FIELD OF THE INVENTION

The present invention relates library of synthetic polynucleotides encoding TCR alpha chain constant regions and TCR beta chain constant regions of a TCR capable of binding to a PRAME peptide having the sequence VLDGLDVLL (SEQ ID NO: 3) for enhanced pairing of recombinant TCR alpha and TCR beta chains and/or enhanced surface expression of recombinant TCRs. The invention further refers to corresponding vector libraries, TCR libraries, cell libraries and methods for isolating TCRs with enhanced TCR alpha and TCR beta chain pairing using said libraries and TCRs isolated from said libraries as well as the medical use thereof.

BACKGROUND OF THE INVENTION

T lymphocytes are part of the adaptive immune response and originate from hematopoietic stem cells located in the bone marrow. T lymphocytes express a unique antigen binding receptor on their membrane, the T cell receptor (TCR), which recognizes antigens in association with major histocompatibility complex (MHC) molecules.

With their central role in the immune system, T cells typically provide protection from pathogens or malignant cells. Each T cell expresses a single form of a T cell receptor, a structure which is used by the T cell to recognize infected or altered cells.

The concept of immunotherapy is based on the specificity of the adaptive immune response for the recognition and elimination of pathogens as well as tumor cells. The aim of a successful immunotherapy is the manipulation or reprogramming of the patient’s immune response in order to specifically target pathogen-infected cells or tumor cells for destruction by the immune system.

DJB:ADW Therapeutic approaches used to reprogram the immune system in the treatment of infectious diseases and cancer include active immunotherapy comprising the use of vaccination strategies, including dendritic cell (DC) vaccines, as well as passive immunotherapy comprising the application of specific antibodies or genetically engineered lymphocytes or the adoptive transfer of T cells specifically recognizing target antigens displayed by pathogen-infected cells or cancers.

The principle of adoptive T cell transfer is based on the ex vivo expansion of autologous or allogeneic target-specific T lymphocytes and the subsequent re-infusion into patients. Cancer regression in patients suffering from metastatic melanoma has been observed after the transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs). The drawback of this therapeutic approach is the requirement for pre-existing tumor-reactive cells that need to be isolated from every individual patient as well as the difficult detection of TILs for cancers other than melanoma. Therefore, other methods were developed that focus on the genetic modification of T cells isolated from patients. These genetically engineered T cells can for example be created by transduction of autologous T cells with the a and [3 chains of target-specific TCRs, i.e. with recombinant TCRs. Likewise, there have also been successful treatments of lifethreatening infections with adoptive transfer of virus-specific T cells, for example to combat Epstein-Barr-Virus- and Cytomegalovirus-driven infections in immunocompromised individuals. This is a pathway that could also be pursued for treatment of COVID-19 patients in dire situations.

An exemplary approach, as developed by Wilde et al. in 2009 and described in WO 2007/017201 , allows the isolation of allo-restricted peptide-specific T cells using autologous DCs co-transfected with RNA species encoding both the target antigen and a selected allogeneic MHC molecule. By co-culturing autologous T cells with DCs presenting self-peptide/allo-MHC complexes, high-avidity T cells that recognize selfantigens can be obtained (Wilde et al., 2009, Dendritic cells pulsed with RNA encoding allogeneic MHC and antigen induce T cells with superior antitumor activity and higher TCR functional avidity. Blood, 114(10), 2131-9). In another exemplary approach, T cells and their corresponding TCRs can be obtained by culturing autologous T cells and autologous DCs expressing the human leukocyte antigens (HLAs) of choice and loaded with foreign antigens, such as those expressed by pathogenic viruses in infected cells. Because T cell cultivation and expansion of individual T cell clones is laborious and requires repeated rounds of re-stimulation, it is an advantage to rapidly acquire the sequences of the TCRs at an early time point in order to allow their rapid characterization by introducing them into recipient peripheral blood lymphocytecontaining T cells. This allows the characterization of the TCRs regarding antigen specificity, peptide/MHC-avidity and functionality before they are selected for further use in therapeutic applications in patients.

However, both safety and efficacy of TCR-T immunotherapy need further to be improved. For efficient immunotherapy strong surface expression of the TCR is vital. A prerequisite for high expression of the TCR on the cell surface is the correct pairing of the TCR alpha and TCR beta chains. In particular, if endogenous TCR chains are present, mispairing of the recombinant TCR chains with the endogenous TCR chains must be avoided.

WO201 7/216324 describes TCRs capable of binding to a preferentially expressed antigen in melanoma (PRAME) peptide having the sequence VLDGLDVLL (SEQ ID NO: 3), also denoted PRAMEVLD herein, or its MHC-bound form.

There is a need for methods to improve the preferential pairing of the recombinant TCR alpha and TCR beta chains in such TCRs targeting PRAMEVLD and improved pairing with the CD3 complex in such TCRs.

