EA042909B1 - T-CELL RECEPTORS THAT RECOGNIZE FRAME-SHIFTING MUTANTS OF TGFβRII - Google Patents

Description

The invention relates to T-cell receptors (TCR) that recognize frameshift mutant variants of the transforming growth factor-β receptor type II (TGFeRII). Such TCRs are used to treat cancer, in particular cancers that contain single frameshift mutations of TGFeRII. The present invention relates to TCR molecules, nucleic acid molecules that encode said TCRs, and vectors containing said nucleic acid molecules. The nucleic acid molecules and vectors of the present invention can be used to modify immune effector cells, including in particular T cells, to express TCR. Nucleic acid molecules and vectors can also be used to modify producing host cells to produce TCR. Such modified immune effector cells can be used in adoptive cell transfer therapy. In particular, the TCRs of the present invention are based on or derived from a particular TCR, defined herein as Radium-1, which was identified in a cytotoxic T lymphocyte (CTL) clone isolated from a clinically responding patient immunized with a TGFeRII peptide carrying a frameshift mutation, and which recognizes the sequence of the neoepitope peptide carrying the frameshift mutation, RLSSCVPVA (SEQ ID NO: 1).

Worldwide, colorectal carcinoma (CRC) is the third most common cancer in men and the second most common cancer in women, with the highest incidence of CRC occurring in the Western Hemisphere. Lynch syndrome, or hereditary non-polyposis colorectal cancer (HNPCC), is an inherited condition in which DNA mismatch repair is impaired, resulting in microsatellite instability (MSI). Individuals with Lynch syndrome are at high risk of developing various types of cancer, including CRC. MSI also occurs in a subgroup of sporadic cancers (ie, non-hereditary cancers), including colorectal and gastric cancers.

MSI results in the insertion or deletion of single nucleotides or dinucleotides in short, repetitive DNA sequences. When such mutations occur in genes encoding proteins, they cause a frameshift of the gene (ie, they are frameshift mutations). Such mutations usually result in the formation of truncated non-functional proteins.

Transforming growth factor-β (TGF-β) proteins bind to TGFeRII receptor proteins on the cell surface, activating a signal transduction pathway that leads to cell cycle arrest. Mutation of TGFeRII can lead to inactivation of this pathway, which promotes carcinogenesis. Frameshift mutations that inactivate TGFeRII occur in approximately 90% of colon cancers characterized by microsatellite instability (MSI+) and approximately 15% of colon cancers characterized by microsatellite stability (MSS, He-MSI ⁺ or MSI ^- ). Such mutations mainly occur in the mutation-vulnerable polyadenine tract in exon 3 of TGFeRII.

MSI+ colon cancers are considered to be more immunogenic than MSS cancers due to the production of neopeptides (i.e., peptides whose sequences do not naturally occur in the individual, which are therefore recognized as non-autologous by the immune system) resulting from shift mutations reading frames in genes containing microsatellite repeats within the coding regions of their transcribed sequences. Patients with Lynch syndrome and patients with the MSI+ subtype of sporadic colon cancer have a better prognosis than patients with other types of sporadic colon cancer. The number and presence of certain frameshift mutations in MSI+ colon cancer correlates with increased density of tumor-infiltrating lymphocytes (IOLs) that characterize these cancers. There is also a well-established correlation between increased IOL density in different types of MSI+ colorectal cancer and improved survival compared to different types of non-MSE colorectal cancer. The enhanced host immune response may, at least in part, explain the improved prognosis for these cancers. These observations indicate that some patients with MSI ⁺ CRC may benefit from immunotherapy that targets products of frameshift mutations in genes such as TGFeRII.

T-cell epitopes have been identified in such neopeptides resulting from frameshift mutations. The TGFeRII -1A mutation, in which one adenine residue is removed from the aforementioned polyadenine tract in TGFeRII exon 3, is an example of a mutation that results in the production of neopeptides that contain T-cell epitopes, including both CD4+ and CD8+ T-cell epitopes.

T cell epitopes are recognized by TCRs, which are protein complexes protruding from the T cell's cell membrane. Most TCRs contain an α-chain and an e-chain, both of which consist of a variable region and a constant region. The variable region is located at the N-terminus of the chain and is completely extracellular; the constant region is located at the C-terminus of the chain and

- 1 042909 consists of an extracellular domain, a transmembrane domain and a short cytoplasmic domain. TCR chains are encoded and synthesized in an immature form with an N-terminal signal (or leader) sequence. This sequence forms the N-terminus of the variable region of the TCR α-chain or β-chain when it is synthesized. After the synthesis of the TCR chain, the signal sequence is cleaved off and, therefore, is not present in the mature TCR located on the cell surface. Recently, soluble TCRs (rTCRs) have been developed that contain the variable regions and extracellular domains of the α- and β-chain constant regions present in native TCRs, but lack the transmembrane and cytoplasmic domains of the constant regions. Soluble TCRs can be expressed and secreted by any cell.

The variable region of the α-chain and β-chain contains three complementarity-determining hypervariable regions (CDRs). These CDRs determine TCR specificity, with CDR3 (ie the third CDR from the N-terminus) being the most important CDR in determining TCR specificity. Regions of the variable regions of the TCR chains that do not form CDRs are known as framework regions. The TCR variable region contains four such framework regions. Framework section 1 is located at the N-terminus of CDR1; frame region 2 connects CDR1 and CDR2; frame region 3 connects CDR2 and CDR3; framework region 3 connects CDR3 to the constant region of the TKR chain. Said framework regions are much less variable than the CDRs and form the backbone for the CDRs. The sequence of the framework regions is important for TCR function because they determine the overall structure of the variable region of the TCR chain. This structure must maintain the CDRs in the correct orientations and relative positions so that they can bind to the target antigen.

The TCR variable region therefore binds to the target antigen, the TCR antigens being proteins. The specific part of the antigen associated with TCR is a T-cell epitope. T cell epitopes are short antigenic fragments, usually peptides, containing 8 to 17 amino acids. The corresponding antigenic fragment is represented by the TCR major histocompatibility complex (MCHC). Upon antigen binding, TKR activates a signal transduction pathway that activates the T cell to initiate an immune response.

There are two classes of SSGC: class I and class II. MHC class I is expressed by all nucleated cells; Class II MHCs are only expressed by specialized antigen-presenting cells (APCs) such as dendritic cells. The function of all MHCs is to present short peptide segments for recognition by T cells. MHC class I presents peptide fragments from the interior of the cell on which it is expressed and is recognized by CD8+ T cells (cytotoxic T cells). If a CD8+ T cell recognizes a peptide presented by MHC class I as an antigen, the T cell initiates apoptosis of the cell on which MHC class I is expressed. MHC class II presents peptide fragments from proteins that have undergone endocytosis by the antigen presenting cells on which it expressed and recognized by CD4+ T cells (helper T cells). If a naive CD4+ T cell recognizes a peptide presented by MHC class II as an antigen, then it will proliferate. Its daughter cells will then differentiate into effector T cells, memory T cells, and regulatory T cells, which co-mediate the immune response of other components of the immune system and confer long-term immunity against infection. In this regard, class I MHCs are generally important in initiating an immune response to virus-infected cells or cells containing mutations that cause the production of pathological proteins in them (such as cancerous or precancerous cells); Class II MHCs are generally important in initiating an immune response to extracellular pathogens.

In humans, MHCs are made up of proteins known as human leukocyte antigens (HLA). Each person has 3 major HLA MHC class I genes (HLA-A, HLA-B and HLA-C) and 6 major HLA MHC class II genes (HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA- DRA and HLA-DRB1). When TCR binds to the MHC-antigen complex, both the antigen and the MHC proteins come into contact with the TCR, which means that the TCR recognizes specific MHC-antigen complexes and not just the antigen. This interaction of TCRs with MHCs is believed to occur via CDR2, meaning that TCRs only recognize antigens if they complex with a particular HLA protein, a feature known as MHC restriction. The HLA genes are highly polymorphic, meaning that different individuals most often carry different HLA alleles and that a particular TCR will not be functional in all individuals (only those carrying the corresponding HLA allele that is restricted to TCR).

TCRs that recognize tumor antigens can be used in cancer therapy, in particular adoptive T cell transfer therapy (June, C., J Clin Invest. 2007 Jun 1; 117(6): 1466-1476). T cells can be retargeted against tumor cells by transferring genes encoding TCRs that recognize tumor antigens. Such retargeted T cells can be administered to patients suffering from cancer in which the corresponding antigen is produced. Said T-cells must then launch an immune response against the cancer cells, which will cause them to die. This is expected to reduce the size of the target tumor, which may result in a cure for the patient, or at least an increase in his life expectancy.

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However, recent clinical trials have shown that adoptive transfer of T cells with retargeted TCRs targeting cancer germline antigens may be associated with severe toxicity, highlighting the need for careful consideration of antigen selection. In one study, three of nine cancer patients who received autologous modified T cells carrying TCR against MAGE-A3 (MAGE-A3, melanoma-associated antigen 3, is a protein of unknown function associated with various types of cancer, including melanoma, but which is also present in healthy cells) experienced severe neurological toxicity (which was fatal in two cases) due to TCR cross-reactivity (Morgan, R.A. et al. (2013), Journal of Immunotherapy, 36(2): 133- 151). A second study targeting MAGE-AZ with HLA-A*01-restricted TCR in patients with myeloma and melanoma found lethal cross-reactivity with myocardial injury (Linette, G.P. et al. (2013), Blood 122 (6):863-871 Cameron, B.J. et al (2013), Science Translational Medicine 5(197):197ra103). Accordingly, true tumor-specific neoantigens may be optimal targets for tumor-targeting TCR therapy without destroying normal tissue. However, this may be associated with certain problems, since most of these neoantigens result from unique mutations that are not similar in patients. Another problem associated with adoptive T cell transfer therapy is that TCRs are limited by MHC as described above. Therefore, an individual TCR functions only in individuals carrying the HLA allele to which it is restricted, or a related isoform.

The authors of the present invention isolated TCR, which is used in therapy based on adoptive T-cell transfer. This TCR is an HLA-A2 restricted TCR specific for TGFeRII carrying a frameshift mutation isolated from a patient with MSI+ colon cancer vaccinated with the TGFeRII peptide. This TCR, known as Radium-1, has been shown to be particularly effective in redirecting T cells to recognize cancer cells carrying a frameshift mutation and reducing cancer growth in an animal model. Radium-1 is an unusually effective TCR with properties that make it particularly useful in medicine. Radium-1 has a very high affinity for the cognate antigen/MHC complex. Its affinity for the cognate antigen/MCGS complex is higher than that of the MART-1-specific TCR, DMF5, for the cognate antigen/MCGS complex. DMF5 has been successfully used clinically in the treatment of melanoma. High affinity for its antigen is an important characteristic of TCR for clinical use, and this high affinity of Radium-1 for its target, combined with a very successful treatment of tumors in animal models, demonstrates its great potential for clinical use. One of the unusual properties of Radium-1 is also that it does not depend on the CD4 and CD8 co-receptors. Most TCRs require CD8 or CD4 co-receptors to interact with the MHC complex on the target cell in order to mediate target cell death, however this is not the case for Radium-1, which means that both CD8+ and CD4+ T cells can functionally express Radium -1 and have been shown to be able to directly mediate the death of target cells.

Radium-1 TCR was isolated from a patient vaccinated with the peptide sequence shown in SEQ ID NO: 49 (SLVRLSSCVPVALMSAMTTSSSQ). The use of such peptides in human cancer vaccination is described in WO 1999/058552. Radium-1 was isolated from a T cell clone obtained from a patient who had been vaccinated in a similar manner to the patients in the study described in WO 1999/058552. However, the specific patient from whom Radium-1 was obtained was not a participant in this particular study, but was a participant in a later clinical study that took place in 2001. Radium-1 recognizes an epitope with the sequence shown in SEQ ID NO: 1 (RLSSCVPVA). It is a neopeptide resulting from the frameshift mutation described above, -1A TGFeRII, which as such is an optimal target for adoptive T cell transfer therapy, since the sequence is not found in the normal human proteome, meaning that TCR toxicity should be minimal. A neoantigen derived from a common frameshift mutation, such as a peptide containing the sequence of SEQ ID NO: 1, is an ideal target for adoptive T cell transfer therapy.

In addition, Radium-1 is HLA-A2 restricted (HLA-A2 is an HLA-A allele). Radium-1 has been shown to recognize antigens presented with the HLA-A*02:01 isoform of HLA-A2 but can recognize other HLA-A2 isoforms. HLA-A2 is one of the most common HLA alleles, with approximately 40-50% of white Americans and Europeans carrying HLA-A*O2:O1 (www.allelefrequencies.net). In this regard, TCR is expected to be functional in a significant portion of the Western Hemisphere population. Accordingly, T cells redirected by Radium-1 or related TCR according to the present invention can be used in cancer therapy. In particular, they can be used in adoptive cell transfer therapy based on

- 3 042909 example, with T cells. Such retargeted T cells or other immune effector cells are particularly useful in the treatment of any cancer that contains the TGFeRII 1A frameshift mutation, including MSI ⁺ CRC variants such as those often seen in individuals suffering from Lynch syndrome.

Neopeptides resulting from the -1A frameshift mutation of TAFeRII have previously been described as possible targets for cancer immunotherapy (see, for example, WO 1999/058552; Saeterdal, I. et al, 2001, Proc. Natl. Acad. Sci USA, Vol.98, pp. 13255-13260; Saeterdal, I. et al, 2001, Cancer Immunol Immunother 50(9):469-476; Linnebacher, M. et al, 2001, International journal of cancer. Journal international du cancer 93(1):6-11). However, it is not possible to design a functional TCR that will bind to a target antigen based solely on information about the corresponding sequence of the antigen. It is not possible to predict which TKR protein sequence will be required to bind to a particular antigen for a particular HLA protein allele. In this regard, while recognizing that a TCR that binds to a neoantigen derived from a frameshift mutation of TGFeRII may be useful for the common HLA allele, the problem has been the lack of such an effective TCR and, in particular, the absence of a known sequence for such a TCR, since one skilled in the art cannot construct such a TCR with currently available tools. It is important to note that not all TCRs that recognize a neoantigen peptide resulting from a frameshift mutation may be effective in practice in stimulating a cellular response against a cancer cell expressing the peptide. Isolation and characterization of the TCR Radium-1 described herein, which is the desired TCR, provides a solution to this problem.

Although Radium-1 TCR was not previously publicly available, the CDR3 sequences of the α- and β-chains of Radium-1 TCR were described in a 2011 Ph.D. thesis Cancer vaccines and T cell-based tumor-specific therapies; Development of new types of cancer immunotherapy by Elsa Marit Inderberg Suso, University of Oslo. However, even with the presence of CDR3 sequences, it is still impossible to predict the functional sequences of complete TCR chains, since two additional CDR sequences in each chain are required to obtain a functional TCR. In view of the foregoing, the present invention provides complete sequence information for TCR Radium-1, and in particular CDR1 and 2 from Radium-1.

Accordingly, the present invention primarily relates to a nucleic acid molecule encoding a TCR molecule directed against a mutant TGFeRII protein that contains the sequence of SEQ ID NO: 1, said TCR molecule being capable of binding to a peptide containing the sequence of SEQ ID NO: 1, when in this case, the specified peptide is represented by MHC class I, and at the same time, the specified TCR molecule contains an α-chain domain and/or a β-chain domain, where each chain domain contains three CDR sequences, while:

a) CDRs 1, 2 and 3 from the α chain domain contain the sequences of SEQ ID NOs: 2, 3 and 4, respectively; And

b) CDRs 1, 2 and 3 from the β-chain domain contain the sequences of SEQ ID NOs: 5, 6 and 7, respectively, and one or more of the specified CDR sequences and, in a more specific embodiment, one or more of the specified CDR1 or CDR2 sequences may optionally be modified by substitution, addition or deletion of 1 or 2 amino acids.

The amino acid sequences of CDR1 and CDR2 of the Radium-1 α chain are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the previously disclosed CDR3 sequence of the Radium-1 α chain is shown in SEQ ID NO: 4. The amino acid sequences of CDR1 and The Radium-1 β chain CDR2 is shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively, and the previously disclosed Radium-1 β chain CDR3 sequence is shown in SEQ ID NO: 7.

The α chain CDR sequences are, or correspond to, the Radium-1 α chain variable region CDR sequences. The Radium1 α chain variable region sequence is set forth in SEQ ID NO: 8. The β chain CDR sequences are, or correspond to, the Radium-1 β chain variable region CDR sequences. The sequence of the variable region of the Radium-1 β chain is shown in SEQ ID NO: 13. SEQ ID NO: 2, 3, and 4, referring to CDRs 1, 2, and 3 of the Radium-1 α chain, respectively, are located at positions 47- 51, 69-73 and 107-117 of the sequence shown in SEQ ID NO: 8, respectively. SEQ ID NOs: 5, 6, and 7, corresponding to CDRs 1, 2, and 3 of the Radium-1 β chain, respectively, are located at positions 46-50, 68-73, and 110-122 of the sequence shown in SEQ ID NO: 13, respectively.

Altering the CDR1 or CDR2 sequence of the TKR chain is less likely to change the specificity of the TKR and may improve the binding affinity of the TKR for its target antigen. Accordingly, in a preferred embodiment, CDR3 is not modified, and in another preferred embodiment, all CDRs are not modified.

The nucleic acid molecule according to the present invention can be used to obtain

- 4 042909 immune effector cells (in particular, modified immune effector cells) directed against cells expressing a mutant TGFeRII receptor, or more specifically representing a peptide resulting from a frameshift mutation containing the sequence of SEQ ID NO: 1. Such (modified) immune effector cells express TCR on their surface and are able to recognize or bind to a target cell representing a peptide containing the sequence of SEQ ID NO: 1, for example, a cancer cell. Accordingly, the nucleic acid molecule can be such that an immune effector cell expressing said TCR (i.e., the TCR encoded by the nucleic acid molecule) has effector activity (e.g., cytotoxic activity) against (e.g., causes death) of the target cell, representing a peptide resulting from a frameshift mutation containing the sequence shown in SEQ ID NO: 1. In other words, the nucleic acid molecule encodes a TCR molecule that, when expressed on the surface of an immune effector cell, is capable of binding a peptide containing the sequence of SEQ ID NO: 1, said peptide being MHC class I. The modified immune effector cell is accordingly a genetically modified or engineered immune effector cell, or in other words, an immune effector cell that has been transduced with a nucleic acid molecule according to the present invention.

Therefore, according to one implementation variant, the present invention relates to a nucleic acid molecule encoding a TCR molecule directed against a mutant TGFeRII protein that contains the sequence of SEQ ID NO: 1, and the specified TCR molecule, when expressed on the surface of an immune effector cell, is able to bind a peptide containing the sequence of SEQ ID NO: 1, if the specified peptide is represented by MHC class I, and at the same time the specified TCR molecule contains an α-chain domain and/or a β-chain domain, where each chain domain contains three CDR sequences, while:

a) CDRs 1, 2 and 3 from the α chain domain contain the sequences of SEQ ID NOs: 2, 3 and 4, respectively; And

b) CDRs 1, 2 and 3 from the β chain domain contain the sequences of SEQ ID NOs: 5, 6 and 7, respectively, and wherein one or more of said CDR sequences and, according to a more specific embodiment of the present invention, one or more of said CDR1 or CDR2 sequences may optionally be modified by substitution, addition or deletion of 1 or 2 amino acids.

In another embodiment, the nucleic acid molecule of the present invention can be used to express a soluble TCR molecule in a host cell. The soluble TCR molecule according to the present invention is capable of binding a peptide containing the sequence of SEQ ID NO: 1 when said peptide is MHC class I, and in particular it can be used to deliver a toxin to a target cell representing a peptide resulting from a mutation with frameshift containing the sequence of SEQ ID NO: 1 to destroy the target cell. Accordingly, the nucleic acid molecule of the present invention may encode a TCR which, when expressed by an immune effector cell, is localized on the cell surface; in another embodiment, the nucleic acid molecule according to the present invention may encode soluble TCR.

Therefore, according to another implementation variant, the present invention relates to a nucleic acid molecule encoding a soluble TCR molecule directed against a mutant TGFeRII protein, which contains the sequence of SEQ ID NO: 1, and the specified TCR molecule, expressed by the host cell, is secreted and is able to bind the peptide , containing the sequence of SEQ ID NO: 1, if the specified peptide is represented by MHC class I, and at the same time the specified TCR molecule contains an α-chain domain and/or a β-chain domain, where each chain domain contains three CDR sequences, while:

a) CDRs 1, 2 and 3 from the α chain domain contain the sequences of SEQ ID NOs: 2, 3 and 4, respectively; And

b) CDRs 1, 2 and 3 from the β-chain domain contain the sequences of SEQ ID NOs: 5, 6 and 7, respectively, and one or more of these CDR sequences and, according to a more specific embodiment of the present invention, one or more of these sequences, CDR1 or CDR2 may optionally be modified by substitution, addition or deletion of 1 or 2 amino acids.

The nucleic acid molecule of the present invention can be introduced into a cell, in particular an immune effector cell such as a T cell or a producing host cell, as mRNA or DNA for expression in the cell. Vectors can be used to transfer a nucleic acid molecule into a cell or to obtain a nucleic acid for transfer (eg, to obtain mRNA for transfer or to obtain a nucleic acid molecule to obtain an expression vector for transfer to a cell).

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Accordingly, according to a further aspect of the present invention, there is provided a vector comprising the nucleic acid molecule of the present invention as defined herein.

The vector may be, for example, an mRNA expression vector, a vector for cloning, or an expression vector for transfer to an immune cell or a producing host cell, such as a viral vector. In case the vector is a viral vector, it may be, for example, a retroviral vector or a lentiviral vector.

According to another aspect of the present invention, there is provided an immune effector cell comprising the nucleic acid molecule or vector of the present invention as defined herein. According to preferred embodiments of the present invention, the immune effector cell may be a T cell or a NK cell.

In a specific embodiment of the present invention, wherein the immune effector cell is a T cell (or more specifically a CD8+ T cell or a human CD8+ T cell), a TCR, or more specifically the nucleic acid molecule is not native to the T cell, i.e. e. the nucleic acid molecule is not present endogenously in the T cell, but is introduced into the T cell. In other words, the T cell is modified with a nucleic acid molecule or a vector, i. modified to express TCR; such a cell is not a native or natural T cell.

The present invention also relates to a producing host cell containing the nucleic acid molecule or vector according to the present invention as defined herein. According to preferred embodiments of the present invention, the production host cell is a mammalian cell, for example, a HEK-293, HEK-293T or CHO cell line.

The present invention also relates to a method for producing an immune effector cell that specifically recognizes a TGFeRII peptide carrying a frameshift mutation containing the sequence of SEQ ID NO: 1, said method comprising introducing into the immune effector cell a nucleic acid molecule or a vector according to the present invention.

Such a method may include stimulating the cell and inducing its proliferation before and/or after administration of the nucleic acid molecule or vector.

The present invention also relates to the TCR molecule as defined herein, in particular the soluble TCR molecule as defined herein. As described above and described in more detail below, soluble TCRs can be used in therapy.

As noted above, soluble TCR and immune effector cells according to the present invention can be used in therapy. Accordingly, additional aspects of the invention include a composition, in particular a therapeutic or pharmaceutical composition, comprising a soluble TCR or an immune effector cell of the present invention as defined herein, and at least one physiologically acceptable carrier or excipient;

soluble TCR, immune effector cell or composition according to the present invention, as defined herein, for use in therapy, in particular, therapy based on adoptive cell transfer;

soluble TCR, immune effector cell or composition according to the present invention, as defined herein, for use in the treatment of cancer, in particular for the treatment of colorectal cancer caused by HNPCC;

a method for treating cancer, in particular colorectal cancer, said method comprising administering to a subject in need thereof a soluble TCR, immune effector cell or composition of the present invention as defined herein, in particular an effective amount of said cell or composition; and the use of a soluble TCR or immune effector cell as defined herein for the manufacture of a medicament (or composition) for use in cancer therapy, in particular for the treatment of colorectal cancer.

In the method for producing a modified immune effector cell according to the present invention, said immune effector cell which is modified by introducing a nucleic acid molecule according to the present invention can be obtained from a subject to be treated (for example, a subject suffering from a cancer such as colorectal cancer). After the immune effector cell has been modified, and optionally expanded in vitro, the modified immune effector cells expressing TCR can be re-introduced (eg, administered) to the subject. Accordingly, autologous immune effector cells can be used in therapeutic methods and uses according to the present invention. Alternatively, heterologous (ie, donor or allogeneic, or syngeneic or xenogeneic) immune effector cells can be used.

An immune effector cell can be any immune cell capable of an immune response against a target cell representing a peptide comprising the sequence of SEQ ID NO: 1. More specifically, an immune effector cell is capable of suppressing, damaging or eliminating

- 6 042909 injure the target cell, i.e. reduce or inhibit the viability of the target cell, preferably causing the death of the target cell (in other words, reducing the viability of the target cell or making it unviable). Accordingly, the immune effector cell is preferably a cytotoxic immune effector cell.

The term cytotoxic is synonymous with cytolytic and is used herein to refer to a cell capable of inducing cell death by lysis or apoptosis in a target cell.

As used herein, the term immune effector cell includes not only mature or fully differentiated immune effector cells, but also progenitor (or progenitor) cells thereof, including stem cells (particularly hematopoietic stem cells, HSCs), or cells derived from HSCs.

Accordingly, the immune effector cell may be a T cell, NK cell, NKT cell, neutrophil, macrophage, or HSC-derived cell contained in a population of CD34+ cells derived from hematopoietic tissue, such as bone marrow, cord blood, or eg, mobilized peripheral blood, which, when administered to a subject, differentiates into mature immune effector cells. As will be described in more detail below, according to preferred embodiments of the present invention, the immune effector cell is a T cell or a NK cell. Primary cells may be used, e.g., cells isolated from a subject to be treated or a donor, optionally with an intermediate cell culture step (e.g., for cell expansion), or other cultured cells or cell lines (e.g., NK cell lines such as line NK92).

The term directed against a peptide containing the sequence of SEQ ID NO: 1 is synonymous with specific for a peptide containing the sequence of SEQ ID NO: 1, i.e. means only that TCR is able to specifically bind to the peptide. In particular, the TCR antigen-binding domain is capable of specifically binding to a peptide (more specifically, if TCR is expressed on the surface of an immune effector cell). Specific binding can be distinguished from non-specific binding to a non-target antigen (in this case, a peptide other than the peptide containing the sequence of SEQ ID NO: 1). Accordingly, an immune effector cell expressing TCR according to the present invention is redirected to specifically bind and exhibit cytotoxicity (e.g., induce death) against a target cell presenting a peptide comprising the sequence of SEQ ID NO: 1. In other words, the immune the effector cell is modified to redirect cytotoxicity preferentially to target cells presenting said peptide or expressing a mutant TGFeRII receptor containing said peptide.

Binding of the TCR antigen-binding domain to a peptide on the surface of the target cell transmits an activating stimulus to the TCR-containing cell, resulting in the induction of effector cell signaling pathways. Binding to a target peptide can thereby trigger proliferation, cytokine production, phagocytosis, lytic activity, and/or production of molecules that can mediate target cell death in an MHC-independent manner.

Soluble TCR according to the present invention can be produced by any suitable producing target cell. A suitable cell is preferably a mammalian cell, for example a human cell or a rodent cell. Any suitable cell line may be used, including HEK-293, HEK-293T and CHO cell lines. Binding of the soluble TCR of the present invention to the peptide on the surface of the target cell results in the internalization of the MHC class I-antigen-TCR complex. Accordingly, soluble TCRs can be used to specifically deliver toxins to target cells, resulting in target cell death.

The TCR molecule of the present invention may comprise an α-chain domain of the present invention and a β-chain domain of the present invention. In another embodiment, the TCR molecule may contain an α-chain domain according to the present invention, but not a β-chain domain according to the present invention; or the TCR molecule may contain a β-chain domain according to the present invention, but not an α-chain domain according to the present invention. In other words, the TCR molecule according to the present invention contains an α-chain domain according to the present invention and/or a β-chain domain according to the present invention. Preferably, however, the TCR molecule of the present invention contains both an α-chain domain as defined herein and a β-chain domain as defined herein.

According to this preferred embodiment of the present invention, wherein the TCR molecule of the present invention comprises both an α-chain domain of the present invention (hereinafter α-chain domain) and a β-chain domain of the present invention (hereinafter β-chain domain), the domain α-chains and β-chain domain may be encoded separately (for example, they are encoded by separate genes or more specifically by separate nucleic acid molecules or by separate parts (in the sense of separately controlled parts) or open reading frames (ORF) can

- 7 042909 nucleic acid molecules and are synthesized as separate proteins). Alternatively, they may be co-encoded by a single gene (ie, one nucleic acid molecule or one ORF, etc.), in which case they are synthesized as a single protein. When the α-chain domain and the β-chain domain are encoded as a single protein, this protein is known as single-stranded TCR (ssTCR). The ssTCR contains an α-chain domain connected to a β-chain domain. As used herein, the term α-chain domain refers to the α-chain of TCR, which is either an individual protein or is part of a protein, and in particular part of ssTCR. As used herein, the term β-chain domain refers to the β-chain of a TCR, which is either an individual protein or is part of a protein, and in particular part of an ssTCR. Expression of the α-chain and β-chain domains in a single ssTCR molecule ensures that the domains of the two chains are expressed at the same time and at similar levels. According to a preferred embodiment of the present invention, the TCR molecule of the present invention is encoded as ssTCR.

In the event that a TCR molecule according to the present invention is encoded as an ssTCR, the α-chain and β-chain domains can be connected using a linker. Such a linker consists of an amino acid sequence between the α chain and β chain domains. Preferably, the α-chain domain is located at the N-terminus of the ssTCR followed by a linker followed by the β-chain domain at the C-terminus of the ssTCR. However, the β-chain domain may alternatively be located at the N-terminus of the ssTCR, with the α-chain domain located at the C-terminus and a linker located between them.

The amino acid sequence of the linker may be of any suitable length. The linker sequence may contain 1-30 amino acids, or more preferably 1-25 or 1-20 amino acids. However, the linker should preferably be cleavable so that the two TCR strands can be separated from each other; if they cannot be separated from each other, then the two strands may not be able to assume the correct conformations and interact correctly, resulting in a non-functional TCR.

According to a preferred embodiment of the present invention, the linker is capable of autosplicing. An autosplicing linker can catalyze cleavage of the ssTCR molecule at the linker position, thereby separating the α- and β-chain domains. This splicing reaction does not require stimulation or induction, and the splicing reaction optimally occurs before transport of the α-chain and β-chain domains to the cell surface. As a result of the cleavage reaction, the linker can be completely excised from the TCR molecule; alternatively, the linker, or part of the linker, may remain attached to one or both of the finished individual TCR strands. The splicing reaction catalyzed by the linker may occur after translation (ie, may be an autocatalytic proteolysis reaction), or it may occur co-translationally. Co-translational splicing can occur by preventing the formation of a peptide bond in the linker or between the linker and one of the chain domains located on either side of it.

A preferred autosplicing linker is a linker derived from the self-cleaving peptide 2A of picornavirus. Peptides 2A contain approximately 20-25 amino acids and end in a conserved sequence motif Asp-Val/Ile-Glu-X-Asn-Pro-GlyPro (SEQ ID NO: 52). Peptides 2A undergo co-translational autosplicing by preventing the formation of a peptide bond between a conserved glycine residue and a terminal proline residue, resulting in efficient protein cleavage between these two amino acids. After cleavage, peptide 2A (with the exception of the C-terminal proline) remains attached to the C-terminus of the protein located before it; the terminal proline residue remains attached to the N-terminus of the protein located after it. A particularly preferred linker sequence derived from the 2A peptide is shown in SEQ ID NO: 18. However, the sequence of the peptide located upstream of the conserved C-terminal 2A sequence motif (SEQ ID NO: 52) can be altered without significant loss of autosplicing activity. Accordingly, the linker may also contain a sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the sequence shown in SEQ ID NO: 18, provided that it ends with the above-described conservative sequence motif and retains autosplicing activity. In particular, autosplicing variants of the sequence shown in SEQ ID NO: 18 can retain at least 70%, 75%, 80%, 85%, 90%, or 95% of the autosplicing activity of a peptide containing the sequence of SEQ ID NO: 18 .

The sequences CDR1, CDR2 and CDR3 from the α chain domain are derived from the sequences shown in SEQ ID NOs: 2, 3 and 4, respectively, while the sequences CDR1, CDR2 and CDR3 from the β chain domain are derived from the sequences shown in SEQ ID NO: 5, 6 and 7, respectively. In other words, CDR1, CDR2 and CDR3 from the α-chain domain contain the sequences of SEQ ID NOs: 2, 3 and 4, respectively, or contain variants of these sequences that have been modified by substitution, addition or deletion of 1 or 2 amino acids. CDR1, CDR2 and CDR3 from the β-chain domain contain the sequences of SEQ ID NOs: 5, 6 and 7, respectively, or contain variants of these sequences that have been modified by substitution, addition or deletion

- 8 042909 lectures of 1 or 2 amino acids. CDR variants are functionally equivalent to their respective native unmodified CDR siblings. By functional equivalence is meant that a protein or amino acid sequence (herein CDR) retains, or substantially retains, the function or activity of the protein or amino acid sequence (herein CDR) from or on which it ( she) is based (i.e., to which he (she) corresponds). In particular, a functionally equivalent variant may retain at least 70%, or more particularly at least 75%, 80%, 85%, 90% or 95% of the activity or function of the corresponding (unmodified) protein or amino acid sequence. In effect, this means that the CDR variant does not adversely affect, or substantially does not adversely affect, the function or activity or properties of the TCR in which it is present (compared to native or unmodified TCR, or compared to a TCR in which sections of the CDR are not modified). First of all, this means that the CDR variant does not adversely affect the specificity of TCR binding; TCR retains the ability to specifically bind to a peptide containing the sequence of SEQ ID NO: 1, when properly presented on the target cell. In addition, the binding affinity of TCR is essentially not reduced compared to native or unmodified TCR or TCR with unmodified CDR regions. However, TCR binding affinity can be improved by modifying CDR regions, in particular CDR 1 and/or 2.

Accordingly, CDR1 and CDR2 sequences modified (or mutated) as described above can have improved binding affinity for their target antigen without loss of binding specificity. Such mutated sequences can therefore be used in the treatment of cancers that produce a neopeptide containing the sequence of SEQ ID NO: 1. Such useful modified TCR sequences can be identified by screening libraries of TCR clones introduced with random mutations in their CDR1 and/or CDR2 regions. . Soluble TCR clones with improved binding affinity for their target antigens can be identified, for example, by surface plasmon resonance or by thermal fluctuation quantification. Insoluble TCR clones with improved binding affinity for their target antigens can be identified, for example, by functional assays in which cytokine release is assayed to monitor immune effector cell activation by TCR. However, according to a preferred embodiment of the present invention, all CDR1 and CDR2 sequences of both the α-chain domain and the β-chain domain are unmodified (i.e., CDRs 1 and 2 from the α-chain domain contain SEQ ID NOs: 2 and 3, respectively, and CDRs 1 and 2 from the β chain domain contain the sequences of SEQ ID NOs: 5 and 6, respectively).

The CDR3 sequences of the α-chain and β-chain domains preferably comprise the sequences of SEQ ID NOs: 4 and 7, respectively, and these sequences are preferably not altered or modified.

The full-length TCR of the present invention (i.e., the insoluble TCR that, when expressed by an immune effector cell, is localized on the cell surface), when expressed on the surface of an immune effector cell, such as a T cell, is able to redirect the cell on which it is expressed such that the cell recognizes a neoantigen containing the sequence of SEQ ID NO: 1 when presented with MHC class I. In other words, such a TCR according to the present invention activates an immune effector cell to direct its action or function, for example, its cytotoxic activity , against a target cell that has undergone a -1A frameshift mutation in TGFeRII, or in fact any similar mutation that results in the production of a neoantigen containing the sequence of SEQ ID NO: 1. of the invention may be any immune effector cell as discussed above and further below, however, in one preferred embodiment, said cell is a CD4+ T cell, CD8+ T cell, or any other type of T cell.

Soluble TCR according to the present invention is able to recognize, i. bind, a neoantigen containing the sequence of SEQ ID NO: 1, when presented with MHC class I, and thus undergo selective internalization by the target cell. Selective internalization of soluble TCR according to the present invention can be identified by any method known in the art. For example, soluble TCR can be conjugated to a fluorophore (eg, a fluorescent protein such as GFP) and therefore TCR internalization can be detected by fluorescence internalization. If soluble TCR is internalized by cells that express a neoantigen containing the sequence of SEQ ID NO: 1, but is not internalized (or internalized to a lower degree) by cells that do not express the sequence of SEQ ID NO: 1, it can be said that soluble TCR is selectively internalized by target cells . As described above, soluble TCRs contain both an α chain and a β chain, but each chain is truncated at its C-terminus by deletion of the transmembrane and intracellular constant region domains. Therefore, soluble TCR chains contain an N-terminal leader sequence (until it is cleaved off during polypeptide maturation), a variable region, and an N-terminus (ie, an extracellular domain) of a constant region. Accordingly, soluble TCR can be said to be truncated TCR with truncated α- and β-chains, while insoluble TCR (such as wild-type TCR) that is expressed on the surface of an immune effector cell, such as a T cell, can be called full-length TCR. with full-length α- and β-chains.

MHC class I, which is a neoantigen containing the sequence of SEQ ID NO: 1, TCR according to the present invention (TCR that is present in solution, i.e. soluble TCR, or a T cell expressing full-length TCR according to the present invention), may contain HLA-A2 allele of HLA-A. More specifically, MHC class I may contain the HLA-A*02:01 isoform of HLA-A2. However, class I MHC is not limited to MHC components containing the HLAA*02:01 isoform, or actually HLA-A2; it can contain any HLA protein that is capable of recognizing TCR. In particular, it may contain an HLA-A2 isoform other than the HLA-A*02:01 isoform. In other words, it may contain any isoform of HLA-A2.

As mentioned above, the CDRs from the α-chain or β-chain of TCR are located within the variable region of the chain. Each variable region contains 3 CDR sequences in a backbone of 4 framework sequences. Based on the CDR sequences alone, it is not possible to predict which framework sequences will hold the CDR sequences together in a functional TCR. As mentioned above, the amino acid sequences of the variable regions of the α- and β-chains of the Radium-1 TCR are shown in SEQ ID NOs: 8 and 13, respectively. However, some modifications to natural framework sequences can generally be made without adversely affecting TCR function. Accordingly, in the TCR of the present invention, the framework regions of the variable regions of the α-chain and/or β-chain domains may be similar to those of the native Radium-1 receptor (i.e., because it has been isolated or found in nature), but not necessarily . Accordingly, the framework regions of the variable regions of the Radium-1 receptor can be modified (for example, by substitution, addition, insertion or deletion of amino acids), including substitutions, for example, with mouse framework regions or framework regions containing fragments of the mouse sequence (respectively, the amino acid sequence of the framework sections can be modified and/or replaced).

According to one embodiment of the present invention, the TCR α chain domain comprises a variable region having, or containing, or consisting of, the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence that is at least 90%, 95%, 97% 98% or 99% identical to the specified amino acid sequence. In the event that the TCR α-chain domain contains a sequence that is a variant of the sequence shown in SEQ ID NO: 8 (i.e., a sequence that is at least 90%, 95%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO: 8, but which is not completely identical), the CDR sequences of the α-chain domain are the sequences according to the present invention, as defined above.

According to another embodiment of the present invention, the TCR β chain domain comprises a variable region having, or containing or consisting of, the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence that is at least 90%, 95%, 97% 98% or 99% identical to the specified amino acid sequence. In the event that the TCR β-chain domain contains a sequence that is a variant of the sequence shown in SEQ ID NO: 13 (i.e., a sequence that is at least 90%, 95%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO: 13, but which is not completely identical), then the CDR sequences of the β-chain domain are the sequences according to the present invention, as defined above.

Based on the foregoing, it follows that the variable regions of the native Radium-1 TCR can be modified. As noted above with respect to the discussion of CDR modifications, functionally equivalent variants of the Radium-1 TCR are included within the scope of the present invention. Such functionally equivalent variants of TCR or α-chain and/or β-chain domains or their variable and/or constant regions, in which the native amino acid sequence of Radium-1 TCR (or chain domain or region) has been modified, retain or substantially retain activity, property or function of a TCR, or chain domain or region, as discussed above. In particular, such a modified functionally equivalent TCR molecule, or a modified functionally equivalent chain domain, or region thereof, as applied to a TCR molecule, retains or substantially retains the activity of a TCR receptor, e.g., as above, retains at least 70% or more of the activity, e.g. , TCR activity aimed at recognition of the target cell (eg, cancer cell) and/or cytotoxic effect against the target cell.

As discussed above, adoptive cell transfer therapy may pose a threat

- 10 042909 for patient safety. As a protective mechanism, it is possible to encode a label in the TCR according to the present invention, which allows targeted killing of cells expressing full-length TCR. Targeted cell killing can be performed if the patient has a negative response to therapy. Targeted killing of cells can be performed using antibodies that recognize the introduced label. Such a mechanism is described in Kieback, E. et al, 2007, Proc. Natl. Acad. sci. USA, Vol. 105, pp. 623-628. According to one implementation variant of the present invention, the TCR contains a sequence of a well-known label. Examples of suitable labels are well known in the art and include the FLAG tag, polyhistidine tag (His tag), HA tag, Strep tag, S tag, and Myc tag. According to a preferred embodiment of the present invention, the TCR according to the present invention contains the Myc label. Several (ie, two or more, eg, 2 to 10, 2 to 8, or 2 to 6) preferably contiguous copies of the tag sequence may be present in the TCR. In a particularly preferred embodiment, the TCR contains a double Myc label. This double Myc tag has the amino acid sequence of SEQ ID NO: 19.

The label can be located in any chain of the TCR. In order to provide optimal access of the antibody to the label, it should preferably be located at the N-terminus of the TCR chain. According to one embodiment of the present invention, the α chain domain comprises a variable region that further comprises a double Myc tag with the amino acid sequence shown in SEQ ID NO: 19. In another embodiment of the present invention, the β chain domain comprises a variable region that further comprises a double a Myc tag with the amino acid sequence shown in SEQ ID NO: 19. According to a further embodiment of the present invention, both α-chain and β-chain domains comprise variable regions containing a double Myc tag.

As mentioned above, the synthesized N-terminus of the TCR chain is a signal peptide. Such a signal peptide is typically located between about 15 and about 30 amino acids. The signal peptide for the Radium-1 α chain is predicted to consist of the first 20 amino acids of SEQ ID NO: 8, which are represented by SEQ ID NO: 50. The Radium-1 β chain signal peptide is predicted to consist of the first 16 amino acids of the sequence SEQ ID NO: 13, which are represented by the sequence of SEQ ID NO: 51. As mentioned above, these leader sequences are absent in Mature TCR.

In order to place a tag at the N-terminus of the mature TCR chain, the tag sequence can be inserted into the variable domain region of the TCR chain immediately after the leader sequence. The label can be inserted into both the α chain domain and the β chain domain. According to a preferred embodiment of the present invention, a double Myc tag containing the sequence of SEQ ID NO: 19 is inserted into the variable region of the α-chain domain, with the sequence of SEQ ID NO: 8 located directly at the C-terminus with respect to the leader sequence shown in SEQ ID NO: 50. According to this embodiment of the present invention, the TCR α-chain variable region of the present invention has, or contains or consists of, the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence that is at least 90% 95%, 97%, 98% or 99% identical to the specified sequence. Where the α-chain variable region of the present invention has, or contains or consists of, a sequence that is a variant of the sequence shown in SEQ ID NO: 20 (i.e., a sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to, but not completely identical to, the indicated sequence), then the CDR sequences are the α-chain domain sequences of the present invention as defined above, and the Myc double tag sequence remains unchanged by compared to the sequence shown in SEQ ID NO: 19.

TCR with an α-chain domain containing a variable region having the amino acid sequence of SEQ ID NO: 20, as described above, remains functional, despite the fact that it may have a slight decrease in activity compared to TCR with an α-chain containing a variable region having the amino acid sequence of SEQ ID NO: 8. This option is preferred for use in therapy, since cells expressing TCR on their surface can be destroyed, if necessary (if the patient suffers a severe negative reaction to treatment), by injecting antibodies to the label Mus.

Both full-length and truncated (soluble) TCRs of the present invention may contain α-chain and/or β-chain domains with the variable regions described above.

As described above, both α- and β-chains of TCR contain variable and constant regions. The sequence of the Radium-1 TCR α-chain constant region is given in SEQ ID NO: 9, and the Radium-1 TCR β-chain constant region sequence is given in SEQ ID NO: 14. Like all full-length TCR chain constant regions, these regions contain an extracellular domain , a transmembrane helix, and a short intracellular domain (as mentioned earlier, the constant regions of the truncated

- 11 042909 chains of soluble TCR contain only the extracellular domain of the corresponding full-length sequence).

The full length TCR α chain domain constant region of the present invention may have, or contain or consist of, the Radium1 α chain constant region sequence. In other words, the α chain domain may contain a constant region having, i. e. containing or consisting of it, the sequence of SEQ ID NO: 9. According to another option, the α-chain domain may contain a constant region having a sequence similar to the sequence shown in SEQ ID NO: 9. In particular, the α-chain domain of TCR according to of the present invention may contain a constant region having, i.e. containing or consisting of it, a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the sequence shown in SEQ ID NO: 9. Accordingly, as above, a functionally equivalent version of the Radium-1 α-chain constant region with a modified sequence is included within the scope of the present invention.

The full length TCR α chain domain of the present invention may, in another embodiment, comprise a constant region that has, or contains or consists of, a mouse sequence equivalent to the Radium-1 α chain constant region. In other words, the α chain domain may comprise a constant region having a sequence that is a murinized variant of SEQ ID NO: 9. Accordingly, in such an α chain domain, a human constant region has been replaced with a mouse constant region.

A mouse TCR α-chain constant domain sequence that is equivalent to the Radium-1 TKR α-chain constant region sequence set forth in SEQ ID NO: 9 is SEQ ID NO: 23. In a specific embodiment of the present invention, wherein the constant region α chain domain is murinized, said murinized constant region has, or contains or consists of, the amino acid sequence of SEQ ID NO: 23. According to another embodiment of the present invention, the murinized constant region has, or contains or consists of, a sequence that at least 95% identical to the sequence shown in SEQ ID NO: 23.

The β chain domain constant region of the full length TCR according to the present invention may have, or contain or consist of, the Radium1 β chain constant region sequence. In other words, the β-strand domain may contain a constant region having, i.e. containing or consisting of the sequence of SEQ ID NO: 14. According to another option, the β-chain domain may contain a constant region, the sequence of which is similar to the sequence shown in SEQ ID NO: 14. In particular, the TCR β-chain domain according to the present of the invention may contain a constant region having, i.e. containing or consisting of it, a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the sequence shown in SEQ ID NO: 14. Accordingly, as above, a functionally equivalent version of the Radium-1 β-chain constant region with a modified sequence is included within the scope of the present invention.

The full length TCR β chain domain of the present invention may, in another embodiment, comprise a constant region that has, or contains or consists of, a murine sequence equivalent to the Radium-1 β chain constant region. In other words, the β chain domain may comprise a constant region having a sequence that is a murinized variant of SEQ ID NO: 14. Accordingly, in such a β chain domain, a human constant region has been replaced with a mouse constant region.

The mouse TCR β chain domain constant region sequence, which is equivalent to the Radium-1 TCR β chain domain constant region sequence set forth in SEQ ID NO: 14, is SEQ ID NO: 29. According to a particular embodiment of the present invention, wherein β-chain domain constant region is murinized, said murinized constant region has, or contains or consists of, the amino acid sequence of SEQ ID NO: 29. According to another embodiment of the present invention, the murinized constant region has, or contains or consists of, a sequence that at least 95% identical to the sequence shown in SEQ ID NO: 29.

In the TCR according to the present invention, although the α-chains and β-chains can be encoded as a single polypeptide chain (ie as ssTCR), in their mature form they form separate chains. The α- and β-chains are each encoded as separate polypeptides, or they are encoded as ssTCR, in which case they are connected by a linker that is cleaved prior to their maturation and transport to the cell membrane. This means that in mature TCR according to the present invention, the α-chain and β-chain form discrete polypeptide chains: i.e. they are no longer linked by peptide bonds.

In all mature αβ-TCR, the α-chain and β-chain are covalently linked by interchain disulfide bonds.

- 12 042909 bonds that form between cysteine residues located in the constant regions of each chain. Interchain disulfide bonds ensure that the two TCR chains are closely linked after TCR formation, which is of great importance for TCR functionality. When TCR is exogenously expressed in a T cell, there is a risk that exogenously encoded TCR chains will form a complex with endogenously encoded TCR chains, leading to the formation of mixed TCR containing an exogenously encoded α-chain and an endogenously encoded β-chain. , or vice versa. In the context of the present invention, this means that the α chain of the present invention (such as the α chain of Radium-1) can complex TCR with the TKR β chain encoded by the T cell in which it is expressed, or vice versa. In such a situation, the activity of the TCR according to the present invention (such as TCR Radium-1) can be reduced compared to the situation in which the TCR chains according to the present invention complex only with each other.

It has been found that it is possible to stimulate preferential pairing of α-chain and β-chain by introducing an additional cysteine residue in the constant regions of the TCR α-chain and β-chain domains according to the present invention. This has been shown to enhance TCR expression and function in some T cells. Accordingly, the α chain domain constant region and/or the β chain domain constant region can be modified by introducing a cysteine residue. Preferably, the constant region domains of both the α chain and the β chain are modified by introducing a cysteine residue. An additional cysteine residue can be introduced by insertion (i.e. by inserting an additional amino acid residue into the constant region of the α-chain or β-chain domain) or by substitution (i.e. by replacing with cysteine an amino acid other than cysteine already present in constant region of the α-chain or β-chain domain). If a cysteine residue is to be introduced into the constant regions of both the α-chain and β-chain domains, then different methods of introducing cysteine can be used for each chain domain. TCR chains or chain domains according to the present invention into which an additional cysteine residue has been introduced may be referred to as modified with cysteine.

According to a preferred embodiment of the present invention, the α chain constant region of the full-length TCR has a cysteine-modified α chain constant region sequence, Radium-1. According to a particularly preferred embodiment of the present invention, the Radium-1 α-chain constant region is modified by replacing T48C. In other words, threonine 48 in the sequence shown in SEQ ID NO: 9 is replaced by cysteine. The sequence of the constant region of the α chain domain, consisting of SEQ ID NO: 9 with the substitution T48C, is given in SEQ ID NO: 10, therefore, according to a particularly preferred embodiment of the present invention, the α chain domain contains a constant region having, i.e. containing or consisting of it, the sequence of SEQ ID NO: 10. According to another option, the α-chain domain may contain a constant region having, ie. containing or consisting of it, an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the sequence shown in SEQ ID NO: 10, provided that the cysteine residue at position 48 (or at the position corresponding to position 48 in the sequence shown in SEQ ID NO: 10) remains unchanged.

According to another preferred embodiment of the present invention, the full-length TCR β-chain constant region has a cysteine-modified β-chain constant region sequence Radium-1. According to a particularly preferred embodiment of the present invention, the Radium-1 β-chain constant region is modified by replacing S57C. In other words, series 57 in the sequence shown in SEQ ID NO: 14 is replaced by cysteine. The sequence of the constant region of the β-chain domain, consisting of SEQ ID NO: 14 with the substitution S57C, is given in SEQ ID NO: 15, therefore, according to a particularly preferred embodiment of the present invention, the β-chain domain contains a constant region having, i.e., containing or consisting of it, the sequence of SEQ ID NO: 15. According to another option, the β-chain domain may contain a constant region having, i. containing or consisting of it, an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the sequence shown in SEQ ID NO: 15, provided that the cysteine residue at position 57 (or at the position corresponding to position 57 in the sequence shown in SEQ ID NO: 15) remains unchanged.

In embodiments in which one or both of the TCR chain domains contain a murinized constant region, said murinized constant region may be modified with cysteine. The preferred sequence of the murine constant region and modified with cysteine from the α-chain domain of the full-length TCR according to the present invention is shown in SEQ ID NO: 24. The sequence of SEQ ID NO: 24 is a cysteine-modified variant of the sequence of SEQ ID NO: 23. The sequence of SEQ ID NO: 24 is obtained by replacing the residue in the murine sequence of SEQ ID NO: 23, which is equivalent to threonine 48 in the human sequence of SEQ ID NO: 9, with a cysteine residue. Accordingly, the domain of the α chain is mo

- 13 042909 may contain a cysteine-modified murine constant region having, i. containing or consisting of it, the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence that is at least 95% identical to the specified sequence in which the cysteine residue at position 47 (or at the position corresponding to position 47 in the sequence shown in SEQ ID NO: 24) remains unchanged.

The preferred sequence of the cysteine-modified murine constant region, from the full-length TCR β-chain domain of the present invention, is shown in SEQ ID NO: 30. The sequence of SEQ ID NO: 30 is a cysteine-modified variant of the sequence shown in SEQ ID NO: 29. The sequence of SEQ ID NO: 30 is obtained by replacing the residue in the murine sequence shown in SEQ ID NO: 29, which is equivalent to serine 57 in the human sequence shown in SEQ ID NO: 14, with a cysteine residue. Accordingly, the β-chain domain may comprise a cysteine-modified murinized constant region having, i. containing or consisting of the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence that is at least 95% identical to the specified sequence in which the cysteine residue at position 56 (or at the position corresponding to position 56 in the sequence shown in SEQ ID NO: 30) remains unchanged.

As described herein, each of the α-chain and β-chain domains in the full-length TCR according to the present invention may contain or consist of a variable region and a constant region, with the sequences of the variable and constant regions defined and described above. Various variable and constant region sequences can be combined in any combination possible, with the most preferred combination of α-chain variable region sequences with α-chain constant region sequences, and combination of β-chain variable region sequences with β-chain constant region sequences.

In one embodiment of the full-length TCR of the present invention, the α-chain domain comprises a variable region and a constant region with the corresponding variable and constant region sequences of the Radium-1 TCR α-chain. In this embodiment, the α chain domain of the present invention may have the α chain sequence of TCR Radium-1 as set forth in SEQ ID NO: 11. Accordingly, in one embodiment of the present invention, the α chain domain has, or contains, or consists of her, the amino acid sequence of SEQ ID NO: 11.

As detailed above, the α-chain domain may contain a label in its variable domain. Such a tag is preferably located directly at the C-terminus of the α chain domain leader so that it forms the N-terminal end of the mature chain domain. The preferred label is a double Myc label. The Radium-1 TCR α-chain with a double Myc tag inserted directly at the C-terminus with respect to the leader sequence has the sequence of SEQ ID NO: 21. Accordingly, according to another embodiment of the present invention, the full-length TCR α-chain domain has, or contains or consists of it, the sequence of SEQ ID NO: 21. According to another option, the α-chain domain of the full-length TCR may have, or contain or consist of, an amino acid sequence that is at least 90% or 95% identical to the sequence shown in SEQ ID NO: 11 or SEQ ID NO: 21, provided that the CDR sequences (and double Myc tag sequence, when present) are those defined above.

As described above, the constant region of the α chain domain according to the present invention may alternatively be murine: in particular, it may be a murine (or murinized) variant of the sequence shown in SEQ ID NO: 9, however, it may be any other constant region mouse TCR α-chains. If the constant region of the α-chain domain is murinized, the variable region may also be murinized (in particular, the framework regions of the variable domain may be murinized), but this is not required. According to a preferred embodiment of the present invention, if the α chain domain constant region is murinized, the variable region is a human variable region. According to preferred embodiments of the present invention, if the full-length TCR α chain domain constant region is murine, it contains the sequence of SEQ ID NO: 23, while the variable region is the Radium-1 α chain variable region. The full length TCR α chain sequence with the Radium-1 α chain variable region and constant region having the sequence of SEQ ID NO: 23 is shown in SEQ ID NO: 25. In one embodiment of the present invention, the TCR α chain domain comprises a murinated constant region and human variable region, and has, or contains or consists of, the sequence of SEQ ID NO: 25.

According to another embodiment of the present invention, the full-length domain of the α chain

- 14 042909 contains a mouse constant region and a variable region that contains a tag, preferably a double Myc tag, preferably directly at the C-terminus relative to the leader sequence. According to this embodiment of the present invention, the constant domain preferably contains the sequence of SEQ ID NO: 23, and the variable domain contains the sequence of SEQ ID NO: 20 (SEQ ID NO: 20 is the variable region of the Radium-1 α-chain with the double Myc tag inserted directly at the C-terminus of the leader sequence). The sequence of the α-chain domain, consisting of the mouse constant region, the sequence of which is shown in SEQ ID NO: 23, and the variable region, the sequence of which is shown in SEQ ID NO: 20, is shown in SEQ ID NO: 27. Accordingly, the α-chain domain of the full-length TCR according to the present invention may have, or contain or consist of, the sequence of SEQ ID NO: 27. % is identical to any of the sequences shown in SEQ ID NO: 25 or SEQ ID NO: 27, provided that the CDR sequences (and the double Myc label sequence, when present) are those defined above.

As described above, the constant regions of the α-chain and β-chain domains can be modified with cysteine to increase the specificity of the interaction of the α-chain with the β-chain of TCR according to the present invention. Such a modified constant region can be paired with any variable region according to the present invention: for example, a cysteine modified constant domain can be paired with a variable region containing a double Myc tag, but this is not required. Accordingly, the α-chain domain may contain a constant region that has been modified with cysteine. Preferred cysteine-modified α-chain domain constant regions have been described above: the full-length cysteine-modified α-chain constant domain Radium-1 contains the sequence of SEQ ID NO: 10, while the cysteine-modified murine equivalent of SEQ ID NO: 10 is shown in SEQ ID NO: 24.

According to preferred embodiments of the present invention, the full-length α chain domain comprises the Radium-1 α-chain variable domain, the sequence of which is given in SEQ ID NO: 8, and a cysteine-modified Radium-1 constant domain, the sequence of which is given in SEQ ID NO: 10, or modified cysteine murinized constant domain, the sequence of which is given in SEQ ID NO: 24. The α-chain domain, which consists of a variable domain, the sequence of which is given in SEQ ID NO: 8, and a constant domain, the sequence of which is given in SEQ ID NO: 10, has the sequence of SEQ ID NO: 12, and an α-chain domain, which consists of a variable domain, the sequence of which is shown in SEQ ID NO: 8, and a constant domain, the sequence of which is shown in SEQ ID NO: 24, has the sequence of SEQ ID NO: 26. Accordingly, according to a preferred embodiment of the present invention, the full-length α-chain domain has, or contains or consists of, the amino acid sequence of SEQ ID NO: 12, or, according to another option, the α-chain domain may have, or contain or consist of her, an amino acid sequence that is at least 90% or 95% identical to the specified sequence, provided that the CDR sequence and modification with cysteine are those defined above. According to another preferred embodiment of the present invention, the α-chain domain has, or contains or consists of, the sequence of SEQ ID NO: 26, or, according to another embodiment, the α-chain domain may have, or contains or consists of, an amino acid sequence that, according to at least 95% identical to the specified sequence, provided that the CDR sequences and modification with cysteine are those defined above.

According to more preferred embodiments of the present invention, the full-length α-chain domain comprises a Radium-1 α-chain variable domain that has been modified by the insertion of a label, preferably a double Myc label, such as that shown in SEQ ID NO: 20. According to the most preferred embodiments of the present invention, the α-chain domain comprises a variable region, the sequence of which is shown in SEQ ID NO: 20, and a cysteine-modified constant domain Radium-1, the sequence of which is given in SEQ ID NO: 10, or a cysteine-modified murine constant domain, the sequence of which is is shown in SEQ ID NO: 24. The α-chain domain, which consists of the variable region, the sequence of which is shown in SEQ ID NO: 20, and the constant domain, the sequence of which is shown in SEQ ID NO: 10, has the sequence of SEQ ID NO: 22 , and the α chain domain, which consists of a variable domain whose sequence is shown in SEQ ID NO: 20 and a constant domain whose sequence is shown in SEQ ID NO: 24, has the sequence of SEQ ID NO: 28.

Accordingly, according to the most preferred embodiment of the present invention, the full-length α-chain domain has, or contains or consists of, the sequence of SEQ ID NO: 2, or, according to another option, the α-chain domain may have, or contain or consist of,

- 15 042909 an amino acid sequence that is at least 90% or 95% identical to the specified sequence, provided that the CDR sequences, the Myc double tag sequence and the cysteine modification are as defined above. According to another most preferred embodiment of the present invention, the α chain domain has, or contains or consists of, the sequence of SEQ ID NO: 28, or, according to another embodiment, the α chain domain may have, or contains or consists of, an amino acid sequence that at least 95% identical to the specified sequence, provided that the CDR sequences, the double Myc tag sequence, and the cysteine modification are as defined above.

Similar to the α-chain domain, according to one embodiment of the present invention, the full-length β-chain domain contains a variable region and a constant region with the corresponding sequences of the variable and constant regions of the Radium-1 TCR β-chain. According to this embodiment of the present invention, the β-chain domain of the present invention may comprise the β-chain sequence of TCR Radium-1 as set forth in SEQ ID NO: 16. Accordingly, in one embodiment of the present invention, the β-chain domain has, or contains, or consists of it, the amino acid sequence of SEQ ID NO: 16. According to another option, the β-chain domain may have, or contain or consist of, an amino acid sequence that is at least 90% or 95% identical to the sequence shown in SEQ ID NO: 16, provided that the CDR sequences are those defined above.

Similar to the α-chain domain, the constant region of the full-length β-chain domain according to the present invention may alternatively be murine: in particular, it may be a murine (or may be murinized) variant of the sequence shown in SEQ ID NO: 14, however, it may be any other mouse TCR β-chain constant region. If the constant region of the β-chain domain is murinated, the variable region may also be murinated (in particular, the framework regions of the variable domain may be murinized), but this is not required. According to a preferred embodiment of the present invention, if the constant region of the β chain domain is murinated, the variable region is a human variable region. According to preferred embodiments of the present invention, if the constant region of the full-length TCR β-chain is murine, it contains the sequence of SEQ ID NO: 29, while the variable region is the Radium-1 β-chain variable region. The sequence of the TKR β chain with the Radium-1 β chain variable region and a constant region containing the sequence of SEQ ID NO: 29 is shown in SEQ ID NO: 31. In one embodiment of the present invention, the TCR β chain domain comprises a murinized constant region, and a human variable region, and has, or contains or consists of, the sequence of SEQ ID NO: 31. According to another option, the β-chain domain may have, or contain or consist of, an amino acid sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 31, provided that the CDR sequences are those defined above.

Similar to the α-chain domain, the full-length β-chain domain may contain a constant region that has been modified with cysteine. Preferred cysteine-modified β-chain domain constant regions have been described above: The cysteine-modified Radium-1 β-chain constant domain contains the sequence of SEQ ID NO: 15, while the cysteine-modified murinized equivalent of SEQ ID NO: 15 is given in SEQ ID NO: thirty.

According to preferred embodiments of the present invention, the full-length β-chain domain comprises the Radium-1 β-chain variable domain, the sequence of which is given in SEQ ID NO: 13, and the cysteine-modified Radium-1 constant domain, the sequence of which is given in SEQ ID NO: 15, or a cysteine-modified murinized constant domain, the sequence of which is given in SEQ ID NO: 30. A β-chain domain, which consists of a variable domain, the sequence of which is given in SEQ ID NO: 13, and a constant domain, the sequence of which is given in SEQ ID NO: 15 has the sequence of SEQ ID NO: 17, and the β-chain domain, which consists of the variable domain, the sequence of which is shown in SEQ ID NO: 13, and the constant domain, the sequence of which is shown in SEQ ID NO: 30, has the sequence of SEQ ID NO: 32. Accordingly, according to a preferred embodiment of the present invention, the full-length β-chain domain has, or contains or consists of, the sequence of SEQ ID NO: 17, or, according to another embodiment, the β-chain domain may have, or contain or consist of of it, an amino acid sequence that is at least 90% or 95% identical to said sequence, provided that the CDR sequences and the cysteine modification are those defined above. According to another preferred embodiment of the present invention, the β-chain domain has, or contains or consists of, the sequence of SEQ ID NO: 32, or, according to another embodiment, the β-chain domain may have, or

- 16 042909 contain or consist of an amino acid sequence that is at least 95% identical to the specified sequence, provided that the CDR sequences and the cysteine modification are those defined above.

As detailed above, the β-chain domain may contain a label in its variable domain. Such a tag is preferably located directly at the C-terminus of the β-chain domain leader so that it forms the furthest portion of the N-terminus of the mature chain domain. The preferred label is the double label Myc.

In the soluble TCR of the present invention, the constant regions of the α-chain and β-chain domains may correspond to truncated versions of the full-length constant regions described above. According to a specific embodiment of the present invention, the truncated α-chain domain constant region corresponds to amino acids 1-95 of the Radium-1 α-chain constant region (i.e., amino acids 1-95 in the sequence shown in SEQ ID NO: 9). The specified sequence is shown in SEQ ID NO: 60. The soluble TCR according to the present invention may contain an α-chain domain containing a constant region containing or consisting of the amino acid sequence shown in SEQ ID NO: 60, or from an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the specified sequence. According to another embodiment of the present invention, the truncated β-chain domain constant region corresponds to amino acids 1-131 of the Radium-1 β-chain constant region (i.e., amino acids 1-131 in the sequence shown in SEQ ID NO: 14). The specified sequence is shown in SEQ ID NO: 62. The soluble TCR according to the present invention may contain a β-chain domain containing a constant region containing or consisting of the amino acid sequence shown in SEQ ID NO: 62, or from an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the specified sequence.

An essential aspect of soluble TCR is that the α-chain and β-chain of mature TCR are connected. If they are not connected, the chains will diffuse from each other in solution and TCR functionality will be insufficient or absent. Chains can be linked covalently or non-covalently. A preferred method by which an α chain and a β chain can be covalently linked involves the formation of one or more disulfide bonds. Such bonds can form between cysteine residues present in native TCR chains, however, according to a preferred embodiment of the present invention, one or more cysteine residues are introduced into the constant regions of each chain, between which disulfide bonds can form. For full-length TCR, according to a preferred embodiment of the present invention, the constant regions of the α-chain and β-chain domains of soluble TCR are modified with cysteine. For full-length TCR chains, each chain can be modified by inserting a cysteine residue or by replacing the native residue with a cysteine residue.

According to a particularly preferred embodiment of the present invention, the truncated Radium-1 α-chain constant region (ie, the sequence shown in SEQ ID NO: 60) is modified by replacing T48C. The sequence of such a cysteine-modified truncated constant region of the α-chain domain is shown in SEQ ID NO: 61 (and corresponds to amino acids 1-95 in the sequence shown in SEQ ID NO: 10, which shows the cysteine-modified sequence of the full-length α-chain constant region of Radium- 1).

Accordingly, according to a preferred embodiment of the present invention, the soluble TCR according to the present invention comprises an α-chain domain containing a constant region containing or consisting of the amino acid sequence shown in SEQ ID NO: 61, or from an amino acid sequence that is at least 60% , 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the specified sequence. In case the α-chain domain is a variant of the sequence shown in SEQ ID NO: 61 (i.e. it has a sequence that is at least 60% but less than 100% identical to the sequence shown in SEQ ID NO: 61), the amino acid at position 48 (or the position corresponding to position 48 in the sequence shown in SEQ ID NO: 61) is cysteine.

According to another preferred embodiment of the present invention, the truncated Radium-1 β-chain constant region (ie, the sequence shown in SEQ ID NO: 62) is modified with the S57C substitution. The sequence of such a cysteine-modified truncated constant region of the β-chain domain is given in SEQ ID NO: 63 (and corresponds to amino acids 1-131 in SEQ ID NO: 15, which shows the cysteine-modified sequence of the full-length β-chain constant region of Radium-1). Accordingly, according to a preferred embodiment of the present invention, the soluble TCR according to the present invention comprises a β-chain domain containing a constant region containing or consisting of the amino acid sequence shown in SEQ ID NO: 63, or from an amino acid sequence that is at least 60% , 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to specified

- 17 042909 sequence. In the event that the α-chain domain is a variant of the sequence shown in SEQ ID NO: 63 (i.e. it has a sequence that is at least 60% but less than 100% identical to the sequence shown in SEQ ID NO: 63), the amino acid at position 48 (or the position corresponding to position 48 in the sequence shown in SEQ ID NO: 63) is cysteine.

Another way of connecting the α-chain and β-chain of soluble TCR involves non-covalent interactions. According to a specific embodiment of the present invention, leucine zippers are used to non-covalently join the chains. According to this embodiment of the present invention, both the α-strand and the β-strand contain leucine zipper domains at the C-terminus of their truncated constant regions (i.e., the α-strand contains a leucine zipper domain at its C-terminus, and the β-strand contains a leucine zipper domain at its C-terminus). Leucine zippers and their sequences are well known in the art and are discussed in, for example, Busch & Sassone-Corsi (1990), Trends Genet 6: 36-40. According to some embodiments of the present invention, both covalent and non-covalent methods can be used to connect the α chain and β chain of soluble TCR, for example, the α chain and β chain can be modified with cysteine and can contain leucine zipper domains.

The soluble TCR of the present invention may suitably contain an α chain domain corresponding to a truncated Radium-1 α chain lacking the 46 C-terminal amino acids. Such a truncated α chain has the sequence of SEQ ID NO: 64 (corresponding to residues 1222 in the sequence shown in SEQ ID NO: 11). If the constant region is modified with cysteine as described above (i.e., the constant region contains a Thr^Cys substitution compared to the wild-type sequence, corresponding to the T175C substitution in the sequence shown in SEQ ID NO: 64), then the truncated α-chain has the sequence of SEQ ID NO: 65. According to a preferred embodiment of the present invention, the domain of the α-chain of soluble TCR contains or consists of the sequence shown in SEQ ID NO: 64 or SEQ ID NO: 65, or from an amino acid sequence that is at least 90 % or 95% identical to the specified sequence. In case the α-strand contains or consists of the sequence variant shown in SEQ ID NO: 64 or 65, the CDR sequences are those defined above, and in the case of the sequence variant shown in SEQ ID NO: 65, the remainder at position 175 (or at the position corresponding to position 175 in the sequence shown in SEQ ID NO: 65) is a cysteine.

The soluble TCR .beta. chain domain of the present invention may correspond to a truncated Radium-1 .beta. chain lacking the 48 C-terminal amino acids. Such a truncated β chain has the sequence of SEQ ID NO: 66 (corresponding to residues 1-262 in the sequence shown in SEQ ID NO: 16). If the constant region is modified with cysteine as described above (i.e., the constant region contains a Ser^Cys substitution compared to the wild-type sequence, corresponding to the S188C substitution in the sequence shown in SEQ ID NO: 66), the truncated β-strand has the sequence SEQ ID NO: 67. According to a preferred embodiment of the present invention, the soluble TCR β-chain domain contains or consists of the sequence shown in SEQ ID NO: 66 or SEQ ID NO: 67, or an amino acid sequence that is at least 90% or 95% identical to the specified sequence. In case the α-strand contains or consists of the sequence variant shown in SEQ ID NO: 66 or 67, the CDR sequences are those defined above, and in the case of the sequence variant shown in SEQ ID NO: 67, the remainder at position 188 (or at the position corresponding to position 188 in the sequence shown in SEQ ID NO: 67) is a cysteine.

In some embodiments of the present invention, the purification label described above is encoded at the C-terminus of the α-chain or β-chain domain. The preferred label is a hexahistidine (His) label. The label may be attached to the C-terminus of the α-chain or β-chain with a linker such as a short Gly-Gly-Gly linker.

In some embodiments of the present invention, the TCR contains only one of the α-chain and β-chain domains described above. For example, it may contain an α-chain domain according to the present invention, but a β-chain domain, which is not included in the scope of the present invention; it may contain a β-chain domain according to the present invention, but an α-chain domain, which is not included in the scope of the present invention. Alternatively, the TCR molecule of the present invention may contain only the α-chain domain of the present invention or only the β-chain domain of the present invention. However, preferably the TCR molecule contains both the α-chain domain of the present invention and the β-chain domain of the present invention.

In the case where the TCR according to the present invention contains both the α-chain domain according to the present invention and the β-chain domain according to the present invention, it may contain any combination of the above-described α-chain and β-chain domains according to the present invention. For example

- 18 042909 measures, an α-chain domain containing a human constant region can be paired with a β-chain containing a mouse constant region, and vice versa. It is generally preferred to pair similar chains such that, for example, an α-chain domain containing a human constant region is paired with a β-chain domain containing a human constant region, and vice versa; an α-chain domain containing a mouse constant region is paired with a β-chain domain containing a mouse constant region, and vice versa; or an α-chain domain containing a constant region that has been modified with cysteine is paired with a β-chain domain containing a constant region that has been modified with cysteine, and vice versa.

As described above, according to preferred embodiments of the present invention, TCR is encoded as ssTCR, in which the C-terminus of the α-chain domain is connected to the N-terminus of the β-chain domain by a linker. The linker must be cleavable: preferably the linker is an autosplicing linker, such as a linker derived from picornavirus peptide 2A. Most preferably, the linker contains the sequence of SEQ ID NO: 18, or a variant thereof.

The full-length TCR according to the present invention may be encoded as ssTCR. According to one such embodiment of the present invention, the ssTCR comprises an α-chain domain with a Radium-1 α-chain sequence and a β-chain domain with a Radium-1 β-chain sequence linked by a linker sequence shown in SEQ ID NO: 18. Such ssTCR has the amino acid sequence of SEQ ID NO: 33.

According to preferred embodiments of the present invention, the constant domain domains of both the α chain and the β chain of ssTCR having the sequence of SEQ ID NO: 33 are modified with cysteine. As described above, a preferred sequence of the cysteine-modified α-chain domain of Radium-1 is SEQ ID NO: 12, and a preferred sequence of the cysteine-modified domain of the β-chain of Radium-1 is SEQ ID NO: 17. ssTCR containing a cysteine-modified α domain -chain of Radium-1, the sequence of which is given in SEQ ID NO: 12, and a cysteine-modified domain of the β-chain of Radium-1, the sequence of which is given in SEQ ID NO: 17, connected by a linker containing the sequence of SEQ ID NO: 18, has an amino acid the sequence of SEQ ID NO: 34.

According to another preferred embodiment, in particular of the full-length TCR according to the present invention, the variable domain region of the α-chain and/or β-chain of the TCR according to the present invention contains a tag sequence, preferably a double Myc tag. Preferably the variable region of only one of the α chain or β chain domains contains a tag sequence, most preferably the variable region of the α chain domain. As described above, the preferred full length α chain domain of the present invention is a domain containing the Radium-1 α chain sequence with a double Myc label inserted directly at the C-terminus of its leader sequence shown in SEQ ID NO: 21. Containing a double the label Myc α-chain domain, the sequence of which is given in SEQ ID NO: 21, can preferably be combined with the full-length β-chain domain of Radium-1 (which has the sequence of SEQ ID NO: 16). An ssTCR comprising a Myc double-labeled α-chain domain, the sequence of which is given in SEQ ID NO: 21, and a Radium-1 β-chain domain, the sequence of which is given in SEQ ID NO: 16, connected by a linker, the sequence of which is given in SEQ ID NO: 21 NO: 18, has the amino acid sequence of SEQ ID NO: 35.

According to another preferred embodiment, in particular the full-length TCR of the present invention, the variable region of the α-chain domain contains a double Myc tag and the constant regions of both the α-chain and β-chain domains are modified with cysteine. As described above, the preferred sequence of the full-length α-chain domain, which contains the double-labeled Myc variable region and the cysteine-modified constant region, is the amino acid sequence of SEQ ID NO: 22. SEQ ID NO: 22 may preferably be combined with a full-length cysteine-modified β-chain domain, the sequence of which is given in SEQ ID NO: 17. ssTCR containing a double-labeled Myc and a cysteine-modified α-chain domain, the sequence of which is given in SEQ ID NO: 22 and a cysteine-modified β-chain domain, the sequence of which is given in SEQ ID NO: 17, connected by a linker, the sequence of which is given in SEQ ID NO: 18, has the amino acid sequence of SEQ ID NO: 36.

Accordingly, according to one embodiment of the present invention, the TCR molecule is an ssTCR whose amino acid sequence is shown in SEQ ID NO: 33, or, according to another embodiment, the ssTCR may have an amino acid sequence that is at least 90% or 95% identical to the sequence, shown in SEQ ID NO: 33, provided that the CDR sequences are as defined above and the linker derived from peptide 2A retains its autosplicing capability.

- 19 042909

According to a preferred embodiment of the present invention, the TCR molecule is an ssTCR whose amino acid sequence is shown in SEQ ID NO: 34, or, in another embodiment, the ssTCR may have an amino acid sequence that is at least 90% or 95% identical to the sequence shown in SEQ ID NO: 34. SEQ ID NO: 34, provided that the CDR sequences and cysteine modifications are as defined above and the 2A peptide derived linker retains its autosplicing capability.

According to another preferred embodiment of the present invention, the TCR molecule is an ssTCR, the amino acid sequence of which is given in SEQ ID NO: 35, or, according to another variant, the ssTCR may have an amino acid sequence that is at least 90% or 95% identical to the sequence shown in SEQ ID NO: 35, provided that the CDR sequences and the double Myc tag sequence are as defined above and the linker derived from peptide 2A retains its autosplicing capability.

According to another preferred embodiment of the present invention, the TCR molecule is an ssTCR with the amino acid sequence shown in SEQ ID NO: 36, or, according to another embodiment, the ssTCR may have an amino acid sequence that is at least 90% or 95% identical to the sequence shown in SEQ ID NO: 36, provided that the sequences of the CDRs, the cysteine modifications, and the double Myc tag sequence are as defined above, and the linker derived from peptide 2A retains its autosplicing capability.

As discussed above, the constant regions of the α-chain and/or β-chain domains of the present invention can be murinized. A preferred full length α chain domain containing the mouse constant region has the sequence SEQ ID NO: 25, and a preferred full length β chain domain containing the mouse constant region has the sequence SEQ ID NO: 31. which is shown in SEQ ID NO: 25, and the β-chain domain, the sequence of which is shown in SEQ ID NO: 31, connected by a linker, the sequence of which is shown in SEQ ID NO: 18, has the sequence of SEQ ID NO: 37.

As described above, according to a preferred embodiment of the present invention, both the α chain and β chain domain constant regions are modified with cysteine. A preferred sequence of the full-length α-chain domain containing a cysteine-modified murinized constant region is shown in SEQ ID NO: 26, and a preferred sequence of a full-length β-chain domain containing a cysteine-modified murinized constant region is shown in SEQ ID NO: 32. ssTCR containing an α-chain domain whose sequence is shown in SEQ ID NO: 26 and a β-chain domain whose sequence is shown in SEQ ID NO: 32 connected by a linker whose sequence is shown in SEQ ID NO: 18 have the sequence of SEQ ID NO: 38.

As described above, according to another preferred embodiment of the present invention, the variable region of the α-chain domain contains a double Myc tag. The preferred sequence of the full-length α chain domain containing the double-tagged Myc variable region and the murinized constant region is shown in SEQ ID NO: 27. ID NO: 27, preferably can be combined with a full-length β-chain domain containing a murinized constant region, the sequence of which is given in SEQ ID NO: 31. ssTCR, containing an α-chain domain, the sequence of which is given in SEQ ID NO: 27, and the β-chain domain, the sequence of which is given in SEQ ID NO: 31, connected by a linker, the sequence of which is given in SEQ ID NO: 18, has the sequence of SEQ ID NO: 39.

According to another preferred embodiment of the present invention, the α-chain domain contains a variable region containing a double Myc tag, and the constant regions of both the α-chain and β-chain domains are modified with cysteine. A preferred full-length α-chain domain that contains a double-labeled Myc variable region and a cysteine-modified murinized constant region has the sequence of SEQ ID NO: 28. which is given in SEQ ID NO: 28, can preferably be combined with a full-length β-chain domain containing a cysteine-modified murinized constant region, the sequence of which is given in SEQ ID NO: 32. ssTCR containing an α-chain domain, the sequence of which is given in SEQ ID NO: 28, and a β chain domain whose sequence is shown in SEQ ID NO: 32 connected by a linker whose sequence is shown in SEQ ID NO: 18 has the sequence of SEQ ID NO: 40.

Accordingly, according to specific embodiments of the present invention, the molecule

- 20 042909

TCR is an ssTCR whose amino acid sequence is shown in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40. Alternatively, an ssTCR may have an amino acid sequence that is at least 90 % or 95% identical to one of the sequences shown in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40, provided that the CDR sequences and double Myc label, in those cases when present and/or cysteine modifications are as defined above and the 2A peptide derived linker retains its autosplicing capability.

The ssTCR polypeptides whose sequences are shown in SEQ ID NOs: 33-40 are encoded by the nucleotide sequences shown in SEQ ID NOs: 41-48, respectively. The nucleic acid molecule of the present invention is a nucleic acid molecule of the present invention that encodes a TCR molecule of the present invention. Accordingly, the nucleic acid molecule according to the present invention may contain a nucleotide sequence that encodes any TCR molecule according to the present invention, as defined above. A nucleic acid molecule according to the present invention that encodes ssTCR with α-chain and β-chain domains that contain human constant regions may specifically comprise the nucleotide sequence of any of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 44. A nucleic acid molecule according to the present invention that encodes ssTCR with α-chain and β-chain domains that contain mouse constant regions, may in particular comprise the nucleotide sequence of any of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48.

Accordingly, the nucleic acid molecule according to the present invention may contain the nucleotide sequence of any of SEQ ID NO: 41-44 or 45-48. In another embodiment, the nucleic acid molecule according to the present invention may contain a nucleotide sequence that is at least 90% or 95% identical to any of the sequences shown in SEQ ID NO: 41-44 or 45-48, or degenerate to the nucleotide sequence of any from the sequences shown in SEQ ID NO: 41-44 or 45-48. A nucleic acid molecule that contains a nucleotide sequence that is inversely complementary to the sequence of any of the above-described nucleic acid molecules of the present invention is also included within the scope of the present invention.

Soluble TCR according to the present invention may be encoded as ssTCR. Preferred ssTCRs for soluble TCRs are as described above for full-length TCRs, using the respective truncated α-chain and β-chain domains. For full-length TCR according to the present invention, ssTCR is preferably encoded with an α chain domain at its N-terminus and a β-chain domain at its C-terminus, the two domains being separated by a linker, preferably a picornavirus 2A peptide, the sequence of which is given in SEQ ID NO: 18, or its derivatives as defined above.

According to one embodiment of the present invention, the soluble ssTCR comprises an α-chain domain with the amino acid sequence shown in SEQ ID NO: 65 (or a variant thereof) and a β-chain domain with the amino acid sequence shown in SEQ ID NO: 67 (or a variant thereof). ), with the two strands separated by a 2A-derived linker. According to a specific embodiment of the present invention, said ssTCR comprises or consists of the sequence shown in SEQ ID NO: 68, or an amino acid sequence that is at least 90% or 95% identical to said sequence. In case the ssTCR contains or consists of the sequence variant shown in SEQ ID NO: 68, the CDR sequences are those defined above and the residues at positions 175 and 436 (or at positions corresponding to positions 175 and 436 in the sequence given in SEQ ID NO: 68) are cysteine residues (these positions correspond to positions 175 and 188 of the α-chain and β-chain domains, respectively).

According to another embodiment of the present invention, the His tag is located at the C-terminus of the ssTCR, the sequence of which is given in SEQ ID NO: 68 (the C-terminus of the ssTCR corresponds to the C-terminus of the β-strand), while the His-tag is separated from the β-strand with using a Gly-Gly-Gly linker. Said ssTCR has the sequence of SEQ ID NO: 69. In another preferred embodiment, the present invention relates to an ssTCR comprising or consisting of the sequence shown in SEQ ID NO: 69 or an amino acid sequence that is at least 90% or 95% identical to said sequences. Where the ssTCR contains or consists of a sequence variant shown in SEQ ID NO: 69, the CDR sequences are those defined above and the residues at positions 175 and 436 (or at positions corresponding to positions 175 and 436 in the sequence given in SEQ ID NO: 69) are cysteine residues (these positions correspond to positions 175 and 188 of the α-chain and β-chain domains, respectively).

The amino acid sequences of SEQ ID NOs: 68 and 69 are encoded by the nucleotide sequences shown in SEQ ID NOs: 70 and 71. A nucleic acid molecule according to the present invention is a nucleic acid molecule that encodes a TCR according to

- 21 042909 of the present invention, including nucleic acid molecules that encode full-length TCRs and nucleic acid molecules that encode soluble TCRs. A nucleic acid molecule according to the present invention, in certain embodiments, is a nucleic acid molecule that contains or consists of the nucleotide sequence shown in SEQ ID NO: 70 or SEQ ID NO: 71, or from a nucleotide sequence that is at least 90% or 95% identical to the sequence shown in SEQ ID NO: 70 or SEQ ID NO: 71. A nucleic acid molecule containing or consisting of a sequence reversely complementary to the sequences shown in SEQ ID NO: 70 and SEQ ID NO: 71, or a nucleotide sequence that is at least 90% or 95% identical to said sequences is also included within the scope of the present invention.

As described above, some embodiments of the present invention relate to polypeptides or polynucleotides with a certain level of sequence identity with a specific defined sequence (reference sequence). In the event that percent sequence identity (%) is presented herein with respect to a specific reference sequence, percent sequence identity (%) is determined over the entire length of the reference sequence. When comparing polypeptide or polynucleotide sequences, two sequences are said to be identical if the nucleotide sequence in the two sequences is the same when compared for maximum match. Methods for determining sequence identity are well known in the art, and any convenient or available method may be used.

Optimal sequence alignment for comparison can be performed using the Megalign program in the Lasergene bioinformatics software package (DNASTAR, Inc., Madison, Wisconsin, USA) using the default parameters. In another embodiment, optimal sequence matching for comparison can be performed using the Smith-Waterman Local Identity Algorithm, Add. APL. Math 2:482 (1981), using the Needleman-Wunsch sequence homologous region alignment algorithm, J. Mol. Biol. 48:443 (1970), by Pearson and Lipman similarity search, Proc. Natl. Acad. sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group (GCG), 575 575 Science Dr., Madison, WI, USA) or by checks.

Similarly, in the case where an amino acid position in a sequence variant is determined to correspond to a particular amino acid position in a reference sequence, the corresponding position can be identified using sequence matching as described above. When a sequence variant is matched to a reference sequence, the amino acids that match at any particular position are defined herein as corresponding. For example, if the sequence variant shown in SEQ ID NO: 10 is matched to the sequence shown in SEQ ID NO: 10, the position in the sequence variant corresponding to position 48 in the sequence shown in SEQ ID NO: 10 is an amino acid that maps to amino acid 48 in the sequence shown in SEQ ID NO: 10. The position in the sequence variant corresponding to the position in the reference sequence may be in the same place as in the reference sequence, for example, position 48 in the sequence shown in SEQ ID NO: 10 may correspond to position 48 in the variant sequence shown in SEQ ID NO: 10. However, the corresponding positions may be in different places. For example, if one amino acid was deleted from the N-terminus of the variant of the sequence shown in SEQ ID NO: 10 (and no other mutations were introduced), then position 48 in the sequence shown in SEQ ID NO: 10 would correspond to position 47 in the variant sequences.

Those skilled in the art will appreciate that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that can encode for each individual TCR molecule described herein.

The nucleic acid molecule of the present invention may be an isolated nucleic acid molecule and may include DNA (including cDNA) or RNA or chemical derivatives of DNA or RNA, including molecules containing a radioactive isotope or a chemical adduct such as a fluorophore, chromophore or biotin (label) . Accordingly, the nucleic acid may contain modified nucleotides. These modifications include base modifications such as bromuridine, ribose modifications such as arabinoside and 2',3'dideoxyribose, and internucleotide bond modifications such as phosphothioate, phosphodithioate, phosphoselenoate, phosphodiselenoate, phosphoanilothioate, phosphoaniladate and phosphoamidate. The term nucleic acid molecule specifically includes single and double stranded forms of DNA and RNA.

Methods for modifying nucleotide sequences to introduce changes in the amino acid sequences of different TCR regions are well known in the art, for example, mutagenesis methods such as site-directed mutagenesis can be used. Semi ways

Values for a nucleic acid molecule encoding a TCR molecule are also well known, for example, conventional polymerase chain reaction (PCR) cloning techniques can be used to construct a nucleic acid molecule.

For example, the nucleic acid molecule can be cloned into a general purpose cloning vector such as pENTR (Gateway), pUC19, pBR322, pBluescript vectors (Stratagene Inc.) or pCR TOPO® from Invitrogen Inc. The resulting nucleic acid construct (recombinant vector) carrying a nucleic acid molecule encoding TCR can then be subcloned into expression vectors or viral vectors for protein expression, for example, in mammalian cells. This step can be performed to produce the TCR protein or to be expressed in immune effector cells, eg human T cells or cell lines or other human immune effector cells. In addition, the nucleic acid can be introduced into mRNA expression vectors to produce mRNA encoding TCR. The mRNA can then be transferred to immune effector cells. Accordingly, according to another aspect of the present invention, there is provided a vector containing a nucleic acid molecule according to the present invention.

The mRNA expression vector may alternatively be transcribed under in vitro conditions to produce mRNA encoding TCR. For in vitro transcription (IVT), a template is first prepared. This may be a linearized mRNA expression vector. The vector can be linearized, for example, using a restriction enzyme. Alternatively, the template may be obtained by PCR amplification of the expression cassette, or by any other method generally known to those skilled in the art. The template is then purified and transcribed. Transcription can be performed using an IVT kit such as the MEGAscript™ kit, the RiboMAX™ kit, or the MAXIscript™ kit. The template DNA can then be removed by DNase digestion of the sample followed by mRNA purification. IVT techniques are well known to those skilled in the art.

The nucleic acid molecule of the present invention may be introduced into a cell in a vector, either as an isolated nucleic acid molecule or as a recombinant construct. Methods for expressing heterologous genes are known in the art, both for the production of a construct/vector and for the introduction of a nucleic acid molecule (vector or construct) into a cell. Accordingly, promoters and/or other expression control sequences suitable for use with mammalian cells, in particular T cells, and corresponding vectors (eg, viral vectors) are well known in the art.

Accordingly, the nucleic acid molecule may be introduced or inserted into a vector. As used herein, the term vector refers to a carrier into which a nucleic acid molecule can be introduced (eg, covalently inserted) so as to cause the expression of a TCR protein or mRNA and/or to allow cloning of the nucleic acid molecule. The vector may be a cloning vector or an expression vector, respectively.

The nucleic acid molecule can be inserted into the vector using any suitable methods known in the art, for example, but not limited to, the vector can be cleaved using suitable restriction enzymes, and then can be ligated to a nucleic acid molecule having matching ends restrictions.

Expression vectors may contain a plurality of control sequences that refer to nucleic acid sequences necessary for transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that regulate transcription and translation, vectors may contain additional nucleic acid sequences that perform other functions, including, for example, functions in replication or functions as selectable markers, etc.

The expression vector must have the necessary 5' and 3' regulatory elements for efficient transcription and translation of genes in the appropriate host cell, such as promoter sequences, examples of which include the CMV, PGK, and EF1a promoters, a TATA box for ribosome recognition, and binding, as well as the transcription termination sequence 3'UTR AATAAA (SEQ ID NO: 53). Other suitable promoters include the simian virus 40 constitutive early promoter (SV40), the mouse mammary tumor virus (MMTV) promoter, the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the Moloney murine leukemia virus (MoMuLV) promoter, the avian leukemia (ALV), immediate early promoter of the virus

Epstein-Barr (EBV) and Rous sarcoma virus (RSV) promoter. Human gene promoters can also be used, including but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In some embodiments of the present invention, inducible promoters are also contemplated as part of the TCR expressing vectors. They provide a molecular switch capable of starting or blocking the expression of a nucleic acid molecule. Examples of inducible promoters include, but are not limited to

- 23 042909 are the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter or the tetracycline promoter.

In addition, the expression vector may contain 5' and 3' untranslated regulatory sequences that may function as enhancer and/or termination sequences that may facilitate or enhance efficient transcription of the nucleic acid molecule.

Examples of vectors include plasmids, autonomously replicating sequences, and transposable elements. Additional exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC), bacteriophages such as lambda -phage or M13 phage, and animal viruses. Examples of categories of animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40 ). Examples of expression vectors include the pCl-neo (Promega) vectors for expression in mammalian cells and pLenti4/V5-DEST™ and pLenti6/V5-DEST™ for lentivirus-mediated gene transfer and expression in mammalian cells.

In some embodiments of the present invention, viral vectors are preferred. The viral vector may be derived from a retrovirus, in particular a lentivirus or a spumavirus/foam virus. As used herein, the term viral vector refers to a nucleic acid vector construct that contains at least one element of viral origin and can be packaged into a viral vector particle. The viral vector may contain a nucleic acid molecule according to the present invention in place of non-essential viral genes. The vector and/or particle can be used to transfer DNA, RNA or other nucleic acids into cells both in vitro and ex vivo.

Accordingly, another aspect of the present invention includes a viral particle containing a nucleic acid molecule as defined and described herein, or a preparation or composition containing such viral particles. Such a composition may also contain at least one physiologically acceptable carrier.

Numerous forms of viral vectors are known in the art. In some embodiments of the present invention, the viral vector is a retroviral vector or a lentiviral vector. The vector may be a self-inactivating vector in which the 3'LTR region of the promoter enhancer, known as the U3 region, has been modified (eg, by deletion or substitution) to prevent transcription of the virus after the first step of viral replication. Accordingly, the vectors are capable of infecting the host and then integrating into its genome only once and cannot be passed on.

Retroviral vectors for use in the present invention can be derived from any known retrovirus, for example type C retroviruses such as Moloney mouse sarcoma virus (MMSV), Harvey mouse sarcoma virus (Ha-MuSV), mouse mammary tumor virus (MMTV), gibbon leukemia virus (GaLV), feline leukemia virus (FLV), spumaviruses, Friend virus, mouse stem cell virus (MSCV), and Rous sarcoma virus (RSV); human T-cell leukemia viruses such as HTLV-1 and HTLV-2; and the lentiviral family of retroviruses such as human immunodeficiency viruses HIV-1 and HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine immunodeficiency virus (EIV), and other classes of retroviruses.

The lentiviral vector is derived from a lentivirus, a group (or genus) of retroviruses that cause a slowly developing disease. Viruses included in this group include HIV (human immunodeficiency virus, including HIV type 1 and HIV type 2).

A retrovirus packaging cell line (typically a mammalian cell line) can be used to generate viral particles which can then be used to transduce T cells. Illustrative viral vectors are described in WO2002087341, WO2002083080, WO2002082908, WO2004000220 and WO2004054512. An exemplary retroviral vector is pMP71 as described in Walchli et al 2011. Other suitable vectors include pBABE, pWZL, pMCs-CAG, pMXs-CMV, pMXs-EF1a, pMXs-IRES, pMXs-SRa, and pMYs-IRES.

Included within the scope of the present invention are gene segments that render immune effector cells carrying a vector or construct of the present invention susceptible to negative selection in vivo. Under negative selection is meant that the cell after the introduction of these agents can be eliminated as a result of a change in the state of the individual under in vivo conditions. A negative selective phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, such as a compound. Negative selection genes are known in the art and include, but are not limited to, the following: herpes simplex virus type 1 thymidine kinase (HSV-I TK) gene (Wigler et al., Cell 11(1):223-232, 1977), which

- 24 042909 ry confers sensitivity to ganciclovir; gene for cellular hypoxanthine phosphoribosyl transferase (HPRT), gene for cellular adenine phosphoribosyl transferase (APRT) and bacterial cytosine deaminase Mullen et al, Proc. Natl. Acad. sci. USA. 89:33-37 (1992)). Accordingly, the vector or construct according to the present invention may contain such a gene.

According to some embodiments of the present invention, it may be useful to include a positive marker in the vector or construct of the present invention that allows selection of cells with a negative selective phenotype in vitro, for example, selection of genetically modified immune effector cells. A positive selectable marker may be a gene which, when introduced into a host cell, expresses a dominant phenotype that positively selects for cells carrying the gene. Genes of this type are known in the art and include but are not limited to the hygromycin B phosphotransferase (hph) gene which confers resistance to hygromycin B, the aminoglycoside phosphotransferase (neo or aph) gene from Tn5 which encodes resistance to the antibiotic G418, the gene dihydrofolate reductase (DHFR), adenosine deaminase (ADA) gene, and multidrug resistance (MDR) gene.

Preferably, the positive selective marker and the negative selective element are connected such that the loss of the negative selective element is also necessarily accompanied by the loss of the positive selective marker. Even more preferably, the positive and negative selectable markers are hybridized such that the loss of one necessarily leads to the loss of the other. An example of a fusion polynucleotide whose expression product is a polypeptide having the desired positive and negative selective features described above is the hygromycin phosphotransferase-thymidine kinase (HyTK) fusion gene. Expression of this gene produces a polypeptide that provides resistance to hygromycin B for positive selection in vitro and sensitivity to ganciclovir for negative selection in vivo (see Lupton S.D., et al, Mol. and Cell. Biology 11:3374- 3378, 1991).

To clone a nucleic acid molecule, the vector can be introduced into a host cell (eg, an isolated host cell), and such cloning host cells containing the cloning vector of the present invention represent a further aspect of the present invention. Suitable cloning host cells may include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells. Suitable prokaryotic cells for this purpose include, but are not limited to, eubacteria such as gram negative or gram positive organisms e.g. Enterobactedaceae such as Escherichia e.g. E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella e.g. Salmonella typhimurium, Serratia, for example, Serratia marcescans and Shigella, as well as Bacilli, such as Bacillus subtilis and Bacillus licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces. In another embodiment, the cloning host cell may contain an mRNA expression vector containing a nucleic acid molecule.

Nucleic acid molecules or vectors are introduced into a host cell (eg, a cloning host cell, a production host cell, or a T cell) using transfection and/or transduction techniques known in the art. As used herein, the terms transfection and transduction refer to processes by which an exogenous nucleic acid sequence is introduced into a host cell. The nucleic acid may be integrated into the DNA of the host cell or may be maintained as an extrachromosomal element. The nucleic acid may be maintained transiently or may be stable. Transfection can be accomplished by various means known in the art, including, but not limited to, DNA-calcium phosphate coprecipitation, DEAE-dextran mediated transfection, polybrene mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection and ballistic transfection. Transduction refers to the delivery of gene(s) using a viral or retroviral vector by viral infection rather than by transfection. In some embodiments of the present invention, retroviral vectors are transduced by packaging the vectors into viral particles or virions prior to contact with a cell.

The present invention also relates to a host cell containing a nucleic acid molecule or a vector according to the present invention. Such a host cell may be any suitable host, including a cloning host, a production host, or an immune effector cell. The host cell can be derived from any species and virtually any kingdom depending on its function.

In one embodiment, the present invention provides an immune effector cell comprising a nucleic acid molecule or a vector of the present invention that encodes a full-length TCR of the present invention. An immune effector cell is any cell of the immune system that has one or more effector functions (eg, cytotoxic activity to kill cells, secretion of cytokines, induction of ADCC and/or CDC). Typical immune effector cells, respectively,

- 25 042909 include T lymphocytes, in particular cytotoxic T cells (CTL; CD8+ T cells) and helper T cells (CTL; CD4+ T cells). Other populations of T cells can also be used in the present invention, such as naive T cells and memory T cells. Other immune effector cells include NK cells, NKT cells, neutrophils, and macrophages. As noted above, immune effector cells also include effector cell progenitors, which progenitor cells can be induced to differentiate into immune effector cells in vivo or in vitro. T cells and NK cells are the preferred immune effector cells of the present invention.

The T cell of the present invention may be any T cell. Said cell may be a cytotoxic T cell (CD8+ T cell), a helper T cell (CD4+ T cell), a naive T cell, a memory T cell, or any other type of T cell. Preferably the T cell is a CD8+ T cell. As defined herein, the T cell of the present invention may also be an immature T cell such as CD47CD8 cell or CD4+/CD8+ cell, or T cell progenitor cell.

The term NK cell refers to a large granular lymphocyte, which is a cytotoxic lymphocyte derived from a common lymphoid progenitor that does not naturally contain an antigen-specific receptor (eg, T cell receptor or B cell receptor). NK cells can be differentiated based on their CD3 ^- , CD56+ phenotype. As used herein, the term includes any known NK cell, or any NK-like cell, or any cell having the characteristics of an NK cell. Accordingly, primary NK cells may be used or, in another embodiment, an NK cell known in the art that has previously been isolated and cultured may be used. Accordingly, the NK cell line can be used. A number of different NK cells are known and described in the literature, and any of them can be used, or a cell line can be obtained from a primary NK cell, for example, by viral transformation (Vogel et al. 2014, Leukemia 28:192-195). Suitable NK cells include, but are by no means limited to, in addition to NK-92, the NK-YS, NK-YT, MOTN-1, NKL, KHYG-1, HANK-1, or NKG cell lines. According to a preferred embodiment of the present invention, the cell is an NK-92 cell (Gong et al. 1994, Leukemia 8:652-658) or a variant thereof. A number of different variants of the original NK-92 cells have been produced, described or available, including NK-92 variants that are non-immunogenic. Any such options can be used and included in the term NK-92. Variants of other cell lines may also be used.

The immune effector cell of the present invention is preferably a human cell. Such an immune effector cell can be obtained from any individual. Preferably, if the immune effector cell is for therapeutic use, it is an autologous immune effector cell: i.e. it is derived from the patient to be treated, which provides histocompatibility and non-immunogenicity, which means that after genetic modification, the specified cell will not induce an immune response in the patient. If the immune effector cell is a non-autologous cell for therapeutic use (i.e., it is a donor cell obtained from a person other than the patient), it is preferably non-immunogenic such that, when administered to a subject, it does not elicit an immune response that has a negative effect , hinders or prevents the use of cells in therapy. Accordingly, the immune effector cell of the present invention may be an ex vivo cell. Alternatively, it may also be a cell under in vitro conditions.

Non-autologous immune effector cells may be non-immunogenic in nature if they match the patient's HLA, ie. they express similar HLA alleles. Non-autologous immune effector cells, including those that do not match the patient's HLA and therefore would be immunogenic, and those that match the patient's HLA and would therefore not be immunogenic, can be modified to abolish the expression of the MHC molecule or to only slightly express MHC molecules on its surface. Alternatively, such cells can be modified to express non-functional MHC molecules.

Any methods of disrupting the expression of a functional MHC molecule are included within the scope of the present invention. Therefore, this may include elimination or suppression of the MHC complex molecule and/or may include a modification that prevents proper transport and/or correct expression of the MHC molecule or the entire complex on the cell surface.

In particular, expression of one or more functional MHC class proteins on the surface of an immune effector cell according to the present invention may be impaired. According to one embodiment of the present invention, immune effector cells can be human cells that are HLA negative, such as cells in which the expression of one or more HLA molecules is disrupted (e.g., eliminated), for example, MHC class I HLA complex molecules.

According to a preferred embodiment of the present invention, disruption of expression

- 26 042909

MHC class I can be accomplished by _eliminating the gene encoding for ₂ -microglobulin (in ₂ -t), a component of the mature _MHC class I complex. -directed mutagenesis of the e ₂ -m promoter (to inactivate the promoter) or within the gene encoding the e ₂ -m protein to introduce an inactivating mutation that prevents expression of the e ₂ -m protein, such as a frameshift mutation or premature codon STOP within the gene. Alternatively, site-directed mutagenesis can be used to generate a non-functional e ₂ -m protein that is unable to form an active MHC protein on the cell surface. Accordingly, the β ₂ m or MHC protein may be retained within the cell, or may be present on the cell surface but non-functional.

Alternatively, the immune effector cells may be irradiated prior to administration to a subject. Without wishing to be bound by any theory, the present inventors believe that irradiation of cells results in cells being present only briefly in the subject, thereby reducing the time available for the subject's immune system to elicit an immune response against the cells. Although such cells can express a functional MHC molecule on their surface, they can also be considered non-immunogenic.

The radiation may be from any source of α-, β- or γ-radiation or may be X-rays or ultraviolet radiation. A radiation dose of 5-10 Gy may be sufficient to abolish proliferation, however other suitable radiation doses may be 1-10, 2-10, 3-10, 4-10, 6-10, 7-10, 8-10 or 9- 10 Gy, or higher doses such as 11, 12, 13, 14, 15 or 20 Gy. In another embodiment, the cells may be modified to express a suicide gene that allows induced cell death or prevents them from replicating in response to an external stimulus.

Accordingly, an immune effector cell according to the present invention can be modified to be non-immunogenic by reducing its ability or ability to proliferate, i.e. by reducing its proliferative capacity.

The modified immune effector cells of the present invention may also be modified in other ways, such as to alter or modify other aspects of cell function or behavior and/or to express other proteins. For example, cells can be modified to express a homing receptor or localization receptor, the function of which is to target or improve the localization of cells in a particular tissue or location in the body.

The present invention also relates to methods for generating immune effector cells that express the TCR described herein. According to one embodiment of the present invention, the method comprises transfecting or transducing T cells isolated from a subject (which may be a patient or a donor) such that the T cells express one or more of the TCRs described herein. In some embodiments of the present invention, T cells are isolated from a subject and modified by introducing a nucleic acid molecule without further manipulation under in vitro conditions. Such cells can then be directly reintroduced into the subject. According to other embodiments of the present invention, T cells are first activated and stimulated to proliferate under in vitro conditions (such activation and stimulation of their proliferation may be referred to as proliferation) before being modified to express TCR. In this regard, T cells can be cultured before or after genetic modification (ie, transduced or transfected to express TCR, as described herein).

T cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumor tissues. In some embodiments of the present invention, T cells may be obtained from a unit of blood collected from a subject using any number of methods known to one of skill in the art, such as Ficoll™ gradient separation. According to one implementation variant of the present invention, cells from the circulating blood of the subject are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. According to one embodiment of the present invention, the cells collected by apheresis can be washed to remove the blood plasma fraction and place the cells in an appropriate buffer or medium for further processing. According to one implementation variant of the present invention, the cells are washed with phosphate-buffered saline (PBS). According to another implementation variant, the wash solution does not contain calcium and/or magnesium, or may not contain many, if not all, divalent cations. Those skilled in the art will appreciate that the washing step can be carried out using methods known to those skilled in the art, such as using a semi-automatic flow centrifuge. For example, using the Cobe 2991 cell processor,

- 27 042909

Baxter CytoMate or the like. After washing, cells can be resuspended in various biocompatible buffers or other saline with or without buffer. According to some embodiments of the present invention, unwanted components of the apheresis sample can be removed in the cell immediately resuspended in culture medium.

In some embodiments of the present invention, T cells are isolated from PBMCs. PBMCs can be isolated from buffy coats obtained by whole blood density gradient centrifugation, for example, by LYMPHOPREP™ gradient centrifugation, PERCOLL™ gradient or FICOLL™ gradient centrifugation. T cells can be isolated from PBMCs by monocyte depletion, for example using CD14 Dynabeads®. In some embodiments of the present invention, the erythrocytes may be lysed prior to density gradient centrifugation.

Specific subpopulations of T cells such as CD28+, CD4 ⁺ , CD8 ⁺ , CD45RA+ or CD45RO+ T cells, if necessary, can be further isolated using positive or negative selection techniques. For example, enrichment of a T cell population by negative selection can be achieved with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method that can be used herein involves sorting and/or selecting cells using negative magnetic immunoadhesion or flow cytometry, which uses a cocktail of monoclonal antibodies directed to cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate cell populations of interest for use in the present invention.

In some embodiments of the present invention, cytotoxic and helper T cells can be sorted into subpopulations of naive T cells, memory T cells, and effector T cells before or after genetic modification and/or expansion. CD8+ T cells can be obtained using the standard methods described above. According to some embodiments of the present invention, CD8+ T cells can be further sorted into naive T cells, central memory T cells, and effector T cells by identifying cell surface antigens that are associated with each of said CD8+ T cell types. Memory T cells can be present in both CD62L+ and CD62L' subgroups of CD8+ T cells in peripheral blood. Cells can be sorted into CD62L-/CD8+ and CD62L+/CD8+ fractions after staining with anti-CD8 and anti-CD62L antibodies. Phenotypic markers of central memory T cells (TMCs) may include the expression of CD45RO, CD62L, CCR7, CD28, CD3 and CD127, as well as the absence of granzyme B expression. MCMs may be CD45RO+/CD62L+/CD8+ T cells. Effector T cells can be negative for CD62L, CCR7, CD28 and CD127 expression, and positive for granzyme B and perforin expression. Naive CD8+ T cells can be characterized based on the expression of naive T cell phenotypic markers, including CD62L, CCR7, CD28, CD3, CD127, and CD45RA.

Isolated immune effector cells can be modified after isolation, or they can be activated and expanded (or differentiated, in the case of progenitor cells) in vitro prior to modification. According to one implementation variant of the present invention, the cells are modified by introducing nucleic acid molecules according to the present invention and then activated and propagated under in vitro conditions. According to another embodiment of the present invention, the cells are activated and propagated under in vitro conditions and then modified by the introduction of nucleic acid molecules according to the present invention. Methods for activating and expanding T cells are known in the art and are described, for example, in US6905874, US6867041, US6797514 and WO2012079000. Typically, such methods involve contacting PBMCs or isolated T cells with a stimulatory agent and a costimulatory agent, such as anti-CD3 antibodies and anti-CD28 antibodies, usually attached to a bead or other surface (eg, in the form of CD3/CD28 Dynabeads®), in culture medium supplemented with appropriate cytokines such as IL-2. The bead with attached antibodies to CD3 and antibodies to CD28 serves as a mimic of an antigen presenting cell (APC). In other embodiments, T cells can be activated and their proliferation can be stimulated with nurse cells and appropriate antibodies and cytokines using methods such as those described in US6040177, US5827642 and WO2012129514.

According to one implementation variant of the present invention, T cells are transduced or transfected using a nucleic acid molecule in accordance with the present invention. Methods of transduction and transfection are described above. The nucleic acid molecule according to the present invention may be a vector containing the nucleic acid molecule according to the present invention (i.e., a molecule that encodes a TCR molecule according to this

- 28 042909 to the present invention). In another embodiment, the nucleic acid molecule may be an mRNA molecule encoding a TCR molecule according to the present invention.

According to another embodiment of the present invention, CD34+ cells are transduced or transfected using a nucleic acid molecule in accordance with the present invention. In some embodiments of the present invention, modified (eg, transfected or transduced) CD34+ cells differentiate into mature immune effector cells under in vivo conditions after administration to a subject, typically the subject from which the cells were originally isolated. According to another embodiment of the present invention, CD34+ cells can be stimulated in vitro before or after administration of the nucleic acid molecule using one or more of the following cytokines: Flt-3 ligand (FL), stem cell factor (SF), multiplication and differentiation factor megakaryocytes (TRO), IL-3 and IL-6 according to methods known in the art.

According to another implementation variant of the present invention relates to a producing host cell that contains a nucleic acid molecule or a vector according to the present invention, which encodes a soluble TCR according to the present invention. Producing host cells are suitable for expression and production of soluble TCR. The producing host cell can be any cell suitable for protein production. For example, the producing host cell may be a prokaryotic cell, in particular a bacterial cell such as a Gram-negative bacterial cell (eg, Escherichia coll) or a Gram-positive bacterial cell (eg, Bacillus subtilis). However, preferably the producing host cell is a eukaryotic cell. The eukaryotic production host cell of the present invention may be a simple eukaryotic cell such as a yeast or fungal cell. Preferably, the eukaryotic producing host cell is an animal cell. The animal cell may be an insect cell or any other animal cell, but preferably a mammalian cell. For example, the mammalian production host cell may be a primate cell, in particular a human cell, or a rodent cell: for example, it may be a HEK-293, HEK-293T, COS (eg, COS7) or CHO cell lines. Mammalian producing host cells, in particular human producing host cells, are preferred because the corresponding post-translational modifications occur when soluble TCR is expressed in a human cell. One skilled in the art will readily be able to select a suitable production host cell.

The present invention also relates to the TCR molecule as defined herein, in particular the soluble TCR molecule as defined herein. Soluble TCR according to the present invention is a protein or protein complex that contains a truncated α-chain domain and a truncated β-chain domain, as defined above. Soluble TCRs are detailed in Walseng et al. (2015), PLoS ONE 10(4): e0119559.

Soluble TCR according to the present invention is preferably encoded as a single strand. Single chain soluble TCRs are described above and, as detailed, such ssTCRs preferably contain self-cleaving linker sequences between the α-chain and β-chain domains, and accordingly form separate α-chains and β-chains.

Accordingly, the soluble TCR according to the present invention is preferably an α-chain-beta-chain TCR complex that is composed of individual polypeptide chains. The soluble TCR of the present invention is the TCR encoded by the nucleic acid molecule of the present invention as defined above. As discussed above, the α- and β-chain variable regions are encoded and synthesized with leader sequences that direct the chains for membrane insertion or, in the case of soluble TCR, for secretion. Such leader sequences are cleaved off during secretion. The TCR strand that contains the leader sequence is known as immature, while the strand from which the leader sequence is cleaved is known as mature. The soluble TCR according to the present invention is preferably mature soluble TCR lacking α and β chain leader sequences.

Soluble TCR according to the present invention is encoded by the nucleic acid molecule as defined above. Accordingly, according to a preferred embodiment of the present invention, the α-chain of soluble TCR is encoded with a variable region containing the sequence of SEQ ID NO: 8. As described above, the leader sequence of the α-chain of Radium-1 is shown in SEQ ID NO: 50, which corresponds to amino acids 1 -20 from the sequence shown in SEQ ID NO: 8. The mature form of the variable region of the Radium-1 α chain (i.e., the variable region without the leader sequence) has the sequence of SEQ ID NO: 72. Preferably, the soluble TCR according to the present invention contains α - a chain containing a variable region containing or consisting of the amino acid sequence shown in SEQ ID NO: 72, or from an amino acid sequence that is at least 90% or 95% identical to that specified after

- 29 042909 consistency. In the case of the sequence variant shown in SEQ ID NO: 72, the CDR sequences are those defined above.

The truncated constant region of the α-chain of soluble TCR preferably has the sequence of SEQ ID NO: 60 or SEQ ID NO: 61 as described above. Accordingly, the α-chain of soluble TCR according to the present invention preferably contains a variable region, the sequence of which is given in SEQ ID NO: 72, and a constant region, the sequence of which is given in SEQ ID NO: 60 or SEQ ID NO: 61. Such α-chain sequences shown in SEQ ID NO: 73 and 74, respectively. Accordingly, the soluble TCR according to the present invention preferably contains an α-chain containing or consisting of the amino acid sequence shown in SEQ ID NO: 73 or SEQ ID NO: 74, or from an amino acid sequence that is at least 90% or 95% identical the specified sequence. In the case of the sequence variant shown in SEQ ID NO: 73 or 74, the CDR sequences are those defined above; in the case of the sequence variant shown in SEQ ID NO: 74, the amino acid at position 155 (or the position corresponding to position 155 in the sequence shown in SEQ ID NO: 74) is a cysteine. Position 155 in the sequence shown in SEQ ID NO: 74 corresponds to position 175 in the sequence of the immature cysteine modified truncated α chain shown in SEQ ID NO: 65.

According to another preferred embodiment of the present invention, the soluble TCR β chain is encoded with a variable region containing the sequence of SEQ ID NO: 13. As described above, the Radium-1 β chain leader is shown in SEQ ID NO: 51, corresponding to amino acids 1- 16 of the sequence shown in SEQ ID NO:13. a chain containing a variable region containing or consisting of the amino acid sequence shown in SEQ ID NO: 75, or from an amino acid sequence that is at least 90% or 95% identical to the specified sequence. In the case of the sequence variant shown in SEQ ID NO: 75, the CDR sequences are those defined above.

The truncated constant region of the β-chain of soluble TCR preferably has the sequence of SEQ ID NO: 62 or SEQ ID NO: 63 as described above. Accordingly, the β-chain of the soluble TCR according to the present invention preferably contains a variable region, the sequence of which is given in SEQ ID NO: 75, and a constant region, the sequence of which is given in SEQ ID NO: 62 or SEQ ID NO: 63. Said sequences of the β-chain presented in SEQ ID NO: 76 and 77, respectively. Accordingly, the soluble TCR according to the present invention preferably contains an α-chain containing or consisting of the amino acid sequence shown in SEQ ID NO: 76 or SEQ ID NO: 77, or from an amino acid sequence that is at least 90% or 95% identical the specified sequence. In the case of the sequence variant shown in SEQ ID NO: 76 or 77, the CDR sequences are those defined above; in the case of the sequence variant shown in SEQ ID NO: 77, the amino acid at position 172 (or the position corresponding to position 172 in the sequence shown in SEQ ID NO: 77) is a cysteine. Position 172 in the sequence shown in SEQ ID NO: 77 corresponds to position 188 in the sequence of the immature cysteine modified truncated α chain shown in SEQ ID NO: 66.

According to another preferred embodiment of the present invention, as discussed above, each of the α-chain and β-chain of soluble TCR contains a leucine zipper sequence at the C-terminus.

According to another preferred embodiment of the present invention, at least one strand of soluble TCR is encoded with a purification label. Such a label may be any suitable label known to those skilled in the art, such as a FLAG tag, a His tag, a HA tag, a Strep tag, an S-tag or a Myc tag, glutathione-S-transferase (GST), maltose-binding protein (MBP), etc. .d. The label is preferably located at the C-terminus of the α chain or β chain, most preferably the β chain. Accordingly, the soluble TCR according to the present invention may contain a label for purification in its α-chain and/or β-chain, preferably at the C-terminus of the chain(s). The TCR chain may be encoded with a linker and/or a protease cleavage site between the main chain sequence (ie, the variable domain and the constant domain segment present, which also contains the leucine zipper domain) and the label for purification. Appropriate protease cleavage sites are well known to those skilled in the art and include thrombin, factor Xa, enterokinase, human rhinovirus 3C (HRV) cleavage sites, and tobacco etch virus (TEV) protease cleavage sites. According to a specific embodiment of the present invention, the β chain of the soluble TCR according to the present invention contains a His tag attached to the chain

- 30 042909 via a Gly-Gly-Gly linker.

The sequence of the α chain shown in SEQ ID NO: 74, with a His tag attached to its C-terminus via a Gly-Gly-Gly linker, is shown in SEQ ID NO: 78; the sequence of the β chain shown in SEQ ID NO: 77 with a His tag attached to its C-terminus via a Gly-Gly-Gly linker is shown in SEQ ID NO: 79. According to preferred embodiments of the present invention, the soluble TCR α chain contains or consists of the amino acid sequence shown in SEQ ID NO: 78, or an amino acid sequence that is at least 90% or 95% identical to the specified sequence, and/or β-chain of soluble TCR contains or consists of the amino acid sequence shown in SEQ ID NO: 79, or from an amino acid sequence that is at least 90% or 95% identical to the specified sequence. In the case of the sequence variant shown in SEQ ID NO: 78, the CDR sequences are as defined above, the amino acid at position 158 (or the position corresponding to position 158 in the sequence shown in SEQ ID NO: 78) is a cysteine, and the C-terminal hexahistidine tag is unchanged; in the case of the sequence variant shown in SEQ ID NO: 79, the CDR sequences are as defined above, the amino acid at position 172 (or the position corresponding to position 172 in the sequence shown in SEQ ID NO: 79) is a cysteine, and the C-terminal hexahistidine tag is unchanged.

As detailed above, according to a preferred embodiment of the present invention, soluble TCR is encoded as ssTCR in which the α and β chain domains are separated by a 2A linker. The 2A linkers are discussed above and, as detailed above, these sequences undergo co-translational cleavage between their terminal proline residue and the penultimate glycine residue. The terminal proline residue of linker 2A therefore forms the N-terminal residue of the polypeptide located after it, while the remaining residues of linker 2A form the C-terminus of the polypeptide located before them. In a preferred ssTCR according to the present invention, the α-chain domain forms the precursor polypeptide and the β-chain domain forms the subsequent polypeptide. As detailed above, the N-terminus of each TCR chain is a leader sequence that is cleaved off during polypeptide maturation. In the ssTCR of the present invention, the proline terminal residue of linker 2A will form the N-terminal residue of the β chain and will be cleaved from the leader sequence chain. Therefore, the residue will not be present in the mature β chain. However, other 2A linker residues will form the C-terminus of the α chain and will be present in the mature α chain.

Accordingly, according to a specific embodiment of the present invention, soluble TCR contains an α-chain, in which the C-terminus is formed from all residues, with the exception of the terminal residue of the 2A peptide. In particular, the C-terminus of the α-chain (i.e., 25 terminal residues) may be amino acids 1-25 of the sequence 2A shown in SEQ ID NO: 18. According to this embodiment of the present invention, the α-chain of soluble TCR, in particular , may contain or consist of the amino acid sequence shown in SEQ ID NO: 81, or an amino acid sequence that is at least 90% or 95% identical to the specified sequence. The amino acid sequence shown in SEQ ID NO: 81 is the sequence of SEQ ID NO: 73 (mature truncated α-chain Radium-1) with the addition of residues 1-25 from the sequence shown in SEQ ID NO: 18 at the C-terminus . In another embodiment, the soluble TCR contains or consists of the amino acid sequence shown in SEQ ID NO: 82 or an amino acid sequence that is at least 90% or 95% identical to said sequence. The amino acid sequence shown in SEQ ID NO: 82 is the sequence of SEQ ID NO: 74 (mature truncated cysteine modified α-chain Radium-1) with the addition of residues 1-25 from the sequence shown in SEQ ID NO: 18, on C -end. Where the α chain is a variant of the sequence shown in SEQ ID NO: 81 or 82, the CDR sequences are those defined above; when the α chain is a variant of the sequence shown in SEQ ID NO: 82, the amino acid at position 155 (or the position corresponding to position 155 in the sequence shown in SEQ ID NO: 82) is a cysteine.

As described above, the α- and β-chains of soluble TCR are connected to each other: this is a necessary condition, since otherwise the complex will separate in solution. As discussed above, the α- and β-chains can be connected covalently, for example, via a disulfide bond, or connected non-covalently via leucine zipper domains located at the C-terminus of the chains.

As stated above, all TCR α and β chains are expressed with an N-terminal leader sequence that directs the polypeptide chain to the insertion/secretion membrane. The α-chain of soluble TCR according to the present invention is encoded by such a sequence, and soluble

- 31 042909

The TCR according to the present invention may or may not contain an N-terminal leader sequence. Similarly, the β-chain of the soluble TCR according to the present invention is encoded with the same sequence, and the soluble TCR according to the present invention may or may not contain an N-terminal leader sequence. Such α- and β-chains and their leader sequences are described above.

Soluble TCR according to the present invention can be obtained using any method known in the art, in particular using a producing host cell as defined above. Soluble TCR can be expressed using standard protein expression techniques under standard conditions. The α- and β-chain leader sequences target the chains for export from the cell in which they are expressed (eg, the producing host cell). Accordingly, the α- and β-chains are exported from the producing host cell to the extracellular environment where the chains are complexed, for example, via disulfide bonding or dimerization of leucine zipper domains. The soluble TCR complex can then be purified: first, the cell culture can be centrifuged and the supernatant isolated. The soluble TCR complex can then be purified from the supernatant by affinity chromatography using a purification label at the C-terminus of the α and/or β chain. If a protease cleavage site is present at the N-terminus of the purification tag, then the tag can be cleaved from the strand using the appropriate protease.

If desired, soluble TCR can be multimerized to form a soluble TCR multimer. Such multimers represent an aspect of the present invention. For example, multimerization can be performed by conjugation of TCR molecules with nanobeads, such as magnetic nanobeads. Methods for such conjugation are well known in the art. According to another embodiment of the present invention, soluble TCR complexes can be biotinylated and conjugated with streptavidin to form tetrameric soluble TCR complexes. For biotinylation of the soluble TCR complex, one of the TCR chains must be expressed with the BirA sequence (SEQ ID NO: 59) at its C-terminus. Biotinylation of the TCR complex at the BirA sequence can then be performed using E. coli BirA (biotin ligase). After biotinylation, soluble TCR complexes can be incubated with streptavidin to obtain soluble TCR tetramers.

According to a particularly preferred embodiment of the present invention, the soluble TCR is conjugated to a toxin. The toxin of choice is a toxin that, individually, is not capable of entering, destroying, or otherwise destroying a human cell, but, when ingested by a human cell via the conjugated molecule, is capable of exerting a toxic effect. Accordingly, such a toxin will only be taken up by, and have its targeted effect on, a soluble TCR-associated cell of the present invention that has been infiltrated by the soluble TCR. The toxin may be any known suitable cytotoxic molecule, ie. it may be any suitable cytotoxin. As used herein, the term cytotoxin refers to any toxin that inhibits cell reproduction and/or viability. Reproduction involves the division of the target cell (ie, the cell into which the toxin enters). Accordingly, a toxin can be any toxin that reduces or adversely affects the viability or survival of a cell, and specifically includes any toxin that induces death of a target cell, for example, a toxin can induce apoptosis or necrosis of a target cell.

Such a toxin may be a peptide toxin lacking a targeting domain. For example, the toxin may be a peptide toxin that does not contain a targeting domain in its native form, or it may be a peptide toxin modified from its native form to remove its targeting domain. Examples of such toxins are saporin and gelonin, which are ribosome-inactivating proteins (RIPs) from the same family as, for example, ricin, but which are unable to cross the plasma membrane of the cell. Similarly, enzymatic domains (eg, catalytic domains) of a cytotoxin from a pathogen can be used, such as an enzymatic domain of a bacterial cytotoxin, for example, an enzymatic domain of diphtheria toxin, Pseudomonas A exotoxin, or a clostridial cytotoxin, for example, TcsL from Clostridium sordellii.

The soluble TCR of the present invention may be encoded as a fusion protein with a toxin located at the C-terminus of either the α chain or the β chain. Alternatively, the toxin may be conjugated to soluble TCR using any suitable method known in the art. For example, a soluble TCR molecule can be biotinylated on both its α-chain and β-chain and conjugated to a streptavidin-conjugated toxin (or vice versa). Other suitable methods are known to those skilled in the art.

The present invention relates to a modified immune effector cell for use in cancer treatment, a modified immune effector cell expressing the TCR described herein. For example, modified immune effector cells can

- 32 042909 can be obtained from PBMCs obtained from a patient diagnosed with MSI+ colorectal cancer. For storage, eg for cryopreservation, of the modified immune effector cells and/or the preparation for use in a human or other subject, standard procedures can be used.

Modified immune effector cells expressing TCR according to the present invention can be used in methods and compositions for adoptive cell transfer immunotherapy in accordance with known techniques. In some embodiments of the present invention, cells are obtained by first collecting them from their culture medium and then washing and concentrating the cells in a medium and container system suitable for administration (pharmaceutically acceptable carrier) in an amount effective for treatment. A suitable infusion medium may be any isotonic medium, usually normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but 5% dextrose in water or lactated Ringer's solution may also be used. The infusion medium can be supplemented with human serum albumin.

The number of cells in the composition that is effective for treatment is at least 2 cells (e.g., at least 1 CD8+ central memory T cell and at least 1 CD4+ helper T cell) or more typically more than 10 ² cells, and up to 10 ⁶ , up to 10 ⁸ or 10 ⁹ cells, and may be more than 10 ¹⁰ cells. The number of cells, as well as the type of cells included in the composition, will depend on the end use for which the composition is intended. For applications according to the present invention, the cells are usually in a volume of 1 liter or less, 500 ml or less, even 250 ml or 100 ml or less. Therefore, the desired cell density is typically greater than 10 ⁶ cells/ml and is typically greater than 10 ⁷ cells/ml, typically 10 ⁸ cells/ml or more. A clinically significant number of immune cells can be distributed over several infusions, which in the aggregate is equal to or greater than 105, 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ or 10 ¹² cells. For example, the patient may be given 2, 3, 4, 5, 6 or more separate infusions at 24 or 48 hour intervals or every 3, 4, 5, 6 or 7 days. The infusions may also be separated by time intervals of a week, two weeks or a month, or by time intervals of 6 weeks or 2, 3, 4, 5 or 6 months. Infusions may also be given once a year. According to some aspects of the present invention, since all injected cells are redirected to a specific target antigen (namely, the TGFeRII peptide containing a frameshift mutation, the sequence of which is given in SEQ ID NO: 1), a smaller number of cells can be introduced, which is in range 10 ⁶ /kg (10 ⁶ -10 ⁸ per patient). Cell compositions can be administered multiple times at doses that are within the indicated ranges. If necessary, treatment may also include the administration of mitogens (eg, PHA) or lymphokines, cytokines and/or chemokines (eg, IFN-γ, IL-2, IL-12, TGF-α, IL-18 and TGF-β, GM -CSF, IL-4, IL-13, Flt3-L, RANTES, MGRT-α, etc.) to enhance the induction of the immune response.

The immune effector cells of the present invention which express the TCR molecule of the present invention may be administered either alone or as a pharmaceutical composition in combination with diluents and/or other components such as IL-2 or other cytokines or cell populations. In general terms, the pharmaceutical compositions of the present invention comprise TKR-expressing immune effector cells, such as the T cells described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may contain buffers such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg aluminum hydroxide); and preservatives. Compositions according to the present invention are preferably formulated for intravenous administration.

As noted elsewhere for in vivo selectable markers for use in TCR-encoding vectors, adverse events can be minimized by transducing TCR-expressing immune effector cells with a suicide gene, such as inducible caspase 9 or thymidine kinase, to, after or during cell modification using the nucleic acid molecule of the present invention. Alternatively, as noted in relation to the TCR according to the present invention, the TCR may contain a label, in particular a double Myc label, which allows targeted killing of T cells according to the present invention using an antibody that recognizes the label used (for example, antibodies to Myc) .

The present invention also relates to the soluble TCR as defined herein and a composition containing such soluble TCR for use in therapy, in particular for use in the treatment of cancer.

Liquid pharmaceutical compositions, whether solutions, suspensions or similar forms,

- 33 042909 may contain one or more of the following: sterile diluents such as water, saline (preferably physiological saline), Ringer's solution, isotonic sodium chloride solution, fixed oils such as synthetic mono- or diglycerides (which can serve as as a solvent or suspending medium), polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; and tonicity adjusting agents such as sodium chloride or dextrose. The preparation for parenteral administration can be enclosed in ampoules, disposable syringes or refillable vials made of glass or plastic. The pharmaceutical composition for injection is preferably sterile.

Pharmaceutical compositions according to the present invention can be administered using a method appropriate to the disease to be treated (or prevented). The number and frequency of administrations will be determined by factors such as the patient's condition and the type and severity of the patient's disease, although appropriate doses may be determined in clinical studies.

An immune response induced in a subject by administration of immune effector cells expressing the TCRs of the present invention as described herein may include cellular immune responses mediated by cytotoxic T cells or NK cells capable of killing target cells (i.e., tumor cells). cells expressing a TGFeRII peptide carrying a frameshift mutation, the sequence of which is shown in SEQ ID NO: 1), regulatory T cells and helper T cells. Humoral immune responses can also be induced, mediated primarily by helper T cells capable of activating B cells and thereby inducing a humoral response.

Administration to a subject of soluble TCR bearing a toxin, as described herein, results in uptake of the TCR/toxin conjugates by the target cells, resulting in direct and selective killing of the target cells by the toxin.

If an effective amount is indicated, then the exact amount of the compositions to be administered can be determined by the physician, taking into account individual differences in age, weight, grade of malignancy and general condition of the patient (subject). The optimal dosage and treatment regimen for a particular patient can be readily determined by one of skill in the medical field by observing the subject for signs of disease and adjusting treatment accordingly.

Accordingly, the present invention relates to the treatment of a subject diagnosed or suspected or at risk of developing cancer that expresses a frameshift mutant variant of TGFeRII, said mutant frameshift variant comprising in its sequence a neopeptide comprising the sequence of SEQ ID NO : 1. Such cancer is likely to be MSI+ cancer, although it may be non-MSE cancer. The cancer can be any cancer that expresses a neopeptide containing the sequence of SEQ ID NO: 1. In particular, non-limiting cancers are colorectal cancer, gastric cancer, liver cancer, ampullary carcinoma, endometrial cancer, pancreatic cancer, or leukemia. Cancer, in particular, may be in a patient suffering from Lynch syndrome/HNPCC. According to a specific implementation variant of the present invention proposed the treatment of colorectal cancer in a subject with HNPCC.

The immune effector cells and/or soluble TCRs of the present invention may be administered in combination with one or more other therapeutic agents, which may include any other known cancer treatment such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormonal therapy, photodynamic therapy. etc. Immune effector cells and soluble TCR according to the present invention can be administered together as a combination. The compositions may also be administered in combination with antibiotics or other therapeutic agents, including, for example, cytokines (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15 and IL-17), growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory drugs, analgesics, chemotherapeutic agents ( for example, auristatin E monomethyl, fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, oxaliplatin, mitomycin, dacarbazine, procarbazine, pozid, tenipozid, campatecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, and 5-fluorouracil), radiotherapy agents, immune checkpoint inhibitors (eg, tremelimumab, ipilimumab, nivolumab, MK-3475, urelumab, bavituximab, MPDL3280A, and MEDI4736 ), small molecular weight inhibitors or other active and auxiliary agents.

Accordingly, immune effector cells, soluble TCRs, and compositions of the present invention are intended for use in therapy or can be used in the manufacture of a medicament for use in therapy, in particular cancer therapy, in particular

- 34 042909 STI, therapies for the treatment of MSI+ cancer or colorectal cancer. The immune effector cells of the present invention (and compositions containing them) are particularly useful in adoptive cell transfer therapy (also known as adoptive cell therapy) as described herein.

The term target cell refers to any cell that is to be killed or neutralized by the modified immune effector cells or soluble TCRs of the present invention. As noted above, it will typically be a cancer (or, in other words, tumor) cell that expresses a frameshift mutant variant of TGFeRII, said mutant frameshift variant containing in its sequence a neopeptide containing the sequence of SEQ ID NO: 1. The target cell may be, in certain embodiments, an MSI+ cancer cell, or a colorectal cancer cell, or a gastric cancer cell, or actually a cancer cell from any of the cancers listed above.

As used herein, cancer is defined broadly to include any neoplastic condition, whether malignant, pre-malignant, or non-malignant. However, it can usually be a malignant condition. Both solid and non-dense tumors are included within the scope of the present invention, and the term cancer cell may be taken as a synonym for tumor cell.

In one embodiment, the cells, soluble TCR, and/or compositions of the present invention may be administered intravenously to a subject. According to another embodiment of the present invention, the cells, soluble TCR and/or composition can be injected directly into the tumor by intratumoral injection.

The subject to be treated using the methods and cells of the present invention may be any mammalian species. For example, the subject may be any kind of domestic animal such as a mouse, rat, rodent, rabbit, guinea pig, hamster, cat, or dog, or livestock such as a goat, sheep, pig, cow, or horse. In another preferred embodiment of the present invention, the subject may be a primate such as an ape, gibbon, gorilla, orangutan, chimpanzee, or bonobo. However, according to a preferred embodiment of the present invention, the subject is a human. It is contemplated that the immune effector cells for use in the present invention may be derived from any mammalian species, however, in a preferred embodiment of the present invention, the immune effector cells will be derived from the same mammalian species as the subject to be treated.

The present invention can be more fully understood based on the non-limiting examples below and with reference to the drawings, which show the following.

In FIG. 1 shows that initially isolated T cell clones are specific for TGFeRII carrying a frameshift mutation, are CD877CD477CD56+, and induce target cell death in a dose dependent manner.

In FIG. 1a shows that the original T cell clone is a CD8-CD4 ^- and CD56 ⁺ T cell.

In FIG. 1b shows the results of quantitative Cr ⁵¹ release studies demonstrating specific lysis induced by T cell clone 26 of colon cancer cell lines loaded with 1 μM of the p573 peptide (which has the sequence of SEQ ID NO: 1) or the control peptide I540 (which has the sequence of SEQ ID NO: 54), at various specified effector-target ratios (E:T).

In FIG. 1c presents the results of quantitative Cr ⁵¹ release studies demonstrating specific lysis induced by a T cell clone, autologous EBV-LCL (Epstein-Barr virus-transformed lymphoregional cell line) or T2 cells loaded with titrated concentrations of the p573 peptide. Also presented are the results of blocking using antibodies to HLA-class I at the maximum concentration of the peptide. The E:T ratio was 25:1. The results shown are representative of three independent experiments.

In FIG. 2 shows TCR expression specific for TGFeRII carrying the -1A frameshift mutation (TGFeRII ^mut ) in transfected T cells expanded in vitro. Expression of TGFeRII ^mut -specific TCR corresponds to T cells that are Ve3 ⁺ (Ve3=TCR β-chain variable domain, TRBV28). Mock transfected T cells, T cells transfected with TCR mRNA, and the original T cell clone were tested.

In FIG. 3 shows that both TKR-transfected CD4+ T cells and TKR-transfected CD8+ T cells produce IFN-γ and TNF-α in response to colon cancer cell lines carrying the TGFeRII-1A frameshift mutation. T cells were transfected with TKR mRNA and incubated overnight prior to co-incubation with colon cancer cell lines LS174T and SW480 expressing mutated TGFeRII. The LS174T line is negative for HLA class I, while SW480 expresses HLA-A2. SW480 cells were loaded (+) or not loaded (-) with the T9FeRII peptide carrying the frameshift mutation, p573. Following overnight stimulation, cells were stained intracellularly to assess cytokine production. On the charts before

- 35 042909 missing populations of CD4 or CD8 T cells, as indicated. The results shown are representative of three independent experiments.

In FIG. 4 shows that CD8+ T cells transfected with TGFβRII ^mut -TCR produce more IFNγ and degranulate more efficiently than the original T cell clone in response to p573 peptide-loaded colon cancer cell lines. TCR-transfected CD8+ T cells and original T cell clone were incubated with colon cancer cell lines for 6 hours prior to CD107a and IFN-γ staining. All colon cancer cell lines carry the TGFeRII-1A frameshift mutation. Both lines, HCT116 and SW480, are HLA-A2+, while the colon cancer line LS174T is HLA-A2 ^- . The graphs show the missing population of CD8 T cells. The results shown are representative of three independent experiments.

In FIG. 5 shows that T-cells transfected with TGFβRII ^mut -TKR induce cell death in target cells carrying the TGFeRII-1A frameshift mutation with an efficiency that is comparable to that of the original T-cell clone. Colon cancer cell lines LS174 and HCT116 were loaded with Cr ⁵¹ . Half of the HCT116 cells were loaded with the T9FeRII peptide carrying the frameshift mutation, p573. The patient's original T cell clone, TKR-transfected CD8+ T cells, and mock-transfected CD8+ T cells were added at the indicated E:T ratio. Cells were co-incubated for 6 hours before measuring the release of Cr ⁵¹ . The results shown are representative of three independent experiments.

In FIG. 6 shows that TGFβRII ^mut -TCR transduced T cells are effective in vitro and in vivo. Donor T cells were transduced using TGFeRII ^mut TCR or, as a negative control, DMF5 (MART-1-specific) TCR.

In FIG. 6A shows that transduction efficiency was found to be about 60% for each of the TCRs when T cells were stained with an anti-Ve3 antibody (TGFβRII ^mut -TCR) or an anti-MART-1 dextramer (DMF5).

In FIG. 6B shows the results of a cognate antigen reactivity test of transduced T cells prior to infusion. HLA-A2+ EBV-LCL were loaded with a long peptide containing a frameshift mutation TGFeRII-1A spanning the epitope of CD8+ T cells (p621, SEQ ID NO: 55) or native peptide 26-35 MART-1 (SEQ ID NO: 56 ). Transduced T cells were co-incubated with EBV-LCL for 5 hours at an E:T ratio of 1:3 and stained for degranulation (CD107a) and TNF-α.

In FIG. 6C NSG mice (TGFeRII ^mut -TKR, n=10, MART-1 TKP, n=10) received intraperitoneal (ip) injections of 106 HCT116 cells expressing firefly luciferase two days before the intraperitoneal injection of 8x106 TKR-transduced T -cells (injections on day 0 and 2, respectively). The treatment was repeated on days 5 and 10 using 2x10 ⁷ TGFeRII ^mut TCR ⁺ T cells. Tumor burden was assessed by bioluminescence imaging at days 2, 9, 16, 24 and 30.

In FIG. The 6D bioluminescence signals (photons/s) for all mice are presented in a scatterplot showing the mean (±SD of the mean).

In FIG. 6E, the results of the Kaplan-Meier survival analysis indicate that mice treated with TGFeRII ^mut TCR ⁺ T cells had a significantly longer survival period compared to control mice (p=0.038; unpaired t-test). The in vivo experiments were repeated three times and the results of one representative experiment are presented.

In FIG. 6F tumors were excised from euthanized mice, single cell suspensions were prepared and stained to detect the presence of transduced T cells using an anti-CD3 antibody and an anti-Ve3 antibody (TGFeRII ^mut -TCR) or an anti-MART-1 dextramer (DMF5). The percentage of MART-1 specific T cells in tumors of the control group was significantly lower than the percentage of TGFeRII ^mut -specific T cells in tumors of mice treated with TGFeRII ^mut -specific TCR (p=0.0038).

In FIG. 7 shows that CD107a expression for TKR-transfected CD8+ T cells was not significantly reduced after stimulation with peptide-loaded HLA-A2+ HEK cells unable to bind CD8. HEK293 cells were transfected with mRNA encoding wild-type HLA-A2 or mutant HLA-A2 unable to bind CD8. FIEK293 cells were loaded with p573 peptide and used to stimulate CD8+ T cells transfected with TGFeRII ^mut specific TCR for 5 hours.

In FIG. 8 shows that the HCT116 colon cancer cell expresses the TRAIL receptor 4 (CD261). The left panel shows the results of staining HCT116 cells with isotype controls; the right panel shows the results of CD261 and CCR6 staining on HCT116 cells.

In FIG. 9 shows that both CD4+ T cells and CD8+ T cells targeted by Radium-1 TCR directly kill target cells presenting the cognate antigen. Purified CD4+ T cells (A) or purified CD8+ T cells (B) electroporated with

- 36 042909 by altering the Radium-1 mRNA can cause death of target cells carrying both the specific frameshift mutation TGFeRII and HLA-A2 (HCT116, colon cancer cells) (no peptide). Addition of exogenous TGFeRII long peptide carrying a frameshift mutation, p621 (SEQ ID NO: 55), which contains SEQ ID NO: 1 (also known as the p573 sequence), resulted in increased cell killing. Purified CD4+ T cells (C) or purified CD8+ T cells (D) were also able to kill HLA-A2 positive cell lines (Granta, a B-cell lymphoma cell line negative for the presence of the TGFeRII frameshift mutation) loaded with exogenous p621 peptide. All data are representative of at least two independent experiments.

In FIG. 10 shows that T cells electroporated with Radium-1 TCR mRNA reduce tumor growth in vivo in mice after multiple infusions. (A) the experimental time scale is shown; (B) presents the tumor load for different groups of mice (n=10 for each group) measured on the basis of bioluminescence. Bioluminescence signals are presented as total flux (photons/s). Mean values for all mice are presented in the graph above; error bars indicate the standard deviation of the mean.

In FIG. 11 compares EC ₅₀ ^CD8 T cells expressing Radium-1 TCR with EC ₅₀ ^CD8 T cells expressing DMF5 TCR, each of these cell types recognizing a different cognate antigen. All data are representative of two independent experiments.

In FIG. 12 shows the results of a flow cytometry study of SupT1 cells to detect binding of soluble TCR Radium-1. Soluble TCR does not bind to HLA-A2 negative cells (A) or HLA-A2 positive cells presenting a non-specific peptide (B). However, soluble TCR has been shown to bind to HLA-A2 positive cells that carry the TGFeRII peptide carrying a frameshift mutation, pc73 (SEQ ID NO: 1).

Examples

Example 1. Materials and methods.

Cell lines, media and reagents.

A CTL (cytotoxic T-lymphocyte) clone reactive for TGFeRII carrying a frameshift mutation and limited by HLA-A2 was isolated from the blood of a patient suffering from MSI+ colon cancer and cloned using the limiting dilution method. The patient was vaccinated with a 23-mer TGFeRII (-1A) peptide carrying a frameshift mutation (peptide containing the sequence of SEQ ID NO: 49). The clinical study was approved by the Norwegian Medicines Agency, the Medical Research Ethics Committee, the Southern Region and the Hospital Review Board. The treatment was carried out in accordance with the Declaration of Helsinki of the World Medical Association. The informed consent of the patient was obtained. An autologous Epstein-Barr virus-transformed lymphoblastic cell line (EBVLCL) was obtained by transforming donor B cells. A T2 cell line incapable of antigen presentation was used as a target T cell in a quantitative flow cytometric assay and a quantitative cytotoxicity assay. Colon cancer cell lines HCT116, SW480 and LS174T, as well as human mesonephros 293 (HEK) cells were obtained from ATCC (Rockville, MD, USA). HEK-Phoenix (Hek-P, collection of the present inventors) was grown in DMEM (PAA, Pasching, Austria) supplemented with 10% fetal calf serum (FBS) HyClone (HyClone, Logan, UT, USA) and 1% antibiotic/antimycotic mixture (penicillin/streptomycin, p/s, RAA).

Unless otherwise indicated, cells were cultured in RPMI-1640 (PAA Laboratories, Pasching, Austria) supplemented with gentamicin, 10% heat-inactivated FST (PAA Laboratories, Pasching, Austria). Colon cancer cell lines were treated with 500 U/ml IFN-γ (PeproTech, Rocky Hill, NJ, USA) overnight prior to use as target cells.

All T cells were grown in CellGro DC medium (CellGenix, Freiburg, Germany) supplemented with 5% heat-inactivated human serum (Trina Bioreactives AG, Nanikon, Switzerland), 10 mM N-acetylcysteine (Mucomyst, AstraZeneca AS, London, UK), 0.01 M HEPES (Life Technologies, Norway) and 0.05 mg/ml gentamicin (Garamycin, Schering-Plough Europe, Belgium), hereinafter referred to as complete medium unless otherwise indicated.

Obtaining T-cell lines and clones specific for TGFeRII peptides carrying a frameshift mutation.

PBMCs collected before and after vaccination were available for analysis. PBMCs were isolated and frozen as previously described (Brunsvig, PF et al. (2006) Cancer Immunol Immunother 55(12): 1553-1564). Thawed PBMCs were stimulated once with the peptide for 10-12 days in vitro and then tested in triplicate in quantitative T cell proliferation studies (H ³ -thymidine) using autologous PBMCs as APCs. PBMCs from different time points were stimulated with TGFeRII peptides carrying a frameshift mutation. pep

- 37 042909 tids included 573 (p573, SEQ ID NO: 1) and 621 (p621, SEQ ID NO: 55) of the TGFeRII protein carrying a frameshift mutation resulting from a 1 bp deletion. (-1A) in the adenosine region (A10) from TGFBRII bases #709-718 (GenBank sequence accession number for wild-type human TGFBRII: NM 003242). The hTERT peptide, I540 (SEQ ID NO: 54) was used as a negative control. Both peptides were provided by Norsk Hydro, ASA, Porsgrunn, Norway.

The MART-1 peptide containing the sequence of SEQ ID NO: 56 (amino acids 26-35 of native MART-1) was manufactured by Prolmmune Ltd, UK. The stimulated T cells were then tested in quantitative proliferation assays against peptide-loaded APCs, either autologous PBMCs or EBV-LCL. Stimulation Index (SI) was defined as the proliferation score with peptide divided by the proliferation score without peptide, and SI>2 was considered a positive response. T cell clones from reactive T cell lines were prepared as previously described (Saeterdal, I. et al. (2001), Cancer Immunol Immunother 50(9):469-476).

Cloning of TCR and HLA-A2.

T cell clones specific for the frameshift mutation (26 and 30) were grown and total RNA was obtained. Cloning was performed using a modified 5'-RACE method. In general terms, cDNA was synthesized using the oligo-dT primer and a region consisting of cytosine residues was added to the 5' end. For amplification of TCR chains, a polyguanosine primer with a primer specific for constant domains was used. The amplicon was cloned and sequenced. An expression construct was obtained by amplifying the TKR α-chain and β-chain separately using specific primers, and a second PCR was performed to hybridize the TKR chains to obtain the TKR-2A construct. The TKR-2A reading frame was cloned into pENTR (Invitrogen) and then recombined into other expression vectors.

For RNA synthesis, the insert was subcloned into a Gateway-modified pCIpA ₁₀₂ variant (SaeboeLarssen, S et al. (2002), J Immunol Methods 259(1-2): 191-203). A detailed description of the method as well as the primer sequence can be found in (Walchli, S et al. (2011), PloS one 6(11):e27930). For retroviral transduction, the insert was subcloned into the pM71 vector. The HLA-A*0201-pCIpA ₁₀₂ construct was cloned as previously described (Stronen, E et al. (2009) Scand J Immunol 69(4):319-328). This construct was used as a template to generate a CD8-incapable mutant variant by targeting residues D227 and T228 and replacing them with K and A, respectively, as described in (Xu, XN et al. (2001), Immunity 14(5):591-602). Standard site-directed mutagenesis was performed using the following primers: 5'-GAGGACCAGACCCCAGAAGGCGGAGCTCGTGGAGAC-3' (SEQ ID NO: 57) and 5'-GTCTCCACGAGCTCCGCCTTCTGGGTCTGGTCCTC-3' (SEQ ID NO: 58). HEK293 cells were transfected with the resulting constructs using FuGENE-6 (Roche, Switzerland) according to the manufacturer's protocol.

Transcription of mRNA under in vitro conditions.

In vitro mRNA synthesis was essentially performed as previously described (Almasbak, H et al. (2011), Cytotherapy 13(5):629-640). An anti-reversal cap analog (Trilink Biotechnologies Inc., San Diego, CA, USA) was used to cap the RNA. mRNA was assessed by agarose gel electrophoresis and Nanodrop device (Thermo Fisher Scientific, Waltham, Massachusetts, USA).

In Vitro Propagation of Human T Cells T cells from healthy donors were expanded using a protocol adapted to obtain T cells using Dynabeads CD3/CD28 according to the NMP requirements essentially as previously described (Almasbak, H. et al. (2011), Cytotherapy 13(5):629-640). In general terms, PBMCs were isolated from buffy coats by density gradient centrifugation and cultured with Dynabeads (Dynabeads® ClinExVivo™ CD3/CD28, courtesy of Dynal Invitrogen, Oslo, Norway) at a 3:1 ratio in CellGro DC complete medium supplemented with 100 U/ml of recombinant human interleukin-2 (IL-2) (Proleukin, Novartis Healthcare, USA) for 10 days. Cells were frozen and aliquots were thawed and left alone in complete medium until transfection.

Electroporation of expanded T cells.

The expanded T cells were washed twice, resuspended in CellGro DC medium (CellGenix GmbH) and resuspended to a concentration of 7 x 10 ⁷ cells/ml. mRNA was mixed with the cell suspension to a concentration of 100 μg/mL and electroporated in a 4 mm lumen cuvette at 500 V with a time constant of 2 ms using a BTX 830 square pulse electroporator (BTX Technologies Inc., Hawthorne, NY, USA) . Immediately after transfection, T cells were transferred to complete culture medium at 37° C. in 5% CO2 and incubated overnight to ensure TCR expression.

Antibodies and flow cytometry.

T cells were washed in staining buffer (SB) consisting of phosphate buffered saline (PBS) containing 0.1% human serum albumin (HSA) and 0.1% sodium azide before staining for 20 minutes at room temperature . The cells were then washed in SB and fixed in SB containing 1% paraformaldehyde. For intracellular staining, T cells were stimulated

- 38 042909 for 6 hours or overnight using APC loaded with p573 or without it, at a ratio of T cells: target 2:1 and in the presence of BD GolgiPlug and BD GolgiStop, diluted in a ratio of 1:1000. Cells were stained both extracellularly and intracellularly using the PerFix-nc kit according to the manufacturer's instructions (Beckman Coulter Inc, USA). The following antibodies were used: VP3-FITC (Beckman Coulter-Immunotech SAS, France), CD3-eFluor 450, CD4-eFluor 450, CD4-PE-Cy7, CD8-APC, CD8-eFluor 450, CD8-PE-Cy7, CD56- PE-Cy5.5 (BD Biosciences, USA), CD107a-PE-Cy5 (BD Biosciences, USA), CXCR2-PE, IFN-y-FITC, IL-2-APC, TNF-a-PE (BD Biosciences, USA ) and CD261/TRAIL-R4-PE (BD Biosciences, USA). Specific for MART-1 (amino acids 26-35) TCR was detected by staining with dextramer (Immudex, Denmark) according to the manufacturer's recommendations. All antibodies were purchased from eBioscience, USA, except where noted. Cells were obtained on a BD LSR II flow cytometer and data was analyzed using FlowJo software (Treestar Inc., Ashland, OR, USA).

Quantitative studies of the release of Cr ⁵¹ .

Quantitative studies of Cr ⁵¹ release to assess cytotoxicity were performed by labeling 2x106 target cells in 0.5 ml FST with Na2Cr ⁵¹ O4 (7.5 MBq) (Perkin Elmer, Waltham, Massachusetts, USA) for 1 h with gentle agitation every 15 min. Cells were washed three times in cold RPMI-1640 medium and plated at a density of 2x103 target cells in 96-well U-bottom microtiter plates. Autologous EBV-LCL cells, T2 target cells or HCT116, SW480 and LS174T colon cancer cells were injected with 10 μM p573 or pI540 for 1 hour at 37°C. The original T-cell clone, TKR-transfected T-cells or mock-transfected T-cells were added at the indicated effector:target (E:T) ratios and the plate was left for 4 hours at 37°C, as indicated. Maximal and spontaneous release of Cr ⁵¹ from target cells was determined after incubation with 5% Triton X-100 (Sigma-Aldrich, Oslo, Norway) or medium, respectively. Supernatants were collected on Luma plates (Packard, Meriden, Conn., USA) and Cr ⁵¹ released from lysed cells was measured using a TopCount microplate scintillation counter (Packard Instrument Company, Meriden, USA). The percentage specific release of chromium was calculated by the formula: [(experimental release-spontaneous release)/(maximum release-spontaneous release)] x 100.

retroviral transduction.

PBMC isolated from healthy donors were cultured and activated in CellGro DC medium (CellGenix GmbH, Germany) supplemented with 5% human serum (HS) and 100 U/ml IL-2 (Proleukin, Novartis Healthcare) for 48 h at 24 -well plate pre-coated with anti-CD3 antibody (OCT-3) and anti-CD28 antibodies (BD Biosciences, USA). After two days of culture, PBMCs were harvested and transduced twice with retroviral supernatant. Spinoculation of PBMCs was performed using 1 volume of retroviral supernatant in a 12-well untreated culture plate (Nunc A/S, Roskilde, Denmark) pre-coated with retronectin (20 μg/ml, Takara Bio. Inc., Shiga, Japan). Two days later, cells were harvested using PBS-EDTA (0.5 mM). Transduced T cells were further expanded with Dynabeads CD3/CD28 as described above.

Studies using a xenograft mouse model.

Mice of the NOD.Cg-Prkdc ^scid Il2rg ^tm1Wj1 /SzJ (NSG) line were grown at our own base in accordance with the approved animal care protocol of the organization and maintained under sterile conditions. Mice aged 6-8 weeks received 1-1.5x106 HCT116 tumor cells ip. HCT116 cells were modified using a retroviral vector (provided by Dr. Rainer Low, EUFETS AG, Idar-Oberstein, Germany) to express firefly luciferase and EGFP. Tumor growth was monitored by bioluminescence imaging using the Xenogen Spectrum system and Living Image v3.2 software. Anesthetized mice were injected ip with 150 mg/kg body weight of D-luciferin (Caliper Life Sciences, Hopkinton, Massachusetts, USA). Animals were examined using imaging methods 10 minutes after injection of luciferin.

Statistical analysis.

Continuous data were described using the median, mean, and range. The Mann-Whitney test was used to analyze tumor burden, while survival was calculated using the Kaplan-Meier method with an unpaired t-test, which was used to compare group survival. All p values given are two-sided values. A p value below 0.05 was considered significant. All statistical analyzes were performed using GraphPad Prism® (GraphPad Software, Inc., USA).

Results.

Isolation of a T cell clone specific for TGFeRII carrying a CTL frameshift mutation, reactive for TGFeRII carrying a frameshift mutation, and restricted to HLA-A2, was isolated from the blood of a patient with MSI+ colon cancer. The patient was vaccinated with a 23-mer TGFeRI peptide carrying a frameshift mutation

- 39 042909 reading, the sequence of which is given in SEQ ID NO: 49. CTL clones were shown to be CD8-CD4- and approximately 50% of the cells expressed CD56 (Fig. 1a). CTL clones were previously thought to be monoclonal because they were shown to express TCR Ve3 (or TRBV 28, IMGT nomenclature) (Kyte, JA (2009), Expert Opin Investig Drugs 18(5):687-694). Molecular cloning revealed that they were indeed sister clones containing the same pair of TCR chains. Specific lysis of colon cancer cell lines HCT116 and SW480 was observed in the absence of exogenously loaded peptide. However, the E:T ratio required to lyse cell lines with the endogenous peptide was higher than when the cell lines were loaded with the exogenous TGFeRII peptide carrying the frameshift mutation (p573, SEQ ID NO: 1). In control experiments, another colon cancer line, LS174T, which is HLAA2 negative but expresses the TGFeRII mutation, was not lysed (FIG. 1b). Importantly, despite CD56 expression on the T cell clone (Fig. 1a), the HLA-A2-negative LS174T cell line did not undergo lysis, indicating that cell death was not mediated by activity similar to that of natural cells. -killer (NK), and was caused by the specific recognition of MHC molecules loaded with the peptide.

To test the relative avidity of T cell clones, TAP-deficient T2 cells were loaded with titrated amounts of the peptide (0.01-1.0 μM). The cell killing activity was found to be dependent on peptide concentration (FIG. 1c). The addition of HLA-specific blocking antibodies reduced cell death (Fig. 1c), confirming the limitation of TCR by HLA class I. Similar observations were made using autologous EBV-LCL cells as APCs. Taken together, the data suggest that TGFβRII ^mut -specific T cells were negative for the presence of co-receptors, peptide-specific, and restricted to HLA class I.

TGFβRII ^mut -TCR is expressed and active in both CD4+ and CD8+ T cells after mRNA electroporation.

The α- and β-chains of TCR from TGFβRII ^mut -reactive sister clones of the T cell were identified and were hereinafter referred to as TCR Radium-1. Two strands were cloned into an mRNA expression vector (see Materials and Methods) and T cells expanded for 10 days in vitro were electroporated to assess their ability to recognize their targets. Radium-1 TCR expression was measured in both CD4+ and CD8+ T cells by surface staining with anti-Ve3 antibody (TRBV 28) (FIG. 2a). Approximately 70% of the transfected T cells expressed the Ve3 chain, with 42% of these T cells being CD8 positive and 32% of the T cells expressing CD4, while less than 5% of the cells naturally express Ve3.

The activity of the Radium-1 transfected T cells was then monitored by intracellular cytokine staining when co-incubated with colon cancer cell lines SW480 and LS174T. The SW480 colon cancer cell line was recognized by both CD8+ and CD4+ T cells in the absence and presence of an exogenously loaded peptide. T cells produced TNF-α and IFNγ (FIG. 3). As expected, the LS174T colon cancer cell line was not recognized. These data confirmed the limitation of Radium-1 TCR on the HLA peptide and its ability to effectively redirect both CD4+ and CD8+ T cells.

Production of CD107a and IFN-γ in CD8+ T cells transfected with Radium-1 TCR.

To determine the cytotoxic potential of TKR-transfected CD8+ T cells against colon cancer cell lines, T cells after mRNA electroporation were co-incubated with colon cancer cell lines for 6 hours and stained with antibodies against degranulation markers, CD107a and IFN-γ (Fig. . 4). Very low levels of IFN-γ production and CD107a expression were detected in the absence of an exogenously loaded peptide. After the addition of p573 peptide (SEQ ID NO: 1), both TCR Radium-1 transfected T cells and the original T cell clone were highly activated. Interestingly, TKR-transfected T cells produced IFN-γ more efficiently and also showed higher levels of degranulation than the original T cell clone, while mock-transfected T cells were not activated. To test for non-dependence of TCR on co-receptors, HEK293 cells were transfected with wild-type (WT) HLA-A2 or mutant HLA-A2 unable to bind CD8, loaded with p573 peptide, and used to stimulate TCR-transfected CD8+ T cells. The number of T cells expressing CD107a was 36% (wild-type HLA-A2) and 26% (HLA-A2 mutant), indicating that this TCR is at least partially independent of co-receptors (Fig. 7) .

T cells transfected with Radium-1 TCR are able to mediate specific lysis of tumor cells.

In addition to cytokine production, the main function requiring adoptive transfer of redirected T cells is the specific killing of tumor cells. To examine the ability of TKR-transfected T cells to lyse target cells, they were tested against colon cancer cell lines in a 6-hour quantitative chromium release assay (Fig.

- 40 042909

5) . TKR-transfected T cells lysed HCT116 cells at levels comparable to those of the original patient clone. As expected, lysis was further increased by the addition of exogenous p573 (SEQ ID NO: 1). Lysis of the HLA-A2-negative LS174T cell line was comparable to that of mock-transfected T cells, suggesting that the low non-specific lysis of HCT116 is likely due to TRAIL-R expression on the target cells (Fig. 8). This cell line has been reported to be susceptible to TRAIL-mediated lysis (Tang, W et al., (2009) Febs J 276 (2): 581-593).

T cells transduced with Radium-1-TCR are effective in vitro and in vivo.

A mouse colon cancer xenograft model was created by intraperitoneal injection of HCT116 (Kishimoto, H et al. (2009), Proc Natl Acad Sci USA 106(34): 14514-14517). T cells were transduced with TCR with a retrovirus and tested for expression, which was approximately 60% for both Radium-1 TCRs and the MART-1-specific TCR (DMF5) used as control (FIG. 6A). Prior to injection, T cells were subjected to functional testing against HLA-A2+ EBV-LCL cells loaded with TGFeRII long peptide carrying a frameshift mutation (p621, SEQ ID NO: 55) or low affinity (wild type) MART1 peptide (SEQ ID NO : 56) (Fig. 6B). All Radium-1 TCR-expressing T cells responded to EBV-LCL loaded with the TGFeRII long peptide carrying a frameshift mutation, while approximately half of the cells expressing TCR MART-1 responded to the low-affinity MART-1 peptide. . NSG mice received ip injections of 106 HCT116 cells on day 0 and on days 2, 5 and 10 mice were injected with 8x106 (day 2) and 2x10 ⁷ (day 5 and 10) T cells (FIG. 6C). Control mice received T cells expressing MART-1-specific TCR.

In vivo imaging studies of mice revealed that tumor burden was significantly lower (p=0.038) in mice treated with TGFeRII ^mut specific T cells compared to MART-1 specific control T cells (Fig. 6D). Mice treated with TGFβRII ^mut -specific T cells also had increased survival compared to control mice (p=0.038, Fig. 6E). Tumors were excised from mice that had to be euthanized due to high tumor burden. Single cell suspensions of tumors were prepared and stained with anti-human CD3 antibody and anti-Ve3 antibody or anti-MART-1 dextramer. The percentage of TCR-expressing T cells in the tumor was found to be significantly higher in mice treated with TGFβRII ^mut -specific T cells (p=0.0038, Fig. 6F), despite the transduction efficiency of the two populations The T cell count was very similar, indicating that TGFβRII ^mut -specific T cells were either more efficiently recruited to the tumor or proliferated in vivo as a result of antigen stimulation. Taken together, these data demonstrate the preclinical efficacy of Radium-1 TCR in vivo.

Example 2

To study target cell killing by CD4+ and CD8+ T cells transduced with Radium-1 TCR, target cells were stably transduced to express luciferase. Two sets of target cells were used: the HCT116 cell line and the Granta cell line. HCT116 cells are described above; the Granta cell line is a human B-cell lymphoma line. Changes in bioluminescence were used to measure changes in the number of target cells during culture with Radium-1 transduced T cells, based on which conclusions were drawn about the destruction of target cells by T cells.

Luciferase-transduced target cells were co-cultured with effector T cells at an effector:target (E:T) ratio of 30:1, and bioluminescence was measured. Cells were cultured for 24 hours and bioluminescence was measured at 1, 2, 3, 4, 5, 8, 11, 20, 21, 22, 23 and 24 hours. Effector T cells were co-cultured with Granta cells with and without exogenous p621 peptide (SEQ ID NO: 55), which contains the sequence of SEQ ID NO: 1.

Purified CD4+ T cells and purified CD8+ T cells transduced with Radium-1 mRNA were found to kill HCT116 cells and Granta cells (FIG. 9). Killing of Granta cells by both CD4+ and CD8+ T cells was significantly higher in the presence of p621 (+TGFeRII peptide) than in its absence (no peptide). This indicates that Radium-1 transduced CD4+ T cells are able to kill target cells without interacting with CD8+ T cells.

The in vivo cytolytic activity of T cells transiently transduced with Radium-1 was further studied in mice. NSG mice were injected ip with 106 HCT 116 cells stably transduced to express luciferase. Two days later (ie, day 2), mice were administered by ip or iv (intravenous) injection of 8-10x106 T cells transfected with Radium-1. Subsequent injections of Radium-1 transfected T cells were given on days 5, 7, 10, 13, 15 and 21 and tumor burden was assessed by bioluminescence imaging on days 2, 7, 17, 29, 45, 53 and 60

- 41 042909 (see Fig. 10A, the last rendering is not indicated).

Mice receiving T cells transfected with Radium-1 TCR had a significantly lower tumor burden than mice receiving mock-transfected T cells (FIG. 10B) (*p=0.01, Wilcoxon-Mann-Whitney test). Due to T cell alloreactivity, mock-transfected T cells have been shown to have some effect on tumor growth after multiple injections. However, this effect was temporary. Because TCR expression was transient in this case, T cells administered by intravenous (IV) injection had no effect on tumor growth.

The efficacy of Radium-1 TCR was compared in vitro with a known high affinity TCR. The MART-1-specific TCR, DMF5, was chosen for comparison. DMF5 has been used clinically to treat melanoma (Johnson, L.A. et al. (2006), J Immunol 177(9):6548-6559).

CD8 T cells were transduced using Radium-1 and MART-1 and sorted. TCR ⁺ T cells were incubated with HLA-A2+ T2 cells (T2 is a human lymphoblastic cell line that does not express MHC class II molecules) loaded with a TGFeRII peptide, p573, carrying a frameshift mutation (SEQ ID NO: 1), and peptide analog 26-35 MART-1, ELAGIGILTV (SEQ ID NO: 80), for 5 hours before staining to evaluate the degranulation marker CD107a as a marker of cytolytic capacity, followed by flow cytometry. The MART-1 peptide analog 26-35, the sequence of which is shown in SEQ ID NO: 80, contains a single amino acid substitution compared to the wild-type peptide containing the sequence of SEQ ID NO: 56, i.e. the alanine at position 2 of the sequence shown in SEQ ID NO: 56 is replaced by leucine. The resulting peptide analog has advantageous properties because it has increased affinity for HLA-A2, resulting in increased presentation of the peptide by HLA-A2-containing MHC class I molecules compared to the wild-type peptide.

It was shown that the EC ₅₀ value of p573 for Radium-1 was 2 nM, compared to the EC ₅₀ value of the MART-1 peptide containing SEQ ID NO: 56, which was 7 nm for DMF5 (Fig. 11). This indicates that Radium-1 has a very high affinity for the cognate antigen/MHC complex and is independent of CD8. In addition, Radium-1 has been shown to have a higher affinity for the cognate antigen/MHC complex than DMF5 TCR, which is known to be clinically effective.

Example 3

Soluble His-tagged Radium-1 TCR encoded by ssTCR, the sequence of which is given in SEQ ID NO: 69, was expressed in HEK cells. The supernatant of expressing HEK cells was isolated. SupT1 cells (HLA-A2 negative cell line) were transduced to express HLA-A2 either hybridized to a non-specific non-target peptide that is not recognized by Radium-1 or hybridized to a TGFeRII peptide carrying a frameshift mutation. Transduced cells were incubated for 30 min at room temperature with soluble TCR Radium-1; non-transduced cells were also incubated with soluble TCR as an additional negative control. After incubation, cells were washed and then stained with allophycocyanin (APC) to identify soluble TCR binding. Staining was performed using primary mouse anti-His followed by incubation with APC-conjugated secondary anti-mouse IgG. The stained cells were then examined by flow cytometry (FIG. 12).

As shown in FIG. 12A and 12B essentially did not stain the negative controls, indicating that Radium-1 soluble TCR did not bind to cells that do not express HLA-A2, or that express HLA-A2 but do not present the TGFeRII peptide carrying the mutation. with frame shift. In FIG. 12C shows that cells expressing HLA-A2 and presenting the TGFeRII peptide carrying the frameshift mutation were recognized by soluble Radium-1 TCR, indicating that it has the expected specificity.

Claims (16)

1. A nucleic acid molecule encoding a T-cell receptor (TCR) molecule directed against a mutated TGFeRII protein that contains the sequence of SEQ ID NO: 1, wherein said TCR molecule is capable of binding the peptide of SEQ ID NO: 1, while said peptide is represented by the main a class I histocompatibility complex (MCHC) containing HLA-A2, and said TCR molecule contains an α-chain domain and an e-chain domain, where the domain of each chain contains three CDR sequences, and: a) CDRs 1, 2 and 3 of the α-chain domain have the sequences of SEQ ID NOs: 2, 3 and 4, respectively; And b) CDRs 1, 2 and 3 of the e-chain domain have the sequences of SEQ ID NOs: 5, 6 and 7, respectively, and the α-chain domain contains: - 42 042909 i) a variable region containing the amino acid sequence shown in SEQ ID NO: 72 or an amino acid sequence that is at least 95% identical to said sequence, and ii) a constant region containing the amino acid sequence shown in SEQ ID NO: 9, or a modified version thereof containing one or more amino acid substitutions or insertions relative to SEQ ID NO: 9 and having at least 95% sequence identity with SEQ ID NO: 9, or a murinized version of SEQ ID NO: 9; and the β-chain domain contains: i) a variable region containing the amino acid sequence shown in SEQ ID NO: 75 or an amino acid sequence that is at least 95% identical to said sequence, and ii) a constant region containing the amino acid sequence shown in SEQ ID NO: 14, or an amino acid sequence that is at least 95% identical to SEQ ID NO: 14, or a murinized variant of SEQ ID NO: 14. 2. The nucleic acid molecule according to claim 1, which is characterized in that the variable region of the α-chain domain and/or the variable region of the β-chain domain contains a double Myc tag with the amino acid sequence shown in SEQ ID NO: 19. 3. A nucleic acid molecule according to claim 1 or 2, which is characterized in that the α-chain domain contains the amino acid sequence of SEQ ID NO: 11, and the β-chain domain contains the amino acid sequence of SEQ ID NO: 16. 4. A nucleic acid molecule according to claim 1 or 2, which is characterized in that the α-chain domain contains the amino acid sequence of SEQ ID NO: 12, and the β-chain domain contains the amino acid sequence of SEQ ID NO: 17. 5. A nucleic acid molecule according to claim 1 or 2, which is characterized in that: (i) the α-chain domain contains the amino acid sequence of SEQ ID NO: 25, and the β-chain domain contains the amino acid sequence of SEQ ID NO: 31; or (ii) the α chain domain contains the amino acid sequence of SEQ ID NO: 26 and the β chain domain contains the amino acid sequence of SEQ ID NO: 32. 6. Nucleic acid molecule according to any one of claims 1 to 5, characterized in that said TCR molecule is encoded as a single-stranded TCR (ssTCR) containing an α-chain domain connected to a β-chain domain by a linker, while preferably ssTCR contains in following order: (i) an α chain domain; (ii) a linker; and (iii) a β chain domain. 7. The nucleic acid molecule according to claim 6, which is characterized in that the specified linker is capable of autosplicing; while preferably the specified linker is a 2A peptide and contains the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that is at least 40% identical to the specified sequence. 8. The nucleic acid molecule according to claim 7, which is characterized in that said ssTCR contains the amino acid sequence of any of SEQ ID NOs: 33-40, while preferably said nucleic acid molecule contains the nucleotide sequence of any of SEQ ID NOs: 41-48. 9. Nucleic acid molecule according to any one of claims 1 to 8, characterized in that said nucleic acid is RNA. 10. Soluble T-cell receptor (TCR) molecule directed against the mutated TGFβRII protein, which contains the sequence of SEQ ID NO: 1, and the specified TCR molecule is capable of binding the peptide of SEQ ID NO: 1, while the specified peptide is represented by the class I major histocompatibility complex (MHC) containing HLA-A2, and at the same time, the specified TCR molecule contains an α-chain domain and a β-chain domain, where the domain of each chain contains three CDR sequences, and: a) CDRs 1, 2 and 3 of the α-chain domain have the sequences of SEQ ID NOs: 2, 3 and 4, respectively; And b) CDRs 1, 2 and 3 of the β-chain domain have the sequences of SEQ ID NOs: 5, 6 and 7, respectively, and the α-chain domain contains: i) a variable region containing the amino acid sequence shown in SEQ ID NO: 72, or an amino acid sequence that is at least 95% identical to said sequence, and ii) a constant region containing the sequence shown in SEQ ID NO: 60, or an amino acid sequence that is at least 95% identical to the specified sequence; and the β-chain domain contains: - 43 042909 i) a variable region containing the amino acid sequence shown in SEQ ID NO: 75 or an amino acid sequence that is at least 95% identical to said sequence, and i) a constant region containing the amino acid sequence shown in SEQ ID NO: 62, or an amino acid sequence that is at least 95% identical to the specified sequence. I. A soluble TCR molecule according to claim 10, characterized in that the constant region of said α-chain domain contains the sequence shown in SEQ ID NO: 61 and the constant region of said β-chain domain contains the sequence shown in SEQ ID NO: 63 . 12. Molecule of soluble TCR by i. 10 or 11, which is characterized by the fact that: i) said α-chain domain contains the sequence shown in SEQ ID NO: 73 or 74; j) said β-chain domain contains the sequence represented by SEQ ID NO: 76 or 77. 13. An immune effector host cell comprising a nucleic acid molecule according to any of pi. 1-9, wherein said immune effector host cell expresses the TCR encoded by said nucleic acid molecule. 14. An immune effector host cell according to claim 13, characterized in that said cell is a T cell or an NK cell. 15. A method of treating cancer, said method comprising administering to a subject in need thereof an immune effector cell according to claim 13 or 14, said cancer expressing a mutated ΤΘΕβΚΙΙ protein which contains SEQ ID NO: 1. 16. The method of claim 15, wherein the cancer is gastric cancer, liver cancer, ampullary carcinoma, endometrial cancer, pancreatic cancer, leukemia, or colorectal cancer. 17. The method according to claim 15 or 16, characterized in that said cancer occurs in a subject with hereditary non-polyposis colorectal cancer (HNPCC).