with the amino acid residue sequence 54-90 of SEQ ID NO: 3 or may have one, two or three insertions, deletions or substitutions in relation to it;

[0034] (vi) amino acids 90-93 can be CVVSGGTDSWGKLQF

[0035] The variable beta-chain domain of a TCR of the invention may comprise the amino acid sequence of SEQ ID NO: 4 or 6 or 9

[0036] or an amino acid sequence in which amino acid residues 1-45, 51-67, 74-109 thereof have at least 90% or 95% identity with the amino acid residue sequence 1-45, Petition 870260068719, dated 10 / 07 / 2026, p. 15 / 117 10 / 44 51-67, respectively 74-109 of SEQ ID NO: 4 or 9, and wherein amino acid residues 46-50, 68-73 and 109-123 have at least 90% or 95% identity with the amino acid residue sequence 4650, 68-73 and 109-123, respectively, of SEQ ID NO: 4 or 9.

[0037] In the variable beta string domain, the sequence of

[0038] (i) its amino acid residues 1-45 may have (a) at least 90% identity with the amino acid sequence of residues 1-45 of SEQ ID NO: 4 or (b) may have one, two or three amino acid residues inserted or deleted relative to the sequence of (a);

[0039] (ii) amino acid residues 46-50 may be KGHDR;

[0040] (iii) amino acid residues 51-67 thereof may have (a) at least 90% identity with the amino acid residue sequence 51-67 of SEQ ID NO: 4 or (b) may have one, two or three amino acid residues inserted or deleted relative to the sequence of (a);

[0041] (iv) amino acid residues 68-73 may be SVFDK;

[0042] (v) its amino acid residues 54-90 may have (a) at least 90% identity with the amino acid residue sequence 54-90 of SEQ ID NO: 4 or (b) may have one, two or three amino acid residues inserted or deleted relative to the sequence of (a);

[0043] (vi) amino acids 109-123 are CATSGQGAYNEQFF or CATSGQGAYREQFF;

[0044] A TCR of the invention may have one of the following combinations of alpha and beta chain-variable domains: Alpha String SEQ ID No. Beta String SEQ ID No. 3 4 3 6 3 9 Petition 870260068719, dated 10 / 07 / 2026, p. 16 / 117 11 / 44 Alpha String SEQ ID No. Beta String SEQ ID No. 5 4 5 6 5 9 7 4 7 6 7 9 8 4 8 6 8 9

[0045] Within the scope of the invention are phenotypically silent variants of any TCR of the invention disclosed herein. As used herein, the term phenotypically silent variants is understood to refer to a TCR that incorporates one or more amino acid alterations in addition to those mentioned above, whose TCR has a phenotype similar to the corresponding TCR without said alteration(s). For the purposes of this application, TCR phenotype comprises antigen-binding specificity (KD and / or binding half-life) and antigen specificity. A phenotypically silent variant may have a KD and / or binding half-life for the HLA-A*0201 GVYDGREHTV complex (SEQ ID NO: 1) within 10% of the measured KD and / or binding half-life of the corresponding TCR without said alteration(s), when measured under identical conditions (e.g., at 25 °C and on the same SPR chip). Suitable conditions are further defined in Example 3. Antigen specificity is further defined below.As is known to those skilled in the art, it may be possible to produce TCRs that incorporate alterations in the constant and / or variable domains of the same, relative to those described above, without altering the affinity for interaction with the HLA-A*0201 GVYDGREHTV complex (SEQ ID NO: 1). In particular, these silent mutations can be incorporated within parts of the sequence that are known. Petition 870260068719, dated 10 / 07 / 2026, page 17 / 117 12 / 44 because they are not directly involved in antigen binding (e.g., outside the CDR). Such trivial variants are included within the scope of the present invention. Those TCRs in which one or more conservative substitutions have been made also form part of this invention.

[0046] Mutations can be performed using any suitable method, including but not limited to those based on polymerase chain reaction (PCR), restriction enzyme-based cloning, or ligation-independent cloning (LIC) procedures. These methods are detailed in many standard molecular biology texts. For more details on polymerase chain reaction (PCR) and restriction enzyme-based cloning, see Sambrook & Russell, (2001) Molecular Cloning - A Laboratory Manual (3rd Ed.) CSHL Press. Further information on ligation-independent cloning (LIC) procedures can be found in Rashtchian, (1995) Curr Opin Biotechnol 6 (1): 30-6.

[0047] The TCRs of the invention have the property of binding to the HLA-A2 peptide complex MAGE-A4, GVYDGREHTV (SEQ ID NO: 1). The TCRs of the invention have been found to be highly specific for MAGE epitopes relative to other, irrelevant epitopes, and are thus particularly suitable as targeting vectors for the delivery of therapeutic agents or detectable markers to cells and tissues that exhibit these epitopes. Specificity in the context of the TCR of the invention refers to its ability to recognize HLA-A*0201 target cells that are positive for the GVYDGREHTV peptide, while having the minimal ability to recognize HLA-A*0201 target cells that are negative for the peptide, or HLA cells that exhibit the MAGE B2 peptide GVYDGEEHSV. To test specificity, TCRs can be in soluble form and / or expressed on the surface of T cells. Recognition can be determined by measuring the level of T cell activation in the presence of a TCR. Petition 870260068719, dated 10 / 07 / 2026, p. 18 / 117 13 / 44 TCR and target cells. In this case, the minimum recognition of MAGE B2 or peptide-negative target cells is defined as a T cell activation level of less than 10%, preferably less than 5%, and more preferably less than 1%, of the level produced in the presence of peptide-positive target cells, when measured under the same conditions. For the TCRs of the present invention, specificity can be determined at a therapeutically relevant TCR concentration. The therapeutically relevant concentration can be defined as a TCR concentration of 10⁻⁹ M or lower, and / or a concentration up to 100, preferably up to 1000 times greater than the corresponding EC50 value. Peptide-positive cells can be obtained by peptide-pulsed or, more preferably, they can be naturally present in said peptide. Preferably, both peptide-positive and peptide-negative cells are human cells.

[0048] Certain TCRs of the invention have been found to be highly suitable for use in adoptive therapy. Such TCRs may have a Kd for the complex of less than 200 μM, for example, from about 0.05 μM to about 20 μM or about 100 μM and / or have a binding half-life (T1 / 2) for the complex in the range of about 0.5 seconds to about 12 minutes. In some embodiments, the TCRs of the invention may have a Kd for the complex starting at about 0.05 μM to about 20 μM, about 0.1 μM to about 5 μM or about 0.1 μM to about 2 μM. Without pretending to be limited by theory, there appears to be an ideal affinity window for TCRs with therapeutic use in adoptive cell therapy. Naturally occurring TCRs that recognize tumor antigen epitopes are generally of very low affinity (20 µM to 50 µM) and very high affinity TCRs (in the nanomolar range or higher) suffer from cross-reactivity issues (Robbins et al (2008) J.Immunol 180 61166131; Zhao et al (2007) J. Immunol 179 5.845-5.854; Scmid et al (2010) J. Immunol 184 4.936-4.946). Petition 870260068719, of 10 / 07 / 2026, p. 19 / 117 14 / 44

[0049] The TCRs of the invention may be αβ heterodimers or may be in single-stranded form. Single-stranded formats include αβ TCR polypeptides of the Va-L-νβ, νβ-L-Va, Va-Ca-L-νβ or VaL-Ve-Ce types, where Va and νβ are variable α and β regions of TCR respectively, Cα and Cβ are constant α and β regions of TCR respectively, and L is a linker sequence. For use as a targeting agent for the delivery of therapeutic agents to the antigen-presenting cell, the TCR may be in soluble form (i.e., not having transmembrane or cytoplasmic domains). For stability, soluble αβ heterodimeric TCRs preferably have a disulfide bond introduced between the residues of the respective constant domains, as described, for example, in WO 03 / 020763. One or both of the constant domains present in an αβ heterodimer of the invention may be truncated at the C-terminal or C-terminals, for example, up to 15, or up to 10, or up to 8 or fewer amino acids.For use in adoptive therapy, a heterodimeric αβ TCR can, for example, be transfected as full-length chains having both cytoplasmic and transmembrane domains. TCRs for use in adoptive therapy may contain a disulfide bond corresponding to that found in nature between the respective constant alpha and beta domains; additionally or alternatively, a non-native disulfide bond may be present.