OBJECTIVES AND SUMMARY OF THE INVENTION

The present invention provides libraries and methods for improving the pairing of the recombinant TCR alpha and TCR beta chains of TCRs targeting PRAMEVLD. Thereby the surface expression of the transgenic TCR chains can be increased. The inventors could show that surprisingly these strategies lead to TCRs with increased functional avidity.

In particular, in TCR-T immunotherapy when recombinant TCRs are expressed in T cells carrying the endogenous TCR, the mispairing of recombinant TCR chains with endogenous TCR chains can thereby be reduced.

The inventors identified specific positions in the constant regions of the TCR alpha and TCR beta chain that impact binding of both chains and collated a library of tailored amino acid substitutions in the constant regions of the TCR alpha and TCR beta chains, which can be introduced to potentially elicit improved binding between the chains. The inventors found that the improved binding by the modified chains resulted in enhanced TCR surface expression and function leading to TCR-Ts expressing such TCRs with improved in vitro efficacy in target cell recognition.

Thus, a first aspect of the invention relates to a library of synthetic polynucleotides encoding TCR alpha chains and TCR beta chains of a TCR capable of binding to a PRAME peptide having the sequence VLDGLDVLL (SEQ ID NO: 3), comprising

- Polynucleotides encoding TCR alpha chains comprising: a) TCR alpha chain constant regions which are selected from the group of TCR alpha constant regions which are at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 1 ; b) TCR alpha chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5 and a CDR3 having the amino acid sequence of SEQ ID NO: 6;

- Polynucleotides encoding TCR beta chains comprising a) TCR beta chain constant regions which are selected from the group of TCR beta constant regions which are at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 15;or

TCR beta chain constant regions which are selected from the group of TCR beta constant regions which are at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 16; b) TCR beta chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 7, a CDR2 having the amino acid sequence of SEQ ID NO: 8 and a CDR3 having the amino acid sequence of SEQ ID NO: 9.

Accordingly, some embodiments relate to a library of synthetic polynucleotides encoding TCR alpha chains and TCR beta chains of a TCR capable of binding to a PRAME peptide having the sequence VLDGLDVLL (SEQ ID NO: 3), comprising

- Polynucleotides encoding TCR alpha chains comprising: a) TCR alpha chain constant regions which are selected from the group of TCR alpha constant regions which are at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 1 ; wherein the amino acid at position 14 is selected from the group consisting of S, V, W, A, C, F, G, I, L, M and P; the amino acid at position 66 is selected from the group consisting of N, Q, S, T and Y; the amino acid at position 92 is selected from the group consisting of S, V, W, A C, F, G, I, L, M and P; the amino acid at position 93 is selected from the group consisting of S, V, W, A C, F, G, I, L, M and P; the amino acid at position 107 is selected from the group consisting of T, V, W, A, C, F, G, I, L, M and P; the amino acid at positionl 15 is selected from the group consisting of S,

V, W, A, C, F, G, I, L, M and P; the amino acid at position 118 is selected from the group consisting of G,

I, L, M, P, V, W, A, C and F; and the amino acid at position 119 is selected from the group consisting of F, G, I, L, M, P, V, W, A and C; b) TCR alpha chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5 and a CDR3 having the amino acid sequence of SEQ ID NO: 6;

- Polynucleotides encoding TCR beta chains comprising a) TCR beta chain constant regions which are selected from the group of TCR beta constant regions which are at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 2 wherein the amino acid at position 4 is selected from the group consisting of K, N; the amino acid at position 5 is selected from the group consisting of N, R,

D, E, H and K; the amino acid at position 18 is selected from the group consisting of E, H, K, R and D; the amino acid at position 22 is selected from the group consisting of S, V,

W, A, C, F, G, I, L, M and P; the amino acid at position 34 is selected from the group consisting of T, D,

E, H, K, N and R; the amino acid at position 37 is selected from the group consisting of Y, F; the amino acid at position 136 is selected from the group consisting of E, V; the amino acid at position 157 is selected from the group consisting of T, V, W, A, C, F, G, I, L, M and P; and the amino acid at position 164 is selected from the group consisting of S,

V, W, A, C, F, G, I, L, M and P; the amino acid at position 177 is selected from the group consisting of S, F; the amino acid at position 178 is R or deleted; the amino acid at position 179 is G or deleted; b) TCR beta chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 7, a CDR2 having the amino acid sequence of SEQ ID NO: 8 and a CDR3 having the amino acid sequence of SEQ ID NO: 9.

In one embodiment the library comprises polynucleotides encoding a TCR alpha chain and a TCR beta chain, each polynucleotide comprising:

- Polynucleotide encoding TCR alpha chains as defined herein; and

- Polynucleotide encoding TCR beta chains as defined herein;

- Optionally a selection marker

- Optionally one or more internal ribosomal entry sites or self-cleaving peptides of the 2A family.

Typically, the library comprises at least 1 x 10 ⁵ , preferably 1 x 10 ⁸ , more preferably 1 x 10 ¹⁰ , even more preferably 1 x 10 ¹¹ , such as 2.25 x 10 ¹³ unique molecules.

Also encompassed is a library of vectors comprising the polynucleotide library as defined herein. Further encompassed is a library of cells, comprising said library of vectors. Moreover, also a library of cells expressing the polynucleotides as defined herein is encompassed. A further aspect relates to a library of polypeptides encoded by the library of synthetic polynucleotides as defined herein.

Another aspect refers to the use of the library as described herein for identifying a TCR receptor with enhanced TCR alpha and TCR beta chain pairing. A further aspect refers to a method for isolating a TCR with enhanced TCR alpha and beta chain pairing, comprising the steps:

- Co-culturing the library of cells defined herein with antigen presenting cells presenting the HLA bound form of the target antigen of the TCR; and at least one of the steps:

- Selecting cells showing elevated expression levels of the heterologous TCR compared to wild type

- Selecting cells showing elevated activation levels compared to wild type.

Accordingly, also a TCR isolated from the TCR library as described herein is encompassed.

Another aspect refers to a library of TCR alpha chains and TCR beta chains, comprising

- TCR alpha chains comprising: a) TCR alpha chain constant regions which are selected from the group of TCR alpha constant regions which are at least about 80% identical to the amino acid sequence as set out in SEQ ID NO 1 ; b) TCR alpha chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5 and a CDR3 having the amino acid sequence of SEQ ID NO: 6;

- TCR beta chains comprising a) TCR beta chain constant regions comprising constant regions which are selected from the group of TCR beta constant regions which are at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 15,

TCR beta chain constant regions comprising constant regions which are selected from the group of TCR beta constant regions which are at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 16; b) TCR beta chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 7, a CDR2 having the amino acid sequence of SEQ ID NO: 8 and a CDR3 having the amino acid sequence of SEQ ID NO: 9.

Also encompassed is a method of preparing a library of synthetic polynucleotides encoding a plurality of TCR alpha chain constant regions and TCR beta chain constant regions, the method comprising:

- Providing sequences of polynucleotides

(i) encoding TCR alpha chain constant regions and TCR beta chain constant regions as defined in herein; or

(ii) encoding the TCR alpha chain and TCR beta chain as defined in herein;

- Assembling the synthetic polynucleotide sequences to produce a library of synthetic polynucleotides.

A further aspect refers to a kit comprising one or more of the libraries described herein.

Another aspect of the invention refers to nucleotide sequences of the synthetic polynucleotides defined herein in computer readable format. Accordingly, the invention also encompasses the amino acid sequences as defined herein in computer readable format.

In a specific embodiment, the invention relates to a TCR capable of binding to a PRAME peptide having the sequence VLDGLDVLL (SEQ ID NO: 3), wherein the TCR comprises:

- A TCR alpha chain comprising

A variable TCR alpha region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5 and a CDR3 having the amino acid sequence of SEQ ID NO: 6; and

- A constant TCR alpha region comprising the sequence SEQ ID NO: 12; and

- A TCR beta chain comprising

- A variable TCR beta region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 7, a CDR2 having the amino acid sequence of SEQ ID NO: 8 and a CDR3 having the amino acid sequence of SEQ ID NO: 9; and - A constant TCR beta region comprising the sequence SEQ ID NO: 13.

FIGURE LEGENDS

Figure legends

Figure 1 Figure 1 A and 1 B show the amino acid sequence of the alpha chain constant region as defined in SEQ ID NO: 1 and the beta chain constant region of the TCR constant region as defined in SEQ: 16 enhancement library, respectively. The introduced mutations are labelled from X01 -X08 in the alpha chain (Figure 1A) and from X09-X14 in the beta chain (Figure 1 B). Donut charts show the distribution at which each amino acid appears in that position. Wild type version of amino acid sequence is highlighted in grey. Figure 1 C visualizes the schematic map of library constructs.

Figure 2 shows a schematic overview of the screening procedure, which was used to identify a promising TCR clone derived from the library.

Figure 3 Comparison of TCR expression on the surface of the promising Jurkat-ieGFP Biosensor library clone (8-9) and Jurkat-ieGFP Biosensor carrying PRAMEVLD-WT- TCR (WT). Cells were stained with an antibody recognizing human monomorphic determinant of the a/|3 chain of the TCR (anti-panTCR-Allophycocyanin, APC) and analyzed with flow cytometry. (A) Dot plots presenting co-expression of blue fluorescent protein (BFP-transduction marker) and TCR (% of BFP ⁺ TCR ⁺ cells is shown). (B) Graph presenting the level of TCR expression as a median fluorescence intensity (MFI) of pan-TCR-APC signal.

Figure 4 Testing TCR specificity of the selected clone. 8-9 clone and WT were cocultured with T2 cells loaded with either relevant (rel.) VLDGLDVLL peptide (T2 + rel. ), irrelevant control (irrel. ) peptide SLLQHLