T cell receptors

Abstract

The present invention relates to T cell receptors capable of binding a HLA-A*02-restricted peptide ROPN1 peptide with the amino acid sequence FLYTYIAKV (SEQ ID NO: 1). The TCRs of the invention are particularly suitable for the treatment of cancer.

Description

T CELL RECEPTORSFIELD

[0001] The present invention relates to binding molecules, such as T cell receptors (TCRs), which bind the HLA-A*02-restricted peptide FLYTYIAKV (SEQ ID NO: 1) derived from Ropporin-1 A (UniProt Q9HAT0 ■ ROP1A_HUMAN) residues 163-171. The binding molecules are characterized by superior binding properties. BACKGROUND

[0002] T cell receptors (TCRs) are protein heterodimers of either alpha and beta chains, or gamma and delta chains. About 95% of T cells comprise alpha-beta TCR, which includes CD4+ and CD8+ T cells. Gammadelta TCRs are usually expressed by CD4- CD8- T cells. TCRs recognize short peptide sequences displayed in a binding groove on the surface of Human Leukocyte Antigen protein complexes (HLA) (DOI:10.1146 / annurev.immunol.16.1.523). CD8+ T cells generally seek out and mediate the killing of diseased cells through recognition of peptides bound to HLA class 1 . CD8+T cells are able to kill cancer and virally infected cells; however, the natural affinity of TCRs fortheir peptide Human Leukocyte Antigens (pHLA) is generally low. This, combined with the often reduced cell surface expression of HLA can result in cancerous cells escaping detection and destruction. Immunotherapeutic approaches designed to enhance T cell recognition of cancerous cells have been FDA approved for the treatment of Unresectable or Metastatic Uveal Melanoma (doi: 10.1056 / NEJMoa2103485).

[0003] Ropporin-1 A (ROPN-1 or ROPN-1A), also known as Rhophilin associated tail protein 1 is normally found in the testis and is important for sperm motility (Human Protein Atlas proteinatlas.org; doi: 10.1126 / science.1260419). Expression of ROPN-1 A has also been reported in fetal liver. Single cell RNA analysis reveals that low levels of RNA transcripts are also found in the breast. Low or non-existing protein expression levels in healthy tissues beyond testis and breast make ROPN1 an attractive potential target cancer antigen. Immune privilege of testis additionally limits the concerns associated with off-target effects resulting from ROPN1 -targeted immunotherapy.

[0004] The Cancer Genome Atlas (TCGA) RNA-Seq dataset, analysed ROPN1 RNA expression levels across 17 cancer types and found ROPN1 expression to be highest in breast cancer and melanoma samples.

[0005] Breast cancer is a heterogenous disease with at least five distinct molecular subtypes, with Luminal A, Luminal B and Normal-like normally hormone receptor positive and Her2 and Basal-like almost exclusively hormone receptor negative. Triple Negative Breast Cancer (TNBC) cases are diagnosed as Basal-like breast cancers (BLBCs), and therefore lack the therapeutic molecular targets used in treating women with the other disease subtypes, (e.g. endocrine therapies (tamoxifen, aromatase inhibitors), and anti-HER2 therapies (trastuzumab)) (Alsabi, Q. (2019). Characterizing Basal-Like Triple Negative Breast Cancer using Gene Expression Analysis: A Data Mining Approach. [Master's thesis, Wright State University]; Klokkerud, SMK (2020) Uncovering key transcription factors in breast cancer subtypes using matrix factorization [Master’sthesis; Norwegian Uni of Life Sciences]). TNBC cases only achieve 19% clinical-complete-response to chemotherapy, highlighting the unmet treatment needs for this disease. The Cancer Genome Atlas (TCGA) breast cancer dataset from 600 non-TNBC and 115 TNBC samples indicated that ROPN1 mRNA was significantly upregulated in TNBC and was extremely overexpressed in the basal-like (BL) subtype (Liu et al. (2020), doi: 10.1096 / fj.201903281 R). ROPN1 upregulation was previously determined to be associated with lower survival in breast cancer patients (Mehta, JP (2010) Gene expression analysis in breast cancer. (2009) PhD Thesis; Dublin City University). Silencing of ROPN1 has been shown to reduce the invasive capacity of TNBC cells, while in in vivo studies, injection of ROPN1 -overexpressing into the BALB / c nude mice led to an increase in the number of lung metastatic nodules (Liu et al. (2020)).

[0006] The expression of ROPN1 has also been shown to progressively increase with disease progression and to be the highest in metastatic growth phase melanoma and lymph node metastasis (Mehta JP (2020) PhD Thesis). In a panel of melanoma cell lines generated in-house, ROPN1 gene expression was detected in 45 out of 55 (81.8%) cell lines and plasma antibodies were detected above noise threshold against ROPN1A / B in 71.2% of patients (n = 74 / 104) with co-reactivity in many cases, indicating ROPN1 as expressed in melanoma and immunogenic (Duarte et al. (2021) Cancers doi: 10.3390 / cancers13081805).

[0007] In multiple myeloma (MM), evaluation of MM cell lines and patient samples revealed evidence of ROPN1 expression in 4 of 5 MM cell lines and 15 of 34 MM cases (44%), with ICC and IF analysis showing that ROPN1 protein was detectable in Permeabilized and Non-Permeabilized patients’ cells, and was detectable at the protein level in 10 of 15 patients (Chiriva-lnternati et al. (2011) J Immunother, doi: 10.1097 / CJI.0b013e31821ca76f).

[0008] The need exists to provide improved binding molecules, including TCRs, which bind the HLA-A*02- restricted peptide FLYTYIAKV (SEQ ID NO: 1) derived from ROPN1 , which are suitable in the treatment or diagnosis of cancer.

[0009] WO2022 / 158977A1 describes TCRs that target ROPN1 . However, there is a requirement for binding molecules for therapeutic use, which have higher levels of affinity to the target antigen, alongside other improved characteristics, such as longer binding half-lives, reduced cross-reactivity and / or yield. These are provided by the present disclosure.SUMMARY

[0010] The present disclosure relates to the above-mentioned technical problem and provides embodiments as described herein, particularly by reference to the claims.

[0011] In one aspect, the disclosure provides a binding molecule capable of binding to FLYTYIAKV (SEQ ID NO: 1) in complex with HLA-A*02 and comprising a TCR alpha chain variable domain and / or a TCR beta chain variable domain, wherein:the alpha chain variable domain comprises an amino acid sequence that has at least 90%, identity, including at least 90%, 91 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, to QKEVEQNSGPLSVPEGAIASLNCTYSDRGSVGFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQ YVSLLIRDSQPSDSATYLCAVNGRDSYKIIFGSGTRLLVRPN SEQ ID NO: 11 and the beta chain variable domain comprises an amino acid sequence that has at least 90%, identity, such as 90%, 91 %, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, toNAGVTQTPKFQVLKTGQSMTLQCAQDMEHEYMSWYRQDPGMGLRLIHYSPTAGLTDQGEVPNGYNVSR STTEDFPLRLLSAAPSQTSVYFCGSSYNLGDGYTFGSGTRLTVVE SEQ ID NO: 15; or the alpha chain variable domain comprises an amino acid sequence that has at least 90%, identity, such as 90%, 91 %, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, to QKEVEQNSGPLSVPEGAIASLNCTYSDRGSVGFFWYRQYSGKSPELIMWIYSNGDKEDGRFTAQLNKASQ YVSLLIRDSQPSDSATYLCAVNGRDSYKIIFGSGTRLLVRPN SEQ ID NO: 33 and the beta chain variable domain comprises an amino acid sequence that has at least 90%, identity, such as 90%, 91 %, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, toNAGVTQTPKFQVLKTGQSMTLQCAQDMEHEYMSWYRQDPGMGLRLIHYSPTAGLTDQGEVPNGYNVSR STTEDFPLRLLSAAPSQTSVYFCGSSYNLGDGYTFGSGTRLTVVE SEQ ID NO: 15 wherein the binding molecule binds the FLYTYIAKV (SEQ ID NO: 1) HLA-A*02 complex with an affinity greater than 10nM as measured by surface plasmon resonance.

[0012] In one embodiment, the binding molecule binds the FLYTYIAKV (SEQ ID NO: 1) HLA-A*02 complex with an affinity greater than 1 nM as measured by surface plasmon resonance.

[0013] In a further embodiment, the binding molecule binds the FLYTYIAKV (SEQ ID NO: 1) HLA-A*02 complex with an affinity greater than 700pM, optionally greater than 160pM as measured by surface plasmon resonance.

[0014] In a further aspect, the binding molecule comprises the alpha chain variable domain sequence of amino acid residues chain CDR1 DRGSVG (SEQ ID NO:12), CDR2 IYSNGD (SEQ ID NO: 13), CDR3 CAVNGRDSYKIIF (SEQ ID NO: 14) (where positions 27-32 is CDR1 , positions 50-55 is CDR2 and positions 89-101 is CDR3) and the beta chain variable domain sequence of amino acid residues CDR1 MEHEY (SEQ ID NO: 16), CDR2 SPTAGL (SEQ ID NO: 17) and CDR3 CGSSYNLGDGYTF (SEQ ID NO:18) (where positions 27-31 is CDR1 , positions 49-54 is CDR2, and positions 91-103 is CDR3).

[0015] In some embodiments, the alpha chain variable region comprises a FR2 region comprising a tryptophan or phenylalanine at position 55 (IMGT) of the alpha chain variable domain. In one embodiment, the alpha chain variable region comprises a FR2 region wherein the amino acid at position 55 (IMGT) is tryptophan. In another embodiment, the alpha chain variable region comprises a FR2 region wherein the nucleic acid at position 55 (IMGT) is phenylalanine.

[0016] In one embodiment, the alpha chain variable region comprises the following framework region sequences: FR1 QKEVEQNSGPLSVPEGAIASLNCTYS (SEQ ID NO: 19), FR2 FFWYRQYSGKSPELIMF (SEQ ID NO: 20), FR3 KEDGRFTAQLNKASQYVSLLIRDSQPSDSATYL (SEQ ID NO: 21) and / or FR4 GSGTRLLVRPN (SEQ ID NO: 22) and / or the beta chain variable region comprises the following framework region sequences: FR1 NAGVTQTPKFQVLKTGQSMTLQCAQD (SEQ ID NO: 23), FR2 MSWYRQDPGMGLRLIHY (SEQ ID NO: 24), FR3 TDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYF (SEQ ID NO: 25) and / or FR4 GSGTRLTVVE (SEQ ID NO: 26).

[0017] In a further embodiment, the alpha chain variable region comprises the following framework region sequences: FR1 QKEVEQNSGPLSVPEGAIASLNCTYS (SEQ ID NO: 19), FR2 FFWYRQYSGKSPELIMW (SEQ ID NO: 34), FR3 KEDGRFTAQLNKASQYVSLLIRDSQPSDSATYL (SEQ ID NO: 21) and / or FR4 GSGTRLLVRPN (SEQ ID NO: 22) and / or the beta chain variable region comprises the following framework region sequences: FR1 NAGVTQTPKFQVLKTGQSMTLQCAQD (SEQ ID NO: 23), FR2 MSWYRQDPGMGLRLIHY (SEQ ID NO: 24), FR3 TDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYF (SEQ ID NO: 25) and / or FR4 GSGTRLTVVE (SEQ ID NO: 26).

[0018] In a further aspect, the alpha chain variable domain and the beta chain variable domain of the binding molecule are alpha chain variable domain set forth in SEQ ID NO: 11 or SEQ ID NO: 33 and beta chain variable domain set forth in SEQ ID NO: 15.

[0019] In one embodiment, the binding molecule is an alpha-beta heterodimer, having an alpha chain TRAC constant domain sequence of SEQ ID NO: 6 and / or a beta chain TRBC2 constant domain sequence of SEQ ID NO: 8.

[0020] In some embodiments, the alpha chain amino acid sequence comprises the sequence set forth in SEQ ID NO: 27 or an alpha chain that has at least 90%, 91%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, to SEQ ID NO: 27, and: the beta chain amino acid sequence comprises an amino acid sequence set forth in SEQ ID NO: 28 or a beta chain that has at least 90%, identity, such as 90%, 91 %, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, to SEQ ID NO: 28.

[0021] In some embodiments, the alpha chain amino acid sequence comprises the sequence set forth in SEQ ID NO: 35 or an alpha chain that has at least 90%, 91%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, to SEQ ID NO: 35, and: the beta chain amino acid sequence comprises an amino acid sequence set forth in SEQ ID NO: 28 or a beta chain that has at least 90%, identity, such as 90%, 91 %, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, to SEQ ID NO: 28.

[0022] A further aspect provides a TCR-anti-CD3 effector molecule (e.g. an antibody) fusion molecule comprising an alpha chain variable domain comprising the sequence set forth in SEQ ID NO: 11 or 33 and abeta chain variable domain comprising the sequences set forth in SEQ ID NO: 15, wherein the anti-CD3 effector molecule is covalently linked to the C-terminus or the N-terminus of the alpha chain or the beta chain via a linker sequence.

[0023] In one embodiment, the anti-CD3 antibody is a scFv. In a further embodiment, the anti-CD3 antibody is UCHT1, optionally comprising the sequence set forth in SEQ ID NO: 32.

[0024] A further aspect provides a nucleic acid encoding a binding molecule as described anywhere herein, or an expression vector comprising a nucleic acid described herein.

[0025] A yet further aspect provides a non-naturally occurring and / or purified and / or engineered cell, such as a T-cell, presenting a binding molecule as described anywhere herein.

[0026] A further aspect provides a pharmaceutical composition comprising a binding molecule as defined anywhere herein, or a nucleic acid as defined anywhere herein, or a cell as defined anywhere herein, with one or more pharmaceutically acceptable carriers or excipients.

[0027] A further aspect provides a binding molecule as defined anywhere herein, or a nucleic acid as defined anywhere herein, or a cell as defined anywhere herein or a pharmaceutical composition as defined anywhere herein for use in medicine, in particular cancer.

[0028] A further aspect provides a method of treating a cancer comprising administration of the binding molecule as defined anywhere herein, or a nucleic acid as defined anywhere herein, or a cell as defined anywhere herein or a pharmaceutical composition as defined anywhere herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Select embodiments of the disclosed technology will now be described in more detail with reference to the following figures:

[0030] Figure 1 provides maps of customised mammalian expression vectors built upon pCDNA3.4 (Thermo Fisher Scientific), allowing for co-expression of both TCR chains. Fig. 1 A depicts the vector for TCR alpha chain and Fig. 1 B depicts the vector for TCR beta chain.

[0031] Figure 2 shows SPR sensorgrams for TCRs of the present disclosure (TCR1 - Fig. 2A; TCR2 - Fig. 2B)

[0032] Figure 3 shows I FNy release assay results for TCRs of the present disclosure.

[0033] Figure 4 shows NFAT reporter cross-reactivity assay results for TCRs of the present disclosure in the presence of peptide mimetics of ROPN 1 peptide of SEQ ID NO: 1 .

[0034] Figure 5 shows I FNy release alloreactivity assay results for TCRs of the present disclosure in the presence of a panel of cell lines expressing various subtypes of HLA-A.DETAILED DESCRIPTION

[0035] DEFINITIONS

[0036] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0037] All publications, including patent documents, scientific articles, and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0038] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present disclosure. The following description illustrates the disclosure and, of course, should not be construed in any way as limiting the scope of contemplated embodiments described herein.

[0039] References to subject-matter disclosed or described “herein” relates to subject-matter disclosed or described anywhere in the present application.

[0040] “Binding affinity” generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g. a TCR) and its binding partner (e.g. an antigen-derived peptide-HLA complex). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 :1 interaction between members of a binding pair (e.g. TCR and an antigen-derived peptide-HLA complex). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured and / or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD), and equilibrium association constant (KA). The KD is calculated from the quotient of koff / kon, whereas KA is calculated from the quotient of kon / koff. kon refers to the association rate constant of, e.g, a TCR to an antigen-derived peptide-HLA complex, and koff refers to the dissociation of, e.g, a TCR from a an antigen-derived peptide-HLA complex. The kon and koff can be determined by techniques known to one of ordinary skill in the art, such as SPR (e.g. BIAcore®) or KinExA.

[0041] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise.

[0042] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation,"e.g." is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[0043] In the specification and claims, the term "about" is used to modify, for example, the quantity of an ingredient in a composition, concentration, volume, process temperature, process time, yield, flow rate, pressure, and like values, and ranges thereof, employed in describing the examples of the disclosure. The term "about" refers to variation in the numerical quantity that can occur, for example, through typical measuring and handling procedures used for making compounds, compositions, concentrates or use formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods, and like proximate considerations. The term "about" also encompasses amounts that differ due to aging of a formulation with a particular initial concentration or mixture and amounts that differ due to mixing or processing a formulation with a particular initial concentration or mixture. Where modified by the term "about" the claims appended hereto include equivalents to these quantities.

[0044] As used herein, “administer” or “administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., a TCR provided herein, or its encoding nucleic acid e.g. in an expression vector, or a cell expressing the TCR provided herein) into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, inhalation e.g. nebulisation and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease, or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

[0045] The sequences defined herein are described with reference to IMGT nomenclature which is commonly used by a skilled person working in the TCR field. For example, see: LeFranc and LeFranc, (2001). "T cell Receptor Factsbook", Academic Press.

[0046] As used herein, the term “carrier” refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.

[0047] As used herein, the term “composition” is intended to encompass a product containing the specified ingredients (e.g. a TCR) in, optionally, the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in, optionally, the specified amounts.

[0048] As used herein the term "comprising" or "comprises" is used with reference to binding molecules, uses, compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.

[0049] The term "consisting of" refers to binding molecules, uses, compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the example.

[0050] As used herein the term "consisting essentially of' refers to those elements required for a given example. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that example.

[0051] An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired effect, including a therapeutic or prophylactic result. A "therapeutically effective amount" refers to the minimum concentration required to effect a measurable improvement or prevention of a particular disorder. A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the TCR to elicit a desired response in the individual. A therapeutically effective amount is also one in which toxic or detrimental effects of the TCR are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at the dosages and for periods of time necessary, to achieve the desired prophylactic result.

[0052] The term “excipients” as used herein refers to inert substances which are commonly used as a diluent, vehicle, preservatives, binders, or stabilizing agent for drugs and includes, but not limited to, proteins (e.g. serum albumin, etc.), amino acids (e.g. aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g. alkyl sulfonates, caprylate, etc.), surfactants (e.g. SDS, polysorbate, nonionic surfactant, etc.), saccharides (e.g. sucrose, maltose, trehalose, etc.) and polyols (e.g. mannitol, sorbitol, etc.). See, also, Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa., which is hereby incorporated by reference in its entirety.

[0053] A binding molecule, TCR, polynucleotide, vector, cell, or composition which is "isolated" is a binding molecule, TCR, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated binding molecules, TCRs, polynucleotides, vectors, cell or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some aspects, a binding molecule, TCR, polynucleotide, vector, cell, or composition which is isolated is substantially pure. As used herein, "substantially pure" refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0054] "Percent (%) amino acid sequence identity" with respect to a peptide, polypeptide or binding molecule sequence, including a TCR sequence, are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence,after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEG ALIGN™ (DNASTAR) software. In one example, the % identity is about 70%. In one example, the % identity is about 75%. In one example, the % identity is about 80%. In one example, the % identity is about 85%. In one example, the % identity is about 90%. In one example, the % identity is about 92%. In one example, the % identity is about 95%. In one example, the % identity is about 97%. In one example, the % identity is about 98%. In one example, the % identity is about 99%. In one example, the % identity is 100%.

[0055] The term “naturally occurring” or “native” when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, refers to those which are found in nature and not manipulated by a human being.

[0056] The term “pharmaceutically acceptable” as used herein means being approved by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.

[0057] As used herein, the term “polynucleotide,” “nucleotide,” nucleic acid” “nucleic acid molecule” and other similar terms are used interchangeable and include DNA, RNA, mRNA and the like.

[0058] As used herein, the terms “prevent”, “preventing”, and “prevention” refer to the total or partial inhibition of the development, recurrence, onset or spread of cancer, and / or symptom related thereto, resulting from the administration of a therapy or combination of therapies provided herein (e.g. a combination of prophylactic or therapeutic agents, such as a TCR or a cell expressing a TCR).

[0059] The term “specificity” refers to selective recognition of a binding molecule for a particular epitope of an antigen.

[0060] The term "subject" or "patient" refers to any animal, including, but not limited to, mammals. As used herein, the term "mammal" refers to any vertebrate animal that suckle their young and either give birth to living young (eutharian or placental mammals) or are egg-laying (metatharian or nonplacental mammals). Examples of mammalian species include, but are not limited to, humans and other primates, including nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats (including cotton rats) and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like.

[0061] As used herein “substantially all” refers to refers to at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.

[0062] As used herein, the term “therapeutic agent” refers to any agent that can be used in the treatment, management or amelioration of a cancer and / or a symptom related thereto. In certain examples, the term “therapeutic agent” refers to binding molecule, particularly a TCR. In certain other examples, the term “therapeutic agent” refers to an agent other than a binding molecule. Preferably, a therapeutic agent is an agent which is known to be useful for treating cancer, or has been or is currently being used for the treatment, management or amelioration of a cancer and / or one or more symptoms related thereto. In specific examples, the therapeutic agent is a binding molecule, particularly a TCR.

[0063] As used herein, the term “therapy” refers to any protocol, method and / or agent that can be used in the prevention, management, treatment and / or amelioration of a cancer. In certain examples, the terms “therapies” and “therapy” refer to a biological therapy, supportive therapy, and / or other therapies useful in the prevention, management, treatment and / or amelioration of cancer known to one of skill in the art such as medical personnel.

[0064] The terms “treat”, “treatment” and “treating” refer to the reduction or amelioration of the progression, severity, and / or duration of a cancer resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents, such as a TCR). In specific examples, such terms refer to the inhibition or reduction of one or more symptoms associated with a cancer.

[0065] Definitions of common terms in cell biology and molecular biology can be found in “The Merck Manual of Diagnosis and Therapy”, 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-911910- 19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10: 0763766321 ); Kendrew et al. (Eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081 -569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds.

[0066] Unless otherwise stated, the present disclosure was performed using standard procedures, as described, for example in Sambrook et al., Molecular Cloning: A Laboratory Manual (4 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1995); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol.152, S. L. Berger and A. R. Kimmel Eds., Academic Press Inc., San Diego, USA (1987); Current Protocols in Protein Science (CPPS) (John E. Coligan, et al., ed., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and DavidBarnes editors, Academic Press, 1st edition, 1998) which are all incorporated by reference herein in their entireties.

[0067] Other terms are defined herein within the description of the various examples of the disclosure.

[0068] The present disclosure relates particularly to binding molecules that specifically bind the HLA-A*02 restricted peptide FLYTYIAKV (SEQ ID NO: 1).

[0069] The peptide FLYTYIAKV (SEQ ID NO: 1) corresponds to amino acids 163-171 of the full length human ROPN1 protein and is presented on the cell surface in complex with HLA-A*02.

[0070] As used herein, the term “binding molecule” refers to a protein that is capable of binding the target antigen, HLA-A*02 restricted peptide FLYTYIAKV (SEQ ID NO: 1) and can be in any appropriate format, such as a T Cell Receptor (TOR) or a TOR bispecific fusion molecule (targeting ROPN1 and CD3, for example). Binding molecules include fragments of binding molecules that can bind the target antigen, HLA- A*02 restricted peptide FLYTYIAKV (SEQ ID NO: 1).

[0071] HLA-A*02 can be HLA-A*02:01, HLA-A*02:02 or HLA-A*02:04.

[0072] In some embodiments, the binding molecule capable of binding to FLYTYIAKV (SEQ ID NO: 1) in complex with HLA-A*02 comprises a TOR alpha chain variable domain wherein: the alpha chain variable domain comprises CDR1 (amino acid residues 27-32), CDR2 (amino acid residues 50-55) and CDR3 (amino acid residues 89-101) sequences of CDR1 DRGSVG (SEQ ID NO: 12), CDR2 IYSNGD (SEQ ID NO: 13), CDR3 CAVNGRDSYKIIF (SEQ ID NO: 14) and a TOR beta chain variable domain wherein: the beta chain variable domain comprises CDR1 MEHEY (SEQ ID NO: 16), CDR2 SPTAGL (SEQ ID NO: 17) and CDR3 CGSSYNLGDGYTF (SEQ ID NO: 18).

[0073] In some embodiments, the binding molecule of the present disclosure comprises the following alpha chain framework region sequences: FR1 QKEVEQNSGPLSVPEGAIASLNCTYS (SEQ ID NO: 19), FR2 FFWYRQYSGKSPELIMF (SEQ ID NO: 20), FR3 KEDGRFTAQLNKASQYVSLLIRDSQPSDSATYL (SEQ ID NO: 21) and / or FR4 GSGTRLLVRPN (SEQ ID NO: 22), or sequences having at least 90% identity to said sequences, including 91 %, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, and / or the following beta chain framework region sequences: FR1 NAGVTQTPKFQVLKTGQSMTLQCAQD (SEQ ID NO: 23), FR2 MSWYRQDPGMGLRLIHY (SEQ ID NO: 24), FR3TDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYF (SEQ ID NO: 25) and / or FR4 GSGTRLTWE (SEQ ID NO: 26). or sequences having at least 90% identity to said sequences, including 91 %, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0074] In some embodiments, the binding molecule of the present disclosure comprises the following alpha chain framework region sequences: FR1 QKEVEQNSGPLSVPEGAIASLNCTYS (SEQ ID NO: 19), FR2 FFWYRQYSGKSPELIMW (SEQ ID NO: 34), FR3 KEDGRFTAQLNKASQYVSLLIRDSQPSDSATYL (SEQ ID NO: 21) and / or FR4 GSGTRLLVRPN (SEQ ID NO: 22), or sequences having at least 90% identity to saidsequences, including 91 %, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, and / or the following beta chain framework region sequences: FR1 NAGVTQTPKFQVLKTGQSMTLQCAQD (SEQ ID NO: 23), FR2 MSWYRQDPGMGLRLIHY (SEQ ID NO: 24), FR3TDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYF (SEQ ID NO: 25) and / or FR4 GSGTRLTWE (SEQ ID NO: 26). or sequences having at least 90% identity to said sequences, including 91 %, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0075] In some embodiments, the alpha chain variable region comprises a FR2 region comprising a tryptophan or phenylalanine at position 55 (IMGT) of the alpha chain variable domain. In one embodiment, the alpha chain variable region comprises a FR2 region wherein the amino acid at position 55 (IMGT) is tryptophan. In another embodiment, the alpha chain variable region comprises a FR2 region wherein the nucleic acid at position 55 (IMGT) is phenylalanine.

[0076] The binding molecules of the present disclosure can incorporate one or more further amino acid changes, including substitutions, insertions and deletions, in addition to those set out above and below, when the binding molecule has a similar phenotype to the corresponding binding molecule without said change. Such a modified binding may have a KD within 20% of the measured KD when measured under identical conditions, such as at the same temperature and / or on the same SPR chip.

[0077] In one aspect, the binding molecule comprises variable domains comprising an alpha chain variable domain set forth in SEQ ID NO: 11 or SEQ ID NO: 33 or an amino acid sequence that has at least 90%, identity, such as 90%, 91 %, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 11 or SEQ ID NO: 33; and beta chain variable domain set forth in SEQ ID NO: 15 or an amino acid sequence that has at least 90%, identity, such as 90%, 91%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, to SEQ ID NO: 15; wherein the binding molecule binds the FLYTYIAKV (SEQ ID NO: 1) HLA-A*02 complex with an affinity greater than 10nM as measured by surface plasmon resonance, including 1 nM, 700pM, 160pM or greater as measured by surface plasmon resonance.

[0078] Any suitable method may be used to determine whether a binding molecule binds the target antigen. Such a method may comprise surface plasmon resonance (SPR), homogenous time resolved fluorescence (HTRF), or bio-layer interferometry.

[0079] The binding molecules disclosed herein specifically bind the target FLYTYIAKV (SEQ ID NO: 1) in complex with HLA-A*02.

[0080] Ability of binding molecule to bind its target antigen, and the affinity of that binding (KD, Kd and / or Ka) can be determined by any routine method in the art, e.g. using surface plasmon resonance (SPR), such as by BiacoreTM (Cytiva Life Sciences) or using the ProteOn XPR36TM (Bio-Rad®), using KinExA® (Sapidyne Instruments, Inc), or using ForteBio Octet (Pall ForteBio Corp.).

[0081] The term "KD", as used herein, is intended to refer to the equilibrium dissociation constant of a particular binding molecule-antigen interaction. Affinity of binding molecule-antigen binding may be determined, e.g., by SPR. Affinity may also be determined by bio-layer interferometry.

[0082] A binding molecule of the invention binds to the FLYTYIAKV (SEQ ID NO: 1) HLA-A*02 complex with an affinity (KD) of high affinity, such as 10nM or greater as determined by SPR, including greater than 9nM, greater than 8nM, greater than 7nM, greater than 6nM, greater than 5nM, greater than 4nM, greater than 3nM, greater than 2nM, greater than 1 nM, greater than 950pM, greater than 900pM, greater than 850pM, greater than 800pM, greater than 750pM, greater than 700pM, greater than 650pM, greater than 600pM, greater than 550pM, greater than 500pM, greater than 450pM, greater than 400pM, greater than 350pM, greater than 300pM, greater than 250pM, greater than 200pM, greater than 190pM, greater than 180pM, greater than 170pM, greater than 160pM, or greater than 155pM as determined by SPR.

[0083] Known TCRs are considered to have a high affinity for the target / HLA complex when affinity is measured in M. See for example, Campillo-Davo et al., Cells 2020 Jul; 9(7), which describes that physiological TCR affinities can range from 1 M to 100pM and that several studies have marked the threshold for affinity for maximal T-cell activity, including antitumour T-cell responses, at 5-1 OpM of peptide epitope. Thus, TCRs with affinity measured in pM are TCRs with extremely high affinity.

[0084] In an example, a binding molecule of the invention binds to the FLYTYIAKV (SEQ ID NO: 1) HLA- A*02 complex with an affinity (KD) of greater than 1 nM as determined by SPR.

[0085] In an example, a binding molecule of the invention binds to the FLYTYIAKV (SEQ ID NO: 1) HLA- A*02 complex with an affinity (KD) of 700pM or greater as determined by SPR.

[0086] In an example, a binding molecule of the invention binds to the FLYTYIAKV (SEQ ID NO: 1) HLA- A*02 complex with an affinity (KD) of greater than 155pM as determined by SPR.

[0087] In one example, the SPR is carried out at 25°C. In brief, the affinity of the binding molecule can be determined using SPR by:1 . Immobilising binding molecules on a biosensor chip (e.g. streptavidin-coated chip).2. Passing the test antigen over the chip’s capture surface at a series of concentrations; and3. Determining the affinity of binding of binding molecule to antigen using surface plasmon resonance.4. KD, Ka and Kd may then be calculated.

[0088] Example 5 describes an example method for determining affinity using SPR. SPR can be carried out using any standard SPR apparatus, such as by BIAcore T200 or using the ProteOn XPR36TM (Bio-Rad®).

[0089] The binding molecules of the disclosure bind FLYTYIAKV in complex with HLA-A*02 with high specificity. A binding molecule may be said to bind its antigen if the level of binding to antigen is at least 2.5 fold greater, e.g. at least 10 fold greater, than binding to a control antigen. Binding between a bindingmolecule and its cognate antigen is often referred to as specific binding. Precise identification of the residues bound can usually be obtained using x-ray crystallography.

[0090] Specificity can be measured, for example, by the BIAcore method of Example 5. The TCR can be assayed for binding to the FLYTYIAKV-HLA-A*02 complex relative to one or more control peptide-HLA complexes. Specificity can be confirmed by binding to the FLYTYIAKV-HLA-A*02 complex at an affinity that is at least 2.5 fold greater, e.g. at least 10 fold greater, than binding to a control peptide HLA complex, optionally at least 20 fold great, 25 fold greater, 30 fold greater or 40 fold greater.

[0091] Specificity can also be measured in vitro cellular assays, where the level of T cell activation is measured in the presence of antigen positive cells e.g. ROPN1 positive cell lines (including e.g. cells engineered to overexpress ROPN-1 ) and antigen negative cells e.g. ROPN1 negative cell line (e.g. BT-549, ATCC Number HTB-122™). Specificity can be confirmed by T cell activation in the presence of antigen positive cells of 50% more than T cell activation in the presence of antigen negative cells, optionally 75% more, optionally 100% more.

[0092] The binding molecules of the disclosure have long binding half-lives (T1 / 2) for the FLYTYIAKV HLA- A*02 complex of from about 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes or 17 minutes. The binding molecules of the disclosure can have an affinity of at least 700 pM and a half-life of at least 5 minutes, optionally an affinity of at least 155pM and a half-life of at least 17 minutes. Half-life can be determined by SPR, for example.

[0093] The binding molecules of the invention have desirable safety profiles, with low levels of crossreactivity. It has been observed that affinity enhancement of TCRs often leads to increased cross-reactivity (see, for example, Spear et al., Cancer Immunol Immunother. 2019 Nov 68(11): 1881-1889) and thus it is particular challenge to generate TCRs with very high affinity levels but acceptable cross-reactivity.

[0094] The inventors have surprisingly found that a single substitution from phenylalanine to tryptophan at position 55 (IMGT) of FR2 region of alpha chain variable domain results in a 4.5-fold improvement in the binding affinity of the binding molecules of the disclosure.

[0095] In one embodiment, the binding molecule comprises alpha and beta variable domains that are heterodimers. In one embodiment, the binding molecule is a heterodimer of alpha and beta chains. In particular, the binding molecule can be a heterodimer comprising the alpha chain TRAC constant domain sequence of SEQ ID NO: 6 or an amino acid sequence that has at least 90%, identity, including at least 90%, 91 %, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, to SEQ ID NO: 6 and / or a beta chain TRBC2 constant domain sequence of SEQ ID NO: 8 or an amino acid sequence that has at least 90%, identity, including at least 90%, 91 %, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, to SEQ ID NO: 8.

[0096] In another embodiment, the binding molecule can be a heterodimer comprising the alpha chain TRAC constant domain sequence of SEQ ID NO: 5 or an amino acid sequence that has at least 90%, identity, such as 90%, 91 %, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, to SEQ ID NO: 5 and / or a beta chain TRBC2 constant domain sequence of SEQ ID NO: 7 or an amino acid sequence that has at least 90%, identity, such as 90%, 91 %, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, to SEQ ID NO: 7.

[0097] In another embodiment, the binding molecule is a single chain TCR with the format of i) alpha variable region-linker-beta variable region, ii) beta variable region-linker-alpha variable region, ill) alpha variable region-alpha constant region-linker-beta variable region, alpha variable region-linker-beta variable regionbeta constant region.

[0098] The binding molecules of the disclosure are preferably soluble. In soluble binding molecules of the disclosure, the alpha and beta chain constant domain sequences can be truncated to exclude the transmembrane region. The alpha and / or beta chain constant sequences can be modified to incorporate an unnatural alpha-beta disulphide bond (see, for example, Boulter et al., Protein Engineering, Design and Selection, 2003 Sept 16(9): 707-711). The alpha and / or beta chain can be modified to substitute a free cysteine.

[0099] In a further aspect of the disclosure, the binding molecule further comprises an anti-CD3 antibody covalently linked to the C-terminus or the N-terminus of the alpha chain or the beta chain. The antibody can be linked via a linker, for example, SSGGGGS (SEQ ID NO: 31 ). In one embodiment, the anti-CD3 antibody is covalently linked to the beta chain via linker SSGGGGS.

[0100] The anti-CD3 antibody can be a scFv, Fab, Fab’, (Fab’)2 or single domain format.

[0101] The anti-CD3 antibody can be UCHTI , OKT3, HIT3a, SP34, BMA-031 or 12F6. The anti-CD3 antibody can be a scFv comprising the sequence set forth in SEQ ID NO: 32.

[0102] In some embodiments, a first signal peptide is present at the N-terminus of the alpha chain variable domain and a second signal peptide is present at the N-terminus of the beta chain variable domain. In some embodiments, the first signal peptide and the second signal peptide are the same. In some embodiments, the first signal peptide and the second signal peptide are not the same. Examples of signal peptides are known in the art. In some embodiments, the first signal peptide and the second signal peptide is Pro-Albumin signal sequence (SEQ ID NO: 4). In some embodiments, the first signal peptide or the second signal peptide is Pro-Albumin signal sequence (SEQ ID NO:4). The signal peptides are cleaved during the secretion of the binding molecule or during the transport of the binding molecule to the cell membrane.NUCLEIC ACIDS, VECTORS, HOST CELLS

[0103] Nucleic acids that encode any one or more of the amino acid sequences (e.g. any one or more CDRs from the alpha and / or beta regions or the full alpha and / or beta variable regions) of any one of the binding molecules described herein are also provided.

[0104] In one example the nucleic acid encodes the variable alpha or beta chain of any one of the binding molecules defined anywhere herein. In another example, the nucleic acid encodes a constant region of any one of the binding molecules described anywhere herein.

[0105] In one example, the nucleic acid is an isolated and purified nucleic acid.

[0106] The term “nucleic acid sequence” is intended to encompass a polymer of DNA or RNA, i.e., a polynucleotide, which can be single-stranded or double-stranded and which can contain non-natural or altered nucleotides. The terms “nucleic acid” and “polynucleotide” as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and thus include double- and single-stranded DNA, and double- and single-stranded RNA. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated and / or capped polynucleotides. Nucleic acids are typically linked via phosphate bonds to form nucleic acid sequences or polynucleotides, though many other linkages are known in the art (e.g. phosphorothioates, boranophosphates, and the like).

[0107] Vectors comprising the nucleic acids described above are also provided. In one embodiment, the vector comprises one or more nucleic acid sequences encoding a binding molecule as defined anywhere herein. In some embodiments, the vector comprises one or more nucleic acids as defined anywhere herein.

[0108] In some embodiments, one vector encodes the alpha and beta chain of a binding molecule described herein. In some embodiments, one vector encodes the alpha chain of a binding molecule described herein and a second vector encodes the beta chain of a binding molecule described herein.

[0109] The vector can be, for example, a plasmid, episome, cosmid, viral vector (e.g., retroviral or adenoviral), or phage. Suitable vectors and methods of vector preparation are well known in the art (see, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994)). In one embodiment, the vector may be a CHO vector. In one embodiment, the vector may be a HEK293 vector.

[0110] In addition to the nucleic acid sequence encoding a binding molecule as defined anywhere herein or a polypeptide as defined anywhere herein, the vector further comprises expression control sequences, such as promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), and the like, that provide for the expression of the coding sequence in a host cell. Exemplary expression control sequences are known in the art and described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

[0111] Host cells comprising one or more of the vectors as described anywhere herein or one or more of the nucleic acids as defined anywhere herein are also provided.

[0112] The vector(s) comprising the nucleic acid(s) encoding the amino acid sequence(s) of the binding molecules described anywhere herein can be introduced into a host cell that is capable of expressing the polypeptides encoded thereby. Preferred host cells are those that can be easily and reliably grown, have reasonably fast growth rates, have well characterized expression systems, and can be transformed or transfected easily and efficiently. The host cell may be a prokaryotic or a eukaryotic cell.

[0113] Suitable eukaryotic cells are known in the art and include, for example, yeast cells, insect cells, and mammalian cells. In one embodiment, the vector is expressed in mammalian cells. A number of suitable mammalian host cells are known in the art, and many are available from the American Type Culture Collection (ATCC, Manassas, VA). Examples of suitable mammalian cells include, but are not limited to, Chinese hamster ovary cells (CHO) (ATCC No. CCL61), CHO DHFR- cells (Urlaub et al, Proc. Natl. Acad. Sci. USA, 97: 4216-4220 (1980)), human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), and 3T3 cells (ATCC No. CCL92). Other suitable mammalian cell lines are the monkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651), as well as the CV-1 cell line (ATCC No. CCL70). The mammalian cell desirably is a human cell. For example, the mammalian cell can be a human lymphoid or lymphoid derived cell line, such as a cell line of pre-B lymphocyte origin, a PER.C6® cell line (Crucell Holland B.V., The Netherlands), or human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573).

[0114] A nucleic acid sequence encoding amino acids of a binding molecule as defined anywhere herein or a polypeptide as defined anywhere herein may be introduced into a cell by “transfection,” “transformation,” or “transduction.” “Transfection,” “transformation,” or “transduction,” as used herein, refer to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods. Many transfection techniques are known in the art and include, for example, calcium phosphate DNA coprecipitation (see, e.g., Murray E.J. (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991 )); DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al, Mol. Cell Biol., 7: 2031-2034 (1987)). Phage or viral vectors can be introduced into host cells, after growth of infectious particles in suitable packaging cells, many of which are commercially available.PHARMACEUTICAL COMPOSITION

[0115] The invention also provides a pharmaceutical composition comprising binding molecule as defined anywhere herein and a pharmaceutically acceptable carrier. Also provided is a composition comprising the nucleic acid sequence encoding binding molecule as defined anywhere herein, or the vector comprising the nucleic acid sequence as defined anywhere herein.

[0116] The pharmaceutical composition comprises an effective amount of the binding molecule as defined herein, nucleic acid as defined anywhere herein or vector as defined anywhere herein. An effective amount of the binding molecule, nucleic acid or vector to be employed therapeutically will depend, for example, uponthe therapeutic objectives, the route of administration, and the condition of the patient. In one embodiment, the effective amount of binding molecule as defined anywhere herein within the pharmaceutical composition is effective to treat or prevent cancer.

[0117] The composition is a pharmaceutically acceptable (e.g., physiologically acceptable) composition, which comprises a carrier, preferably a pharmaceutically acceptable (e.g., physiologically acceptable) carrier. The pharmaceutically acceptable carrier may include one or more excipients. Pharmaceutically acceptable excipients are known and include carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Any suitable carrier can be used within the context of the invention, and such carriers are well known in the art.

[0118] The composition optionally can be sterile. The composition can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use. The compositions can be generated in accordance with conventional techniques described in, e.g., Remington: The Science and Practice of Pharmacy, 21 st Edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001 ).THERAPEUTIC USE

[0119] We have discovered improved binding molecules that bind FLYTYIAKV (SEQ ID NO: 1) in complex with HLA-A*02 with high affinity and have advantageous properties suitable for development as medicaments for treating or preventing cancer.

[0120] Binding molecules described herein or pharmaceutical compositions thereof may be used in therapy. In particular, binding molecules or pharmaceutical compositions thereof may be used in treating cancer. Binding molecules or pharmaceutical compositions thereof may also be used in preventing cancer.

[0121] Therefore the disclosure provides a binding molecule as defined anywhere herein or a pharmaceutical composition as defined anywhere herein for use in therapy.

[0122] The disclosure also provides a binding molecule as defined anywhere herein or a pharmaceutical composition as defined anywhere herein for use in treating cancer.

[0123] The disclosure also provides a method of treating cancer, wherein the method comprises administering an binding molecule as defined anywhere herein or a pharmaceutical composition as defined anywhere herein to a subject in need thereof.

[0124] The disclosure also provides the use of a binding molecule as defined anywhere herein or a pharmaceutical composition as defined anywhere herein for the manufacture of a medicament for the treatment of cancer.

[0125] In preferred embodiments, the subject is a human.

[0126] In one embodiment, one or more symptoms of cancer are reduced. In one example, the progression of cancer is reduced. In one example, the risk of developing cancer is reduced.

[0127] Therefore, in some embodiments, the binding molecule is for use in treating cancer. The cancer expresses ROPN1.

[0128] In some embodiments, the cancer is primary cancer. In some embodiments, the cancer is metastatic cancer. In some embodiments the cancer is melanoma, multiple myeloma or breast cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is triple negative breast cancer (TNBC). In some embodiments the cancer is Basal-like breast cancer (BLBC). In some embodiments, the cancer is HER2 breast cancer (i.e. the breast cancer is ER-negative, PR-negative and HER2-positive).DIAGNOSTIC USE

[0129] The soluble binding molecules disclosed herein can also be associated with a label.

[0130] "Label" or "labelled" as used herein refers to the addition of a detectable moiety to a binding molecule, for example, a radiolabel, fluorescent label, enzymatic label, chemiluminescent label or a biotinyl group or gold. Radioisotopes or radionuclides may include 3H, 14C, 15N, 35S, 90Y, 99Tc, 115ln, 1251, 1311, fluorescent labels may include rhodamine, lanthanide phosphors or FITC and enzymatic labels may include horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase. Additional labels include, by way of illustration and not limitation: enzymes, such as glucose-6-phosphate dehydrogenase ("G6PDH"), alpha- D-galactosidase, glucose oxydase, glucose amylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase and peroxidase; dyes (e.g. cyanine dyes, e.g. Cy5TM, Cy5.5TM. or Cy7TM); additional fluorescent labels or fluorescers include, such as fluorescein and its derivatives, fluorochrome, GFP (GFP for "Green Fluorescent Protein"), other fluorescent proteins (e.g. mCherry, mTomato), dansyl, umbelliferone, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fiuorescamine; fluorophores such as lanthanide cryptates and chelates e.g. Europium etc (Perkin Elmer and Cisbio Assays); chemoluminescent labels or chemiluminescers, such as isoluminol, luminol and the dioxetanes; sensitisers; coenzymes; enzyme substrates; particles, such as latex or carbon particles; metal sol; crystallite; liposomes; cells, etc., which may be further labelled with a dye, catalyst or other detectable group; molecules such as biotin, digoxygenin or 5-bromodeoxyuridine; toxin moieties, such as for example a toxin moiety selected from a group of Pseudomonas exotoxin (PE or a cytotoxic fragment or mutant thereof), Diptheria toxin or a cytotoxic fragment or mutant thereof, a botulinum toxin A, B, C, D, E or F, ricin or a cytotoxic fragment thereof e.g. ricin A, abrin or a cytotoxic fragment thereof, saporin or a cytotoxic fragment thereof, pokeweed antiviral toxin or a cytotoxic fragment thereof and bryodin 1 or a cytotoxic fragment thereof.

[0131] In this manner, the binding molecules can also be useful in diagnostic purposes by binding to the target antigen.SEQUENCESEXAMPLES

[0132] Here we describe TCRs with enhanced affinity for FLYTYIAKV in complex with HLA-A*02 as exemplary binding molecules of the disclosure. The Examples should not be construed as in any way limiting.EXAMPLE 1 - TCR CONSTRUCT DESIGN

[0133] Genes encoding TCR chains were cloned into a customised mammalian expression vector built upon pCDNA3.4 (Thermo Fisher Scientific), and the TCR alpha and beta chains were co-expressed from two separate plasmids (Figure 1). The construct design encodes: pro-albumin signal peptides (for both alpha and beta chains), a decahistidine tag (1 Ox His) to the C-terminus of the TCR alpha chain, and an AviTag (GLNDIFEAQKIEWHE (SEQ ID NO:3)) at the C-terminus of the beta chain.

[0134] To produce soluble secreted TCRs, sequences were truncated to exclude the transmembrane region and modified through incorporation of an unnatural alpha-beta disulphide bond between the constant domains (https: / / doi.org / 10.1093 / protein / gzg087) and substitution of a free cysteine.

[0135] The TCR alpha constant domain (UNIPROT P01848; SEQ ID NO: 5) was modified at position 47; a threonine replaced with a cysteine and the domain terminates at position 94 (as in SEQ ID NO: 6 - TRAC1).

[0136] The TCR beta constant domain (UNIPROT A0A5B9; SEQ ID NO: 7) was modified at position 9; a lysine replaced by a glutamic acid, position 56; a serine replaced by a cysteine, position 74; a cysteine replaced by an alanine, position 88; an asparagine replaced by an aspartic acid and the domain terminates at position 130 (as in SEQ ID NO: 8 - TRCB2_1).EXAMPLE 2 - TCR EXPRESSION

[0137] TCRs constructs were expressed in Chinese hamster ovary cells (CHO) using the commercially available transient ExpiCHO™ Expression System (ThermoFisher) following manufacturers high-titre protocol. The expression media was supplemented with 100 pM biotin dissolved in PBS (137 mM NaCI, 2.7 mM KCI, 10 mM Na2HPO4) and the two plasmids (one for each TCR chain) was co-transfected with an expression plasmid encoding biotin ligase (pDisplay-BirA-ER; DOI: 10.1038 / nmeth.1206) at a ratio of 4:1 and a total of 0.4 pg / ml plasmid. 2.5 ml conditioned media was harvested 7 days post transfection and clarified by centrifugation at 2800 x g.EXAMPLE 3 - TCR PURIFICATION

[0138] Secreted recombinant TCR was purified using immobilised metal affinity chromatography (IMAC), via the 10x His tag on the TCR alpha chain. Clarified media was supplemented with 50 mM Na phosphate pH8, 0.5M NaCI and 10 mM imidazole before purification using 20 pl (of a 25% slurry) PureCube Ni-INDIGO Magbeads (Cube Biotech). TCR-bound beads were subjected to a 3 ml PBS wash and eluted with 200 pl PBS containing 300 mM imidazole.EXAMPLE 4 ■ HUMAN LEUKOCYTE ANTIGEN (HLA) AND BETA 2 MICROGLOBULIN (B2M) CONSTRUCT DESIGN

[0139] The Novagen pET21 (+) expression plasmid was used for bacterial expression using E.coli (BL21 DE3) as a host organism. Due to the nature of the target protein domains (HLA and B2M), both are expressed as insoluble inclusion bodies (IB). The total protein expressed can be more than 50% of the total cell protein.IB preparation, solubilisation and refolding of pHLA are well established methods (htps: / / doi.Org / 10.1073 / pnas.89.8.3429), and often yield highly pure, homogenous protein preparations (free of N-linked glycosylation).

[0140] Bacterial expression constructs used for the production of pHLA protein complex were produced in the pET21 a vector using the start codon of the Ndel restriction site to initiate transcription. The HLA A*02:01 construct (pET21 |HLA_A2_25-300) codes for HLA A*02:01 amino acids 25-300 (regions alpha 1 , 2 and 3) the construct terminates before the transmembrane region (SEQ ID NO: 9). The beta 2 microglobulin construct (pET21 |B2M_21-119), matches the naturally secreted protein: amino acids 21-119 (SEQ ID NO: 10).

[0141] HLA and B2M expression, IB isolation, protein refolding in the presence of the ROPN1 peptide FLYTYIAKV residues 163-171 (SEQ ID NO: 1) purification were performed using established methods known to those in the art (https: / / doi.Org / 10.1073 / pnas.89.8.3429).

[0142] Briefly, HLA and B2M plasmids were transformed separately into the E. coli strain Rosetta 2(DE3)pLysS. Individual colonies were used to inoculate LB (+ ampicillin; 100 g / ml) medium and grown at 37°C until an ODeoo of 0.5 was reached. The culture was then used to inoculate auto induction medium (AIM) - Super Broth (ForMedium) supplemented with ampicillin at 100 pg / ml), grown at 37°C for 5 hours and 30°C for a further 16 hours. Cells were harvested by centrifugation. Cell pellets were lysed using a Cell Disruptor (Constant System) and inclusion bodies were recovered by centrifugation. Inclusion bodies were washed three times using Triton wash buffer (50 mM Tris pH 8.1 , 100 mM NaCI, 1 % Triton-X100 and 10 mM EDTA) and twice in resuspension buffer (50 mM Tris pH 8.1, 100 mM NaCI, and 10 mM EDTA) and finally resuspended in the same buffer. Inclusion body protein concentration was estimated by solubilising in solubilisation buffer (6M guanidine hydrochloride, 50 mM Tris pH 8.1 , 100 mM NaCI, 10 mM EDTA and 20 mM DTT) and measuring the QD280 and calculated considering the extinction coefficient. Inclusion body purity was assessed by solubilising ~2 g protein in 8M urea and SDS-PAGE under reducing conditions. Purity was estimated by densitometry (GelDoc, Bio-Rad). Inclusion bodies were stored at +4°C or -80°C for short or long storage, respectively. Solubilised and reduced HLA, B2M and peptide (at a molar ratio of 1 :2:4) were refolded by rapid dilution into refold buffer (0.4 M L-arginine-HCI, 100 mM Tris pH 8.0, 2 mM EDTA, 6.6 mM cysteamine hydrochloride and 3.7 mM cystamine dihydrochloride). Refolded material was dialysed into 10 mM Tris-HCI pH 8.1 and purified by anion exchange and further purified by size exclusion chromatography.EXAMPLE 5 - BINDING ANALYSIS SURFACE PLASMON RESONANCE

[0143] Affinity analysis for soluble TCRs binding to pHLA complexes was carried out by surface plasmon resonance (SPR) using a BIAcore T200 instrument. All measurements were carried out at 25°C.

[0144] Purified TCRs were immobilised on a sensor chip preloaded with streptavidin (Sensor Chip SA). As the TCRs were biotinylated via an AviTag on the TCR beta chain, any potential alpha chain homodimers would not bind to the sensor surface - allowing only monodispersed heterodimer TCRs to bind to the streptavidin coated sensor surface.

[0145] A Biacore T200 instrument was primed into a running buffer consisting of HEPES Buffered Saline containing 0.05% Tween-20. A five-point, two-fold titration of ROPN1 FLYTYIAKV pHLA, from low to high concentration with a maximum concentration of 2 nM was passed of the flow cells at 60 pl / min. Successive 100 second injections were followed by a 3600 second dissociation phase. Specific responses were determined by subtracting the non-specific response of a flow cell immobilised with only streptavidin from the response of each TCR-loaded flow cell.

[0146] The Kd value was determined by nonlinear curve fitting using software provided with Biacore T200 and the binding isotherm equation equivalent to the Langmuir isotherm for the absorption of a gas onto a surface.

[0147] Y = Bmax*X / (Kd+X)

[0148] Kd is the equilibrium dissociation constant, in the same units as X (Molar). It is the concentration needed to achieve a half-maximum binding at equilibrium. Bmax is the maximum specific binding in the same units as Y (RU). Y is the equilibrium binding in RU at injected TCR concentration X.

[0149] TCR affinity is provided in Table 1 and the SPR sensorgrams presented in Figure 4. A TCR comprising alpha chain CDR1 DRGSVG (SEQ ID NO: 12), CDR2 IYSNGD (SEQ ID NO: 13), CDR3 CAVNGRDSYKIIF (SEQ ID NO: 14) and beta chain CDR1 MEHEY (SEQ ID NO: 16), CDR2 SPTAGL (SEQ ID NO: 17) and CDR3 CGSSYNLGDGYTF (SEQ ID NO: 18) has an affinity of 697pM (Figure 2A). A TCR with the same CDRs but with tryptophan residue in FR2 position 55 (IMGT) of alpha chain variable domain has an affinity of 151 pM (Figure 2B).EXAMPLE 6 - T-CELL ACTIVATION ASSAY (CYTOKINE RELEASE)

[0150] The binding molecules of the disclosure were formatted into a bispecific format. The heterodimer TCRs comprising alpha chain SEQ ID NO: 27 (TCR1) or SEQ ID NO: 35 (TCR2) and beta chain SEQ ID NO: 28 were fused to the anti-CD3 scFv (SEQ ID NO: 32) using a GGGGS linker (SEQ ID NO: 29).

[0151] The binding molecules in the bispecific format were then added to a co-culture of ROPN1 positive target cells (MCF7, HepG2 and BT-549 engineered to overexpress ROPN1) and effector T cells (isolated from healthy blood donors, NHS Blood and Transplant ref. number T366). Release of the effector T cell cytokine I FNy into culture supernatant was then measured to provide an indication of the efficacy. It was demonstrated that both bispecific molecules formatted with TCR1 and TCR2 were able to stimulate strong release of I FNy from effector cells in the co-culture. The results are presented in Figure 3. When targeting MCF7 cells expressing ROPN1, the release of the IFNy in the presence of bispecific molecule with TCR1was greater than the release of the IFNy in the presence of bispecific molecule with TCR2, demonstrating improved stimulation of effector T cells. When targeting BT549 cells expressing ROPN1 , the release of the IFNy in the presence of bispecific molecule with TCR2 was greater than the release of the IFNy in the presence of bispecific molecule with TCR1 , demonstrating improved stimulation of effector T cells. When targeting HepG2 cells expressing ROPN1 , the release of the IFNy in the presence of bispecific molecule with TCR1 was comparable to the release of the IFNy in the presence of bispecific molecule with TCR2, demonstrating similar stimulation of effector T cells. Presence of irrelevant TCR in the bispecific format resulted in no IFNy release, regardless of the cell line used. No release of IFNy was observed when either of the TCRs in the bispecific format was added to a co-culture of ROPN negative cells (wild-type BT-549, HepG2 or BT549) and the effector T cells.EXAMPLE 7 - SPECIFICITY (NFAT REPORTER ASSAY)

[0152] Engineered T cell line (e.g., Jurkat cells), was exposed to antigen presenting cells (e.g., T2 cells) pulsed with various peptide antigens of interest, including target ROPN1 peptide (SEQ ID NO: 1 ; +ve control), peptide mimetics with varying degree of sequence similarity to the (SEQ ID Nos: 40;Mimetic A to E), as well as a peptide derived from a protein unrelated to ROPN1 (-ve control) and then co-incubated with TCR1 and TCR2 formatted into a bispecific as described in Example 6. The level of NFAT activation was measured by luminometry.

[0153] The results of the NFAT reporter assay (presented in Figure 4) demonstrated that both TCR1 and TCR2 in the bispecific format are specific for the ROPN1 peptide-HLA-A*02 complex, with either no or negligible cross-reactivity to peptide mimetics (natural peptides with high degree of sequence similarity to the target peptide).EXAMPLE 8 - ALLOREACTIVITY

[0154] In order to confirm that TCR1 and TCR2 are not reactive to other HLA-A subtypes in the absence of ROPN-1 peptide, the investigators assessed the reactivity of TCR1 and TCR2 in the bispecific format as described in Example 6 in a panel of cell lines expressing different HLA-A subtypes. T2 cells (which are HLA- A*02:01 positive) pulsed with either the ROPN1 peptide (SEQ ID NO: 1) or a peptide derived from a protein unrelated to ROPN1 were used as positive and negative controls, respectively. The positive and negative control T2 cells, as well as cell lines of the panel were then co-cultured with effector T cells. TCR1 and TCR2 in the bispecific format as described in Example 6 were added to the co-cultures.

[0155] Release of the effector T cell cytokine IFNy into culture supernatant in the presence and absence of the respective TCR bispecific was then measured to provide an indication of potential alloreactivity. The results are depicted in Figure 5.

[0156] No release of I FNy above background (i.e. in the absence of bispecific) was observed when either of the TCR bispecifics were added to the co-cultures, indicating a lack of reactivity to HLA-A subtypes in the absence of ROPN1 peptide (SEQ ID NO: 1).able 1 - Affinity

Claims

CLAIMS1 . A binding molecule capable of binding to FLYTYIAKV (SEQ ID NO: 1) in complex with HLA-A*02 and comprising a TOR alpha chain variable domain and / or a TOR beta chain variable domain, wherein: the alpha chain variable domain comprises an amino acid sequence that has at least 90%, identity, including at least 90%, 91 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, to QKEVEQNSGPLSVPEGAIASLNCTYSDRGSVGFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQ YVSLLIRDSQPSDSATYLCAVNGRDSYKIIFGSGTRLLVRPN (SEQ ID NO: 11) and the beta chain variable domain comprises an amino acid sequence that has at least 90%, identity, such as 90%, 91 %, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, to NAGVTQTPKFQVLKTGQSMTLQCAQDMEHEYMSWYRQDPGMGLRLIHYSPTAGLTDQGEVPNGYNVSR STTEDFPLRLLSAAPSQTSVYFCGSSYNLGDGYTFGSGTRLTVVE (SEQ ID NO: 15); or : the alpha chain variable domain comprises an amino acid sequence that has at least 90%, identity, such as 90%, 91%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, to QKEVEQNSGPLSVPEGAIASLNCTYSDRGSVGFFWYRQYSGKSPELIMWIYSNGDKEDGRFTAQLNKASQ YVSLLIRDSQPSDSATYLCAVNGRDSYKIIFGSGTRLLVRPN (SEQ ID NO: 33) and the beta chain variable domain comprises an amino acid sequence that has at least 90%, identity, such as 90%, 91 %, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, to NAGVTQTPKFQVLKTGQSMTLQCAQDMEHEYMSWYRQDPGMGLRLIHYSPTAGLTDQGEVPNGYNVSR STTEDFPLRLLSAAPSQTSVYFCGSSYNLGDGYTFGSGTRLTVVE (SEQ ID NO: 15), wherein the binding molecule binds the FLYTYIAKV (SEQ ID NO: 1) HLA-A*02 complex with an an affinity greater than 10nM as measured by surface plasmon resonance.

2. The binding molecule of claim 1 that binds to FLYTYIAKV (SEQ ID NO: 1) HLA-A*02 complex with an affinity greater than 1 nM, optionally with affinity of 700pM, 160pM or greater as measured by surface plasmon resonance.

3. The binding molecule of claim 1 or claim 2, wherein: the alpha chain variable domain sequence of amino acid residues 27-32 (CDR1), 50-55 (CDR2) and 89- 101 (CDR3) and the beta chain variable domain sequence of amino acid residues 27-31 (CDR1), 49-54 (CDR2), and 91-103 (CDR3) are: alpha chain CDR1 DRGSVG (SEQ ID NO: 12), CDR2 IYSNGD (SEQ ID NO: 13), CDR3 CAVNGRDSYKIIF (SEQ ID NO: 14) and beta chain CDR1 MEHEY (SEQ ID NO: 16), CDR2 SPTAGL (SEQ ID NO: 17) and CDR3 CGSSYNLGDGYTF (SEQ ID NO: 18).

4. The binding molecule of any preceding claim, wherein the alpha chain variable region comprises a FR2 region comprising a tryptophan or phenylalanine at position 55 (IMGT) of the alpha chain variable domain.

5. The binding molecule of any preceding claim, wherein the alpha chain variable region comprises the following framework region sequences: FR1 QKEVEQNSGPLSVPEGAIASLNCTYS (SEQ ID NO: 19), FR2 FFWYRQYSGKSPELIMF (SEQ ID NO: 20), FR3 KEDGRFTAQLNKASQYVSLLIRDSQPSDSATYL (SEQ ID NO: 21) and / or FR4 GSGTRLLVRPN (SEQ ID NO: 22) and / or the beta chain variable region comprises the following framework region sequences: FR1 NAGVTQTPKFQVLKTGQSMTLQCAQD (SEQ ID NO: 23), FR2 MSWYRQDPGMGLRLIHY (SEQ ID NO: 24), FR3 TDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYF (SEQ ID NO: 25) and / or FR4 GSGTRLTVVE (SEQ ID NO: 26).

6. The binding molecule of any preceding claim, wherein the alpha chain variable region comprises the following framework region sequences: FR1 QKEVEQNSGPLSVPEGAIASLNCTYS (SEQ ID NO: 19), FR2 FFWYRQYSGKSPELIMW (SEQ ID NO: 34), FR3 KEDGRFTAQLNKASQYVSLLIRDSQPSDSATYL (SEQ ID NO: 21) and / or FR4 GSGTRLLVRPN (SEQ ID NO: 22) and / or the beta chain variable region comprises the following framework region sequences: FR1 NAGVTQTPKFQVLKTGQSMTLQCAQD (SEQ ID NO: 23), FR2 MSWYRQDPGMGLRLIHY (SEQ ID NO: 24), FR3 TDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYF (SEQ ID NO: 25) and / or FR4 GSGTRLTVVE (SEQ ID NO: 26).

7. The binding molecule of any preceding claims, wherein the binding molecule is an alpha-beta heterodimer, having an alpha chain TRAC constant domain sequence of IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDF ACANAFNNSIIPEDTFFPSPESSC (SEQ ID NO: 6) and / or a beta chain TRBC2 constant domain sequence of DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALND SRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC (SEQ ID NO: 8).

8. The binding molecule of any preceding claim, wherein the alpha chain amino acid sequence comprises the sequence set forth in QKEVEQNSGPLSVPEGAIASLNCTYSDRGSVGFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQ YVSLLIRDSQPSDSATYLCAVNGRDSYKIIFGSGTRLLVRPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQ TNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSC (SEQ ID NO: 27) or an alpha chain that has at least 90%, 91%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, to SEQ ID NO: 27; and: wherein the beta chain amino acid sequence comprises an amino acid sequence set forth in NAGVTQTPKFQVLKTGQSMTLQCAQDMEHEYMSWYRQDPGMGLRLIHYSPTAGLTDQGEVPNGYNVSR STTEDFPLRLLSAAPSQTSVYFCGSSYNLGDGYTFGSGTRLTVVEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQ VQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC SEQ ID NO: 28 or a beta chain that has at least 90%, identity, such as 90%, 91 %, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, to (SEQ ID NO: 28).

9. The binding molecule of any preceding claim, wherein the alpha chain amino acid sequence comprises the sequence set forth in QKEVEQNSGPLSVPEGAIASLNCTYSDRGSVGFFWYRQYSGKSPELIMWIYSNGDKEDGRFTAQLNKASQ YVSLLIRDSQPSDSATYLCAVNGRDSYKIIFGSGTRLLVRPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQ TNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSC (SEQ ID NO: 35) or an alpha chain that has at least 90%, 91%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, to SEQ ID NO: 35; and: wherein the beta chain amino acid sequence comprises an amino acid sequence set forth in NAGVTQTPKFQVLKTGQSMTLQCAQDMEHEYMSWYRQDPGMGLRLIHYSPTAGLTDQGEVPNGYNVSR STTEDFPLRLLSAAPSQTSVYFCGSSYNLGDGYTFGSGTRLTVVEDLKNVFPPEVAVFEPSEAEISHTQKAT LVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQ VQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC (SEQ ID NO: 28) or a beta chain that has at least 90%, identity, such as 90%, 91 %, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, to SEQ ID NO: 28.

10. The binding molecule of any preceding claim, further comprising an anti-CD3 effector molecule (e.g. an antibody), wherein the anti-CD3 effector molecule is covalently linked to the C-terminus or the N- terminus of the alpha chain or the beta chain via a linker sequence.11 . The binding molecule of claim 10, wherein the anti-CD 3 antibody is a scFv.

12. The binding molecule of claim 8 or claim 9 where the anti-CD 3 antibody is UCHT 1 or a variant thereof, optionally comprising the sequence set forth in SEQ ID NO: 32.

13. The binding molecule of any one of claims 10 to 12, wherein the binding molecule further comprises a human IgG Fc region, optionally wherein the human IgG Fc region is a modified region with reduced effector function compared to a non-modified human IgG Fc region.

14. A nucleic acid encoding a binding molecule as claimed in any preceding claim.

15. An expression vector comprising the nucleic acid of claim 14.

16. A non-naturally occurring and / or purified and / or engineered cell, such as a T-cell, presenting a binding molecule as claimed in any one of claims 1 to 9.

17. A pharmaceutical composition comprising a binding molecule as claimed in any one of claims 1 to13, or a nucleic acid of claim 14, or an expression vector of claim 15, or a cell of claim 16, with one or more pharmaceutically acceptable carriers or excipients.

18. The binding molecule as claimed in any one of claims 1 to 13, or a nucleic acid of claim 14, or a cell of claim 16 or pharmaceutical composition of claim 17 for use in medicine.

19. The binding molecule as claimed in any one of claims 1 to 13, or a nucleic acid of claim 14, or an expression vector of claim 15, or a cell of claim 16 or pharmaceutical composition of claim 17 for use in treating a Ropporin-1 A expressing cancer.

20. The binding molecule for the use of claim 19, wherein the cancer is melanoma, multiple myeloma, or breast cancer.21 . The binding molecule for the use of claim 19, wherein the breast cancer is HER 2 breast cancner or triple negative breast cancer (TNBC, for example basal-like breast cancer (BLBC)).

22. A method of treating a Ropporin-1 A expressing cancer comprising administration of the binding molecule as claimed in any one of claims 1 to 13, or a nucleic acid of claim 14, or a cell of claim 16 or a pharmaceutical composition of claim 17 to a patient in need thereof.

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