[0050] As will be obvious to those skilled in the art, it may be possible to truncate the sequences provided at the C-terminal and / or N-terminal thereof by 1, 2, 3, 4, 5 or more residues without substantially affecting the binding characteristics of the TCR. All such trivial variations are encompassed by the present invention.

[0051] Alpha-beta heterodimeric TCRs of the invention typically comprise an alpha-chain TRAC constant-domain sequence and a TRBC1 or TRBC2 constant-domain sequence. Petition 870260068719, dated 10 / 07 / 2026, p. 20 / 117 15 / 44 beta chain. The alpha and beta chain constant domain sequences can be modified by truncation or substitution to eliminate the native disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2. The alpha and beta chain constant domain sequences can also be modified by substituting cysteine ​​residues for Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2, the aforementioned cysteines forming a disulfide bridge between the alpha and beta constant domains of TCR.

[0052] Some TCRs of the invention have a binding affinity and / or a binding half-life for the GVYDGREHTV-HLA-A2 complex that is substantially higher than that of the reference MAGE-A4 TCR. The increased binding affinity of a native TCR often reduces the specificity of the TCR for its MHC-peptide ligand, and this is demonstrated in Zhao Yangbing et al., The Journal of Immunology, The American Association of Immunologists, US, vol. 179, No. 9, November 1, 2007, 5845-5854. However, the TCRs of the invention, which are derived from the parental TCR, remain specific for the GVYDGREHTV-HLA-A2 complex despite having a substantially higher binding affinity than the parental TCR. Furthermore, they are significantly more selective (e.g., at least ten times) for MAGE-A4 over MAGE-B2 than the parental TCR.

[0053] Binding affinity (inversely proportional to the equilibrium constant Kd) and binding half-life (expressed as T1 / 2) can be determined using the Surface Plasmon Resonance (BIAcore) method of Example 3 here. Measurements can be performed at 25 °C and a pH between 7.1 and 7.5 using a soluble version of the TCR. It will be appreciated that doubling the affinity of a TCR results in a halving of Kd. T1 / 2 is calculated as In2 ​​divided by the dissociation rate (koff). Thus, doubling T1 / 2 results in a halving of koff. Kd and koff values ​​for TCR are Petition 870260068719, dated 10 / 07 / 2026, page 21 / 117 16 / 44 generally measured for soluble forms of TCR, i.e., forms that are truncated to remove residues from the hydrophobic transmembrane domain. Therefore, it should be understood that a given TCR satisfies the requirement that it has a binding affinity for, and / or a binding half-life for, the GVYDGREHTV-HLA-A2 complex if a soluble form of the TCR satisfies this requirement. Preferably, the binding affinity or a binding half-life of a given TCR is measured multiple times, for example, 3 or more times, using the same assay protocol, and an average of the results is taken. The reference MAGE-A4 TCR has a KD of approximately 65 μM as measured by this method, and its koff is approximately 0.73 s⁻¹ (i.e., T₁ / ₂ is about 0.95 s).

[0054] In another aspect, the present invention provides nucleic acid encoding a TCR of the invention. In some embodiments, the nucleic acid is cDNA. In some embodiments, the invention provides nucleic acid comprising a sequence encoding an α-strand variable domain of a TCR of the invention. In some embodiments, the invention provides nucleic acid comprising a sequence encoding a β-strand variable domain of a TCR of the invention. The nucleic acid may be non-naturally occurring and / or purified and / or genetically engineered.

[0055] In another aspect, the invention provides a vector comprising nucleic acid of the invention. Preferably, the vector is a TCR expression vector.

[0056] The invention also provides a cell that hosts a vector of the invention, preferably a TCR expression vector. The vector may comprise the nucleic acid of the invention encoding in a single open reading frame, or two distinct open reading frames, the alpha strand and the beta strand, respectively. Another aspect provides a cell that hosts an expression vector that Petition 870260068719, dated 10 / 07 / 2026, page 22 / 117 17 / 44 comprises a first nucleic acid encoding the alpha chain of a TCR of the invention, and a second expression vector comprising the nucleic acid encoding the beta chain of a TCR of the invention. Such cells are particularly useful in adoptive therapy. The cells of the invention may be isolated and / or recombinant and / or non-naturally occurring and / or genetically engineered.

[0057] As the TCRs of the invention have utility in adoptive therapy, the invention includes a non-naturally occurring and / or purified and / or genetically engineered cell, especially a T cell, exhibiting a TCR of the invention. The invention also provides an expanded population of T cells exhibiting a TCR of the invention. There are a number of suitable methods for transfecting T cells with nucleic acid (such as DNA, cDNA, or RNA) encoding the TCR of the invention (see, for example, Robbins et al., (2008) J Immunol. 180: 6116-6131). T cells expressing the TCRs of the invention will be suitable for use in cancer treatment based on adoptive therapy. As will be known to those skilled in the art, there are a number of suitable methods by which adoptive therapy can be performed (see, for example, Rosenberg et al., (2008) Nat Rev Cancer 8(4): 299-308).

[0058] The soluble TCRs of the invention are useful for delivering detectable markers or therapeutic agents to antigen-presenting cells and tissues containing antigen-presenting cells. They can therefore be associated (covalently or otherwise) with a detectable marker (for diagnostic purposes, where the TCR is used to detect the presence of cells presenting the GVYDGREHTV-HLA-A2 complex); a therapeutic agent; or a PK modifier radical (e.g., by PEGylation).

[0059] Detectable markers for diagnostic purposes include, Petition 870260068719, dated 10 / 07 / 2026, p. 23 / 117 18 / 44 for example, fluorescent markers, radioactive markers, enzymes, nucleic acid probes and contrast reagents.

[0060] Therapeutic agents that can be associated with the The TCRs of the invention include immunomodulators, radioactive compounds, enzymes (perforin, for example) or chemotherapeutic agents (cisplatin, for example). To ensure that the toxic effects are exerted at the desired site, the toxin could be contained within a liposome bound to the TCR so that the compound is released slowly. This will prevent harmful effects during transport in the body and ensure that the toxin has a maximum effect after binding of the TCR to the relevant antigen-presenting cells.

[0061] Other suitable therapeutic agents include:

[0062] · Small molecule cytotoxic agents, that is, compounds with the ability to kill mammalian cells having a molecular weight of less than 700 Daltons. Such compounds may also contain toxic metals capable of having a cytotoxic effect. Furthermore, it should be understood that these small molecule cytotoxic agents also include prodrugs, that is, compounds that decompose or are converted under physiological conditions to release cytotoxic agents. Examples of such agents include cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer fotofrin II sodium, temozolomide, topotecan, trimetreate glucuronate, auristatin E, vincristine and doxorubicin;

[0063] · Peptide cytotoxins, that is, proteins or fragments thereof with the ability to kill mammalian cells. For example, ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNase and RNase;

[0064] • radionuclides, that is, unstable isotopes of elements that decay with the simultaneous emission of one or more α and β particles Petition 870260068719, dated 10 / 07 / 2026, p. 24 / 117 19 / 44 β or γ rays. For example, iodine 131, rhenium 186, indium 111, yttrium 90, bismuth 210 and 213, actinium 225 and astatine 213; chelating agents can be used to facilitate the association of these radionuclides to high-affinity TCRs, or multimers thereof;

[0065] · immunostimulants, that is, immune effector molecules that stimulate the immune response. For example, cytokines, such as IL-2 and IFN-γ,

[0066] • Superantigens and their mutants;

[0067] · TCR-HLA mergers;

[0068] · chemokines such as IL-8, platelet factor 4, melanoma growth-stimulating protein, etc;

[0069] • antibodies or their fragments, including anti-T cell-determining antibodies or NK cell-determining antibodies (e.g. anti-CD3, anti-CD28 or anti-CD16);

[0070] • alternative protein scaffolds with antibody-like binding features

[0071] · complement activators;

[0072] • xenogeneic protein domains, allogeneic protein domains, viral / bacterial protein domains, viral / bacterial peptides

[0073] A preferred embodiment is provided by a TCR of the invention associated (generally by fusion with an N- or C-terminal of the alpha or beta chain) with an anti-CD3 antibody, or a functional fragment or variant of said anti-CD3 antibody. Antibody fragments and variants / analogs suitable for use in the compositions and methods described herein include minibodies, Fab fragments, F(ab')2 fragments, dsFv and scFv fragments, Nanobodies™ (these constructs, marketed by Ablynx (Belgium), comprise a variable domain of single synthetic immunoglobulin heavy chain derived from a camelid antibody (e.g., camel or Petition 870260068719, dated 10 / 07 / 2026, page 25 / 117 20 / 44 llama)) and domain antibodies (Domantis (Belgium), comprising a variable heavy chain domain of single-dimmed immunoglobulin affinity-matured variable light chain domain of immunoglobulin) or alternative protein scaffolds that exhibit antibody-like binding features, such as affibodies (Affibody (Sweden), comprising a genetically modified protein A scaffold) or anticalins (Pieris (German), comprising genetically modified anticalins) to mention just a few examples.

[0074] For some purposes, the TCR of the invention can be aggregated into a complex comprising several TCRs to form a multivalent TCR complex. There are a number of human proteins that contain a multimerization domain that can be used in the production of multivalent TCR complexes. For example, the tetramerization domain of p53 has been used to produce tetramers of scFv antibody fragments that have shown increased serum persistence and significantly reduced the dissociation rate compared to the monomeric scFv fragment. (Willuda et al. (2001) J. Biol. Chem. 276 (17) 14385-14392). Hemoglobin also has a tetramerization domain that could be used for this type of application. A multivalent TCR complex of the invention may have greater binding capacity to the GVYDGREHTV HLA-A2 complex compared to a non-multimeric wild-type or heterodimer T cell receptor of the invention.Thus, the multivalent TCR complexes of the invention are also included within the scope of the invention. Such multivalent TCR complexes according to the invention are particularly useful for screening or targeting particular antigen-presenting cells in vitro or in vivo, and are also useful as intermediates for the production of new multivalent TCR complexes having such uses. Petition 870260068719, dated 10 / 07 / 2026, p. 26 / 117 21 / 44

[0075] As is well known in the art, TCRs can undergo post-translational protein modification. Glycosylation is one such modification, which comprises the covalent attachment of oligosaccharide moieties to defined amino acids in the TCR chain. For example, asparagine or serine / threonine residues are well-known sites for oligosaccharide attachment. The glycosylation state of a particular protein depends on a number of factors, including the protein sequence, the protein conformation, and the availability of certain enzymes. Furthermore, the glycosylation state (i.e., oligosaccharide type, covalent linkage, and total number of linkages) can influence protein function. Therefore, when producing recombinant proteins, control of glycosylation is often desirable. Controlled glycosylation has been used to improve antibody-based therapeutics. (Jefferis R., Nat Rev Drug Discov. March 2009; 8(3): 226-34.).For the soluble TCRs of the invention, glycosylation can be controlled in vivo, using particular cell lines, for example, or in vitro, by chemical modification. Such modifications are desirable, since glycosylation can improve pharmacokinetics, reduce immunogenicity, and more closely mimic a native human protein (Sinclair AM and Elliott S., Pharm Sci. August 2005; 94(8): 1626-35).

[0076] For administration to patients, the TCRs, nucleic acids and / or cells of the invention (generally associated with a detectable marker or a therapeutic agent) can be provided in a pharmaceutical composition together with a pharmaceutically acceptable vehicle or excipient. Therapeutic or imaging TCRs in accordance with the invention will normally be provided as part of a sterile, pharmaceutical composition that will normally include a pharmaceutically acceptable vehicle. This pharmaceutical composition may be in any appropriate form. Petition 870260068719, dated 10 / 07 / 2026, page 27 / 117 22 / 44 (depending on the desired method of administration to a patient). It may be supplied in unit dose form, will generally be dispensed in a sealed container, and may be dispensed as part of a kit. Such a kit would typically (though not necessarily) include instructions for use.

[0077] The pharmaceutical composition can be adapted for administration by any appropriate route, preferably a parenteral route (including subcutaneous, intramuscular, or preferably intravenous). Such compositions can be prepared by any method known in the field of pharmacy, for example, by mixing the active ingredient with the vehicle(s) or excipient(s) under sterile conditions.

[0078] The dosages of the substances of the invention may vary within wide limits, depending on the disease or disorder to be treated, the age and condition of the individual to be treated, etc., and a physician will determine the appropriate dosages to be used.

[0079] TCRs, pharmaceutical compositions, vectors and nucleic acids and cells of the invention may be provided in a substantially pure form, for example, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% pure.

[0080] The following are also provided by the invention:

[0081] · A TCR, nucleic acid or cell of the invention for use in medicine, preferably for use in a method of treating cancer, such as solid tumors (e.g., lung, liver and gastric metastases) and / or squamous cell carcinomas.

[0082] • the use of a TCR, nucleic acid or cell of the invention in the manufacture of a drug to treat cancer.

[0083] • a method of treating cancer in a patient, Petition 870260068719, dated 10 / 07 / 2026, page 28 / 117 23 / 44 comprising administering to the patient a TCR, nucleic acid or cell of the invention.

[0084] The preferred features of each aspect of the invention are the same for each of the other aspects mutatis mutandis. The prior art documents referred to herein are incorporated to the fullest extent permitted by law.

[0085] The invention is further described in the following non-limiting examples.

[0086] Reference is made to the attached sequences, in which:

[0087] SEQ ID NO: 1 is the peptide MAGE A4

[0088] SEQ ID NO: 2 is the peptide MAGE B2

[0089] SEQ ID NO: 3 is the amino acid sequence of the extracellular alpha chain portion of a specific parental MAGE-A4 TCR, and SEQ ID NO: 4 shows the amino acid sequence of the extracellular beta chain portion of a specific parental MAGE-A4 TCR beta chain amino acid sequence.

[0090] SEQ ID NO: 5 shows the amino acid sequence of the alpha chain of a native Lenti TCR (herein referred to as the reference TCR). The sequence is the same as that of the parental TCR except that one cysteine ​​is replaced by T162 (i.e., T48 of the TRAC constant region). SEQ ID NO: 6 is the beta chain of a native Lenti TCR (herein referred to as the reference TCR). The sequence is the same as the parental TCR, except that the cysteine ​​is replaced by S169 (i.e., S57 of the TRBC2 constant region) and A187 is replaced by C187 and D201 is replaced by N201.

[0091] SEQ ID NO: 7 and 8 shows the alpha chain sequences that may be present in the TCRs of the invention. The subsequences that form the CDR regions, or substantial parts of the CDR regions, are underlined.

[0092] SEQ ID NO: 9 shows the sequence of the beta string which can Petition 870260068719, dated 10 / 07 / 2026, p. 29 / 117 24 / 44 is present in the TCRs of the invention. The subsequences that form the CDR regions, or substantial parts of the CDR regions, are underlined.

[0093] SEQ ID Nos: 11 to 15 show the soluble beta and alpha chain sequences of TCRs A, B, and C. None of these TCRs could be developed into a functional TCR according to the present invention. Examples Example 1 - Cloning of alpha and beta chain variable region sequences of reference TCR MAGE-A4 into pGMT7-based expression plasmids

[0094] The alpha variable domains of the parental MAGE-A4 TCR and the beta variable domains of the TCR from SEQ ID NOs: 3 and 4, respectively, were cloned into pGMT7-based expression plasmids containing either Ca or Ce by conventional methods described in (Molecular Cloning: A Laboratory Manual, Third Edition by Sambrook and Russell). The plasmids were sequenced using an Applied Biosystems 3730x1 DNA Analyzer. The alpha variable domains of the reference MAGE-A4 TCR and the beta variable domains of the TCR from SEQ ID NOs: 4 and 5, respectively, were cloned in the same manner.

[0095] The DNA sequence encoding the alpha strand variable region of the TCR was ligated into pEX956, which was cleaved with restriction enzymes. The DNA sequence encoding the beta strand variable region of the TCR was ligated into pEXb21, which was also cleaved with restriction enzymes.

[0096] Bound plasmids were transformed into competent XL1Blue E. coli cells and plated onto LB / agar plates containing 100 pg / mL ampicillin. After overnight incubation at 37 °C, individual colonies were harvested and grown in 5 ml of LB containing 100 pg / mL ampicillin overnight at 37 °C with Petition 870260068719, dated 10 / 07 / 2026, page 30 / 117 25 / 44 agitation. Cloned plasmids were purified using a Miniprep kit (Qiagen), and the plasmids were sequenced using an Applied Biosystems 3730x1 DNA Analyzer. Example 2 - Expression, refolding and purification of soluble reference MAGEA4 TCR

[0097] Expression plasmids containing the α chain and β chain of reference TCRs, respectively, as prepared in Example 1, were separately transformed into the BL21 pLysS strain of E. coli, and isolated ampicillin-resistant colonies were grown at 37 °C in TYP medium (100 pg / mL ampicillin) at an ODg0 of ~0.6-0.8 before induction of protein expression with 0.5 mM IPTG. Cells were harvested three hours post-induction by centrifugation for 30 minutes at 4000 rpm in a Beckman J-6B. Cell pellets were lysed with 25 mL of Bug Buster (Novagen) in the presence of MgCl2 and DNasel. Inclusion body pellets were recovered by centrifugation for 30 minutes at 13000 rpm in a Beckman J2-21 centrifuge. Three washes with detergents were then performed to remove cellular debris and membrane components.Every hour, the inclusion body sediment was homogenized in Triton buffer (50 mM Tris-HCl at pH 8.0, 0.5% Triton-X100, 200 mM NaCl, 10 mM NaEDTA) before being sedimented by centrifugation for 15 minutes at 13,000 rpm in a Beckman J2-21. The detergent and salt were then removed by a similar wash in the following buffer: 50 mM Tris-HCl at pH 8.0, 1 mM NaEDTA. Finally, the inclusion bodies were divided into 30 mg aliquots and frozen at -70 °C. The protein yield of the inclusion body was quantified by solubilization with 6M guanidine-HCl, and an OD measurement was performed on a Hitachi L-2001 spectrophotometer. The protein concentration was then calculated using the extinction coefficient. Petition 870260068719, dated 10 / 07 / 2026, page 31 / 117 26 / 44

[0098] Inclusion bodies solubilized in approximately 15 mg of TCR α chain and 15 mg of TCR β chain were thawed from frozen stocks and diluted in 10 ml of a guanidine solution (6M guanidine hydrochloride, 50 mM Tris-HCl at pH 8.1, 100 mM NaCl, 10 mM EDTA, 10 mM DTT) to ensure complete chain denaturation. The guanidine solution containing completely reduced and denatured TCR chains was then injected into 0.5 liters of the following refolding buffer: 100 mM Tris at pH 8.1, 400 mM L-arginine, 2 mM EDTA, 5 M urea. The redox pair (cysteamine hydrochloride and cystamine dihydrochloride) with final concentrations of 6.6 mM and 3.7 mM, respectively, was added approximately 5 minutes before the addition of the denatured TCR chains. The solution was left for ~30 minutes. The redoxed TCR was dialyzed on a Spectrapor 1 membrane (Spectro; Product No. 132670) against 10 L of H2O for 18-20 hours.After this time, the dialysis buffer was changed twice to 10 mM fresh Tris at pH 8.1 (10 L) and dialysis continued at 5 °C ± 3 °C for another ~8 hours.

[0099] Soluble TCR was separated from degradation products and impurities by loading the dialyzed refold onto a POROS 50HQ anion exchange column and eluting the bound protein with a 0-500 mM NaCl gradient in 10 pM Tris at pH 8.1 over 50 column volumes using an Akta purifier (GE Healthcare). The peak fractions were pooled and a protease inhibitor cocktail (Calbiochem) was added. The pooled fractions were then stored at 4 °C and analyzed by Coomassie-stained SDS-PAGE before being pooled and concentrated. Finally, the soluble TCR was purified and characterized using a GE Healthcare Superdex 75HR gel filtration column pre-equilibrated in PBS buffer (Sigma). The peak that eluted with a relative molecular weight of approximately 50 kDa was pooled and concentrated before... Petition 870260068719, dated 10 / 07 / 2026, page 32 / 117 27 / 44 characterization by BIAcore surface plasmon resonance analysis. Example 3 - Bond characterization BIAcore Analysis

[00100] A surface plasmon resonance biosensor (BIAcore 3000™) can be used to analyze the binding of a soluble TCR to its peptide-MHC ligand. This is facilitated by producing soluble biotinylated peptide-HLA (pHLA) complexes that can be immobilized onto a streptavidin-coated binding surface (sensor chip). The sensor chips comprise four individual flow cells, allowing simultaneous measurement of T cell receptor binding to four different pHLA complexes. Manual injection of the pHLA complex allows the precise level of immobilized class I molecules to be easily manipulated.

[00101] Biotinylated HLA-A*0201 class I molecules were refolded in vitro from bacterially expressed inclusion bodies containing the constituent subunit proteins and the synthetic peptide, followed by purification and enzymatic biotinylation in vitro (O'Callaghan et al. (1999) Anal. Biochem. 266: 9-15). The HLA-A*0201 heavy chain was expressed with a C-terminal biotinylation marker that replaces the transmembrane and cytoplasmic domains of the protein in a suitable construct. Inclusion body expression levels of ~75 mg / liter of bacterial culture were obtained. The MHC light chain or e2-microglobulin was also expressed as inclusion bodies in E. coli from a suitable construct, at a level of -500 mg / liter of bacterial culture.

[00102] E. coli cells were lysed and inclusion bodies purified to approximately 80% purity. Inclusion body protein was denatured in 6 M guanidine-HCl, 50 mM Tris at pH 8.1, 100 mM NaCl, 10 mM DTT, 10 mM EDTA, and was Petition 870260068719, dated 10 / 07 / 2026, p. 33 / 117 28 / 44 refolded to a concentration of 30 mg / liter of heavy chain, 30 mg / liter of β2ιτ in 0.4 M L-arginine, 100 mM Tris at pH 8.1, 3.7 mM cystamine dihydrochloride, 6.6 mM cysteamine hydrochloride, 4 mg / L of the MAGE-A4 GVYDGREHTV or MAGE-B2 GVYDGEEHSV peptide required to be carried by the HLAa*02 molecule, by adding a single pulse of denatured protein in refolding buffer at < 5 °C. Refolding was allowed to reach completion at 4 °C for at least 1 hour.

[00103] The buffer was changed by dialysis in 10 volumes of 10 mM Tris at pH 8.1. Two buffer changes were necessary to reduce the ionic strength of the solution sufficiently. The protein solution was then filtered through a 5 μm cellulose acetate filter and loaded onto a POROS 50HQ anion exchange column (8 ml bed volume). The protein was eluted with a linear gradient of 0-500 mM NaCl in 10 mM Tris at pH 8.1 using an Akta purifier (GE Healthcare). The HLA-A*0201-peptide complex eluted at approximately 250 mM NaCl, and peak fractions were harvested, a protease inhibitor cocktail (Calbiochem) was added, and the fractions were cooled on ice.

[00104] Biotin-labeled PHLA molecules were buffered to 10 mM Tris at pH 8, 1.5 mM NaCl using a GE Healthcare fast desalting column balanced in the same buffer. Immediately after elution, the protein-containing fractions were cooled on ice and protease inhibitor cocktail (Calbiochem) was added. Then, biotinylation reagents were added: 1 mM biotin, 5 mM ATP (buffered at pH 8), 7.5 mM MgCl2 and 5 pg / mL BirA enzyme (purified according to O'Callaghan et al. (1999) Anal. Biochem 266: 9-15). The mixture was then incubated at room temperature overnight.

[00105] Biotinylated pHLA-A*0201 molecules were purified Petition 870260068719, dated 10 / 07 / 2026, page 34 / 117 29 / 44 using gel filtration chromatography. A GE Healthcare Superdex 75 HR 10 / 30 column was pre-equilibrated with filtered PBS, and 1 ml of biotinylation reaction mixture was applied. The column was then developed with PBS at 0.5 ml / min using an Akta purifier (GE Healthcare). Biotinylated pHLA-A*0201 molecules eluted as a single peak at approximately 15 ml. Protein-containing fractions were pooled, cooled on ice, and protease inhibitor cocktail was added. Protein concentration was determined using a Coomassie binding assay (PerBio), and aliquots of biotinylated pHLA-A*01 molecules were stored frozen at -20 °C.

[00106] These immobilized complexes are capable of binding to both T cell receptors and CD8aa coreceptors, both of which can be injected in the soluble phase. The pHLA binding properties of soluble TCRs are observed to be qualitatively and quantitatively similar whether the TCR is used in the soluble or immobilized phase. This is an important control for the partial activity of soluble species and also suggests that biotinylated pHLA complexes are as biologically active as non-biotinylated complexes.

[00107] Measurements of the BIAcore 3000™ surface plasmon resonance (SPR) biosensor show changes in refractive index expressed in response units (RU) near a sensor surface within a small flow cell, a principle that can be used to detect receptor ligand interactions and to analyze their affinity and kinetic parameters. BIAcore experiments were performed at a temperature of 25 °C, using PBS buffer (Sigma, pH 7.1–7.5) as the running buffer and in the preparation of protein sample dilutions. Streptavidin was immobilized with the flow cells by conventional amine coupling methods. pHLA complexes were immobilized using the biotin marker. The assay was then performed via the Petition 870260068719, dated 10 / 07 / 2026, page 35 / 117 30 / 44 passage of soluble TCR over the surfaces of different flow cells at a constant flow rate, measuring the SPR response while doing so. Equilibrium bond constant

[00108] Previous BIAcore analysis methods were used to determine equilibrium binding constants. Serial dilutions of the disulfide-linked soluble heterodimeric form of the reference TCR MAGE-A4 were prepared and injected at a constant flow rate of 5 μL min-1 over two different flow cells; one coated with ~1000 RU of specific GVYDGREHTV HLA-A*0201 complex, the second coated with ~1000 RU of non-specific complex. The response was normalized for each concentration using the measurement from the control cell. The normalized data response was plotted against the TCR sample concentration and fitted to a nonlinear curve fitting model to calculate the equilibrium binding constant, KD. (Price & Dweck, Principles and Problems in Physical Chemistry for Biochemists (2nd Edition) 1979, Clarendon Press, Oxford).The disulfide-linked soluble form of reference TCR MAGE-A4 (Example 2) showed a Kd of approximately 2.00 μM. From the same BIAcore data, T1 / 2 was approximately 0.95 s. Kinetic Parameters

[00109] The BIAcore analysis methods above were also used to determine equilibrium binding constants and rates.

[00110] For high-affinity TCRs (see Example 4 below) KD was determined by experimentally measuring the dissociation rate constant, koff, and the association rate constant, kon. The equilibrium constant KD was calculated as koff / kon.

[00111] TCR was injected onto two different cells, one coated with ~1000 RU of HLA-A*0201 GVYDGREHTV complex. Petition 870260068719, dated 10 / 07 / 2026, p. 36 / 117 31 / 44 specific, the second coated with ~1000 RU of non-specific complex. The flow rate was fixed at 50 μL / min. Typically, 250 pL of TCR at a concentration of ~1 μM were injected. The buffer was then poured until the response had returned to baseline values ​​or > 2 hours had elapsed. Kinetic parameters were calculated using BIAevaluation software. The dissociation phase was fitted to an exponential decay equation that allows the calculation of the half-life. Example 4 - Preparation of high-affinity TCRs of the invention

[00112] Expression plasmids containing the α chain and β chain of TCR, respectively, were prepared as in Example 1: TCR ID Alpha String SEQ ID No. Beta String SEQ ID No. TCR1 (parental) 3 4 TCR2 3 9 TCR3 7 9 TCR4 8 9

[00113] Plasmids were separately transformed into the BL21pLysS strain of E. coli, and individual ampicillin-resistant colonies were grown at 37 °C in TYP medium (100 pg / mL ampicillin) at 0.6–0.8 °C before induction of protein expression with 0.5 mM IPTG. Cells were harvested three hours post-induction by centrifugation for 30 minutes at 4000 rpm in a Beckman J-6B. Cell pellets were lysed with 25 mL of Bug Buster (Novagen) in the presence of MgCb and DNasel. Inclusion body pellets were recovered by centrifugation for 30 minutes at 13000 rpm in a Beckman J2-21 centrifuge. Three detergent washes were then performed to remove cell debris and membrane components. Every hour the sediment of inclusion bodies was homogenized in a Triton buffer (50 mM Tris-HCl at pH 8.0, 0.5% Triton-X100, 200 mM NaCl, 10 mM NaEDTA) before... Petition 870260068719, dated 10 / 07 / 2026, page 37 / 117 32 / 44 were sedimented by centrifugation for 15 minutes at 13,000 rpm in a Beckman J2-21. The detergent and salt were then removed by a similar wash in the following buffer: 50 mM Tris-HCl at pH 8.0, 1 mM NaEDTA. Finally, the inclusion bodies were divided into 30 mg aliquots and frozen at -70 °C. Protein yield from inclusion bodies was quantified by solubilization with 6M guanidine-HCl, and an OD measurement was performed on a Hitachi U-2001 spectrophotometer. The protein concentration was then calculated using the extinction coefficient.

[00114] Inclusion bodies solubilized to approximately 10 mg of TCR α chain and 10 mg of TCR β chain for each TCR of the invention were diluted in 10 mL of a guanidine solution (6M guanidine hydrochloride, 50 mM Tris-HCl at pH 8.1, 100 mM NaCl, 10 mM EDTA, 10 mM DTT) to ensure complete chain denaturation. The guanidine solution containing completely reduced and denatured TCR chains was then injected into 0.5 liters of the following refolding buffer: 100 mM Tris at pH 8.1, 400 mM L-arginine, 2 mM EDTA, 5 M urea. The redox pair (cysteamine hydrochloride and cystamine dihydrochloride) to final concentrations of 6.6 mM and 3.7 mM, respectively, was added approximately 5 minutes before the addition of the denatured TCR chains. The solution was left for ~30 minutes. The redoxed TCR was dialyzed on a Spectrapor 1 membrane (Spectro; Product No. 132670) against 10 L H2O for 18–20 hours.After this time, the dialysis buffer was changed twice to 10 mM fresh Tris at pH 8.1 (10 L) and dialysis continued at 5 °C ± 3 °C for another ~8 hours.

[00115] Soluble TCR was separated from degradation products and impurities by loading the dialyzed refold onto a POROS 50HQ anion exchange column and eluting the bound protein with a 0-500 mM NaCl gradient in 10 mM Tris at pH 8.1 over 15 Petition 870260068719, dated 10 / 07 / 2026, page 38 / 117 33 / 44 column volumes were used with an Akta purifier (GE Healthcare). The pooled fractions were then stored at 4 °C and analyzed by Coomassie-stained SDS-PAGE before being pooled and concentrated. Finally, the soluble TCRs were purified and characterized using a GE Healthcare Superdex 75HR gel filtration column pre-equilibrated in PBS buffer (Sigma). The peak that eluted with a relative molecular weight of approximately 50 kDa was pooled and concentrated before characterization by BIAcore surface plasmon resonance analysis.

[00116] The TCR affinity profiles thus prepared for the MAGE-A4 epitope or MAGE-B2 epitope were evaluated using the method of Example 3, and compared with the reference TCR. The results are presented in the following table: MAGE A4 Kd (μΜ) MAGE-B2 Kd (μΜ) Reference (TCR1) 65.1 17 TCR2 17.2 197.5 TCR3 2.6 27.6 TCR4 5.1 52.6

[00117] Attempts were also made to prepare high-affinity TCRs based on combinations of SEQ IDs NOS 10 / 11, 12 / 13, 14 / 15.

[00118] In the case of TCR A, which combines the alpha chain of SEQ ID NO: 10 and the beta chain of SEQ ID NO: 11, cross-reactivity was noted between MAGE-A1, MAGE-A10, and PRAME. It was not possible to remove this cross-reactivity by mutation and selection.

[00119] TCR B combines the alpha chain of SEQ ID NO: 12 and the beta chain of SEQ ID NO: 13. TCR B cannot be unfolded to form a soluble TCR, so no linkage characterization was possible.

[00120] TCR C combines alpha string of SEQ ID NO: 14 and string Petition 870260068719, dated 10 / 07 / 2026, p. 39 / 117 34 / 44 beta of SEQ ID NO: 15. This soluble TCR was expressed and could deliver to the antigen. However, when expressed on T cells, TCR C showed no activity. Example 5 - Transfection of T cells with parental and variant MAGE-A4 TCRs (a) Lentiviral vector preparation by Express-In mediated transient transfection of 293T cells

[00121] A third-generation lentiviral packaging system was used to package lentiviral vectors containing the gene encoding the desired TCR. 293T cells were transfected with 4 plasmids (one lentiviral vector containing the TCR alpha-chain P2A ORF gene - single TCR beta-chain ORF described in Example 5c (below), and 3 plasmids containing the other components needed to construct infectious but non-replicative lentiviral particles) using Express-In mediated transfection (Open Biosystems).

[00122] For transfection, a T150 flask of 293T cells in the exponential growth phase was taken, with cells evenly distributed across the plate and slightly more than 50% confluent. Aliquots of Express-In were placed at room temperature. 3 mL of serum-free medium (RPMI 1640 + 10 mM HEPES) were placed in a sterile 15 mL conical tube. 174 μL of Express-In reagent were added directly to the serum-free medium (this provides a weight ratio of 3.6:1 of DNA reagent). This was mixed well by inverting the tubes 3-4 times and incubated at room temperature for 5-20 minutes.

[00123] In a separate 1.5 ml microtube, 15 pg of plasmid DNA were added to aliquots of premixed pack mixtures (containing 18 pg of pRSV.REV (Rev expression plasmid), 18 pg of pMDLg / p.RRE (Gag / Pol expression plasmid), 7 pg Petition 870260068719, dated 10 / 07 / 2026, page 40 / 117 35 / 44 pVSV-G (VSV glycoprotein expression plasmid), usually ~22 pl, were pipetted up and down to ensure homogeneity of the DNA mixture. Approximately 1 mL of Serum-Free Express-In Medium was added dropwise to the DNA mixture and gently pipetted in before transferring back to the remaining Serum-Free Express-In Medium. The tube was inverted 3-4 times and incubated at room temperature for 15-30 minutes. The old culture medium was removed from the cell flask. The Express-In / medium / DNA complex (3 mL) was added directly to the bottom of a vertical 293T cell flask. The flask was slowly flattened to cover the cells and very gently shaken to ensure even distribution. After 1 minute, 22 mL of fresh culture medium (R10 + HEPES: RPMI 1640, 10% heat-inactivated FBS, 1% Pen / Strep / L-glutamine, 10 mM HEPES) were added and the flask was carefully returned to the incubator.This was incubated overnight at 37 °C / 5% CO2. After 24 hours, the medium containing the packaged lentiviral vectors was harvested.

[00124] To harvest the packaged lentiviral vectors, the cell culture supernatant was filtered through a 0.45 micron nylon syringe filter, the culture medium centrifuged at 10,000 g for 18 hours (or 112,000 g for 2 hours), most of the supernatant removed (taking care not to disturb the pellet), and the pellet resuspended in the remaining few mL of supernatant (generally about 2 mL from a volume of 31 mL per starting tube). This was flash-frozen on dry ice in 1 mL aliquots and stored at -80 °C. (b) Transduction of T cells with packaged lentiviral vectors containing gene of interest

[00125] Before transduction with packaged lentiviral vectors, human T cells (CD8+ or CD4+ or both, depending on the Petition 870260068719, dated 10 / 07 / 2026, page 41 / 117 36 / 44 requirements) were isolated from the blood of healthy volunteers. These cells were counted and incubated overnight in R10 containing 50 U / mL of IL-2 at 1 x 10⁶ cells per mL (0.5 mL / well) in 48-well plates with microspheres coated with pre-washed anti-CD3 / CD28 antibody (Dynabeads® T-cell expander, Invitrogen) at a ratio of 3 spheres per cell.

[00126] After overnight stimulation, 0.5 mL of packaged liquid lentiviral vector was added to the desired cells. This was incubated at 37 °C / 5% CO2 for 3 days. 3 days post-transduction, the cells were counted and diluted to 0.5 x 10⁶ cells / mL. Fresh medium containing IL-2 was added as needed. The beads were removed 57 days post-transduction. The cells were counted and fresh medium containing IL-2 was replaced or added at 2-day intervals. The cells were maintained between 0.5 x 10⁶ and 1 x 10⁶ cells / mL. Cells were analyzed by flow cytometry from day 3 and used for functional assays (e.g., ELISpot for IFNγ release, see Example 6) from day 5. From day 10, or when cells are slowly dividing and reduced in size, cells are frozen in aliquots of at least 4 x 10⁶ cells / flask (at 1 x 10⁷ cells / mL in 90% FBS / 10% DMSO) for storage. Example 6 - Activation of genetically modified TCR cells MAGE B2

[00127] The following assay was performed to demonstrate the activation of TCR-transduced cytotoxic T lymphocytes (CTLs) in response to tumor cell lines. IFN-γ production, as measured using the ELISPOT assay, was used as a readout for cytotoxic T lymphocyte (CTL) activation. ELISPOTS Reagents

[00128] Test media: 10% FCS (Gibco, Cat # 2011-09), 88% Petition 870260068719, dated 10 / 07 / 2026, p. 42 / 117 37 / 44 of RPMI 1640 (Gibco, Cat # 42401), 1% glutamine (Gibco Cat # 25030) and 1% penicillin / streptomycin (Gibco Cat # 15070-063).

[00129] Wash buffer: PBS at 0.01M / 0.05% Tween 20

[00130] PBS (Gibco Cat # 10010)

[00131] The human IFNy ELISPOT kit (BD Bioscience; Cat # 551849) containing capture and detection antibodies and 96-well PVDF ELISPOT plates of human IFN-γ, with associated AEC substrate set (BD Bioscience, Cat # 551951) Methods Target cell preparation

[00132] The target cells used in this method were natural epitope-presenting cells: human melanoma cells A375, which are both HLA-A2+MAGE A10+. Human colon cancer cells HCT116, which are ALH-A2+MAGE A10-, were used as a negative control. Sufficient target cells (50,000 cells / well) were washed by centrifugation three times at 1200 rpm for 10 min in a Megafuge® 1.0 (Heraeus). The cells were then resuspended in assay medium at 10⁶ cells / mL. Effector Cell Preparation

[00133] The effector cells (T cells) used in this method were peripheral blood lymphocytes (PBLs), obtained by negative selection using CD14 and CD25 microsphere kits (Miltenyi Biotech Cat # 130050-201 and 130-092-983, respectively) from freshly isolated peripheral blood mononuclear cells (PBMCs) from the venous blood of healthy volunteers. The cells were stimulated with anti-CD3 / CD28 coated beads (Dynabeads® T cell expanders, Invitrogen), transduced with lentivirus carrying the gene encoding the full αβ TCR of interest (based on the construct described in Example 5), and expanded in assay medium containing 50 U / mL IL-2 for up to 10–13 days post-transduction. Petition 870260068719, dated 10 / 07 / 2026, page 43 / 117 38 / 44 These cells were then placed in assay medium before washing by centrifugation at 1200 rpm for 10 min in a Megafuge® 1.0 (Heraeus). The cells were then resuspended in assay media at a final required concentration of 4X.

[00134] The plates were prepared as follows: 100 μL of anti-IFN-γ capture antibody was diluted in 10 ml of sterile PBS per plate. 100 μL of diluted capture antibody was then distributed into each well. The plates were then incubated overnight at 4 °C. After incubation, the plates were washed (Program 1, Plate Type 2, Ultrawash Plus 96-well plate washer; Dynex) to remove the capture antibody. The plates were then blocked by adding 200 μL of assay media to each well and incubated at room temperature for two hours. The assay media was then washed from the plates (Program 1, Plate Type 2, Ultrawash Plus 96-well plate washer, Dynex) and any remaining material was removed by gently shaking and tapping the ELISPOT plates on a paper towel.

[00135] The assay components were then added to the ELISPOT plate in the following order:

[00136] 50 pL of target cells 106 cells / mL (giving a total of 50,000 target cells / well)

[00137] 50 pL of media (test media)

[00138] 50 pL of effector cells (20,000 PBL TCR transduced cells / well)

[00139] The plates were then incubated overnight (37 °C / 5% CO2). The following day, the plates were washed three times (program 1, plate type 2, 96-well Ultrawash Plus plate washer, Dynex) with washing buffer and gently tapped and dried on paper towels to remove excess washing buffer. 100 µl of primary detection antibody were then added to each Petition 870260068719, dated 10 / 07 / 2026, page 44 / 117 39 / 44 well. The primary detection antibody was diluted in 10 mL of dilution buffer (the volume required for a single plate), using the dilution specified in the manufacturer's instructions. The plates were then incubated at room temperature for at least 2 hours before being washed three times (program 1, plate type 2, 96-well Ultrawash Plus plate washer, Dynex) with washing buffer; excess washing buffer was removed by gently tapping the plate on a paper towel.

[00140] Secondary detection was performed by adding 100 μL of diluted streptavidin-HRP to each well and incubating the plate at room temperature for 1 hour. Streptavidin-HRP was diluted in 10 mL of dilution buffer (the volume required for a single plate), using the dilution specified in the manufacturer's instructions. The plates were then washed three times (program 1, type 2 plate, Ultrawash Plus 96-well plate washer, Dynex) with washing buffer and gently tapping on a paper towel to remove excess washing buffer. The plates were then washed twice with PBS, adding 200 μL to each well, agitating the buffer and gently tapping on a paper towel to remove excess buffer. No more than 15 minutes before use, one drop (20 μL) of AEC chromogen was added to each 1 mL of AEC substrate and mixed. 10 mL of this solution were prepared for each plate; 100 µL were added per well.The plate was then protected from light using foil, and local development was monitored regularly, typically occurring within 5–20 min. The plates were washed in tap water to terminate the development reaction and shaken dry before being disassembled into three constituent parts. The plates were then allowed to air dry at room temperature for at least 2 hours before counting the spots using an Immunospot® plate reader (CTL; ​​Cellular Technology Limited). Example 7 - Identifying the reason for the substitution link Petition 870260068719, dated 10 / 07 / 2026, page 45 / 117 40 / 44 with all alternative amino acids

[00141] Variants of the native MAGE-A4 peptide were obtained in which the amino acid residue at each position was sequentially replaced by all 19 alternative naturally occurring amino acids, such that 171 peptides were prepared in total. The amino acid-substituted and native peptides were pulsed onto antigen-presenting cells, and interferon γ (IFNγ) production, as measured using the ELISpot assay, was used as a readout for the activation of TCR1-transduced T cells. Essential positions were defined by a greater than 50% reduction in T cell activity compared to the native peptide.

[00142] The ELISPOT tests were performed as described in Example 6.

[00143] The tolerated residues at each position in the peptide are shown below. The underlined amino acids represent the native residue at the corresponding position in the peptide. Position Tolerated Residues 1 GH 2 VIL 3 YVF 4 DN 5 GN 6 KARGSCHTQMFVNLYI 7 SHQMEILWPYFATCNDGRKV 8 DSHQMEILWPYFATCNGRKV 9 SHQMEILWPYFATCNDGRKV 10 VFMAILT Ό0144] It is therefore evident that TCR4 MAGE A4 enters in contact with at least V2, Y3 and D4 of the peptide (SEQ ID NO: 1) when in complex with HLA-A*0201 on the surface of antigen-presenting cells. Petition 870260068719, dated 10 / 07 / 2026, page 46 / 117 41 / 44 SEQ ID NO: 1 MAGE Epitope A4 GVVDGREHTV SEQ ID NO: 2 MAGE B2 GVYDGEEHSV Epitope SEQ ID NO: 3 string variable alpha MKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTRGGPV SLTIMTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVS ggtdswgklqf SEQ ID NO: 4 variable string beta masllffcgafyllgtgsmdadvtqtprnritktgkrimlecsqtkg hdrmywyrqdpglglrliyysfdvkdinkgeisdgysvsrqaqakfs lslesaipnqtalyfcatsgqgayneqff SEQ ID NO: 5 soluble form of alpha chain mkkhlttflvilwlyfyrgngknqveqspqsliilegknctlqcnytv spfsnlrwykqdtgrgpvsltimtfsentksngrytatldadtkqss lhitasqlsdsasyicvvsggtdswgklqfgagtqvvvtpdiqnpdp avyqlrdskssdksvclftdfdsqtnvsqskdsdvyitdktvldmrs MDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS SEQ ID NO: 6 soluble form of beta chain masllffcgafyllgtgsmdadvtqtprnritktgkrimlecsqtkg hdrmywyrqdpglglrliyysfdvkdinkgeisdgysvsrqaqakfs lslesaipnqtalyfcatsgqgayneqffgpgtrltvledlknvfpp evavfepseaeishtqkatlvclatgfypdhvelswwvngkevhsg vstdpqplkeqpalndsryclssrlrvsatfwqnprnhfrcqvqfy Petition 870260068719, dated 10 / 07 / 2026, page 47 / 117 42 / 44 GLSENDEWTQDRAKPVTQIVSAEAWGRAD SEQ ID NO: 7 variable string alpha mutant mknqveqspqsliilegknctlqcnytvspfsnlrwykqdtgrgpv SLTIVTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSG GTDSWGKLQF SEQ ID NO: 8 alpha mutant variable chain MKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPV SLTILTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSG GTDSWGKLQF SEQ ID NO: 9 mutant beta variable string MASLLFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKG HDRMYWYRQDPGLGLRLLIYYSFDVKDINKGEISDGYSVSRQAQAKFS LSLESAIPNQTALYFCATSGQGAYEEQFF SEQ ID NO: 10 soluble form of alpha chain METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSA IYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYI AASQPGDSATYLCAVGGYSTLTFGKGTVLLVSPDNIQNPDPAVYQLR DSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKS NSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFET DTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 11 soluble form of beta chain MSISLLCCAAFPLLWAGPVNAGVTQTPKFRILKIGQSMTLQCAQDMN HNYMYWYRQDPGMGLKLIYYSVGAGITDKGEVPNGYNVSRSTTEDF PLRLELAAPSQTSVYFCASSYSRWSPLHFGNGTRLTVTEDLNKVFPP EVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSG Petition 870260068719, dated 10 / 07 / 2026, page 48 / 117 43 / 44 VSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFY GLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATI LYEILLGKATLYAVLVSALVLMAMVKRKDF SEQ ID NO: 12 soluble form of alpha chain MQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPE LIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVKM ANQAGTALIFGKGTTLSVSSNIQNPDPAVYQLRDSKSSDKSVCLFTDF DSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACA NAFNNSIIPEDTFFPSPESS SEQ ID NO: 13 soluble form of beta chain MQDGGITQSPKFQVLRTGQSMTLLCAQDMNHEYMYWYRQDPGMG LRLIHYSVGAGITDQGEVPNGYNVSRLNKREFSLRLESAAPSQTSVY FCASLGGLADEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHT QKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALN DSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAK PVTQIVSAEAWGRAD SEQ ID NO: 14 soluble form of alpha chain MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTD SSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLS LRIADTQTGDSAIYFCAERNSGAGSYQLTFGKGTKLSVIPNIQNPDPA VYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRS MDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVE KSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 15 soluble form of beta chain MGTSLLCWMALCLLGADHADTGVSQNPRHKITKRGQNVTFRCDPIS EHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGS Petition 870260068719, dated 10 / 07 / 2026, page 49 / 117 44 / 44 FSTLEIQRTEQGDSAMYLCASSLFSGVNTEAFFGQGTRLTVVEDLNK VFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEV HSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQ VQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGV LSATILYEILLGKATLYAVLVSALVLMAMVKRKDF

Claims

1. T cell receptor (TCR), characterized in that it comprises a variable alpha chain domain of TCR and a variable beta chain domain of TCR, wherein: A) the variable alpha chain domain of TCR comprises the following CDRs: Alpha CDR1 is VSPFSN; Alpha CDR2 is LTIMTF or LTIVTF or LTILTF; and Alpha CDR3 is CVVSGGTDSWGKLQF; and B) the variable beta chain domain of TCR comprises the following CDRs: Beta CDR1 is KGHDR; Beta CDR2 is SFDVK; and Beta CDR3 is CATSGQGAYEEQFF.

2. TCR, according to claim 1, characterized in that it has the property of binding to GVYDGREHTV (SEQ ID NO: 1) in complex with HLA-A*0201 with a dissociation constant of 0.05 μM to 20.0 μM when measured with surface plasmon resonance at 25 °C and a pH between 7.1 and 7.5 using a soluble form of the TCR, and has at least a tenfold selectivity of binding to SEQ ID NO: 1 in complex with HLA-A*0201 over binding to GVYDGEEHSV (SEQ ID NO: 2) in complex with HLA-A*0201, wherein the TCR comprises an alpha chain variable domain of TCR and a beta chain variable domain of TCR, and wherein the variable domains of TCR form contacts with at least residues V2, Y3 and D4 of GVYDGREHTV (SEQ ID NO: 1).

3. TCR, according to claim 1 or 2, characterized in that it is an alpha-beta heterodimer, having an alpha-chain constant domain sequence TRAV10 + TRAC and a beta-chain constant domain sequence BV24-1 + TRBC-2. Petition 870260068719, dated 10 / 07 / 2026, p. 51 / 117 2 / 3 4. TCR, according to any one of claims 1 to 3, characterized in that it is in the form of a single chain of the type Va-L-Ve, Ve-L-Va, Va-Ca-L-Ve, or Va-L-Ve-Ce, wherein Va and νβ are variable and β regions of TCR, respectively, Ca and Ce are constant and β regions of TCR, respectively, and L is a linker sequence.

5. TCR, according to any one of claims 1 to 4, characterized in that it is associated with a detectable marker, a therapeutic agent or a PK modifying moiety.

6. TCR, according to any one of claims 1 to 5, characterized in that the alpha string variable domain consists of the alpha string variable domain of SEQ ID NO: 3 or 7 to 8.

7. TCR, according to any one of claims 1 to 6, characterized in that the alpha string variable domain consists of the alpha string variable domain of SEQ ID NO: 7 or 8.

8. TCR, according to any one of claims 1 to 7, characterized in that the variable beta string domain consists of the variable beta string domain of SEQ ID NO:

9.

9. TCR, according to any one of claims 1 to 8, characterized in that the alpha string variable domain consists of the alpha string variable domain of SEQ ID NO: 8, and the beta string variable domain consists of the beta string variable domain of SEQ ID NO:

9.

10. Pharmaceutical composition, characterized in that it comprises a TCR as defined in any one of claims 1 to 9, together with one or more pharmaceutically acceptable vehicles or excipients.

11. Pharmaceutical composition, characterized in that it comprises an isolated or non-natural cell, wherein the cell is defined as having a TCR as defined in any of claims 1 to 9, in conjunction with dimethyl sulfoxide.

12. TCR, according to any one of claims 1 to 9, characterized in that it is for use in medicine.

13. Use of a TCR as defined in any of claims 1 to 9, or of an isolated or non-natural cell, wherein the cell is defined as having a TCR as defined in any of claims 1 to 9, characterized in that it is in the preparation of a medicament for the treatment of cancer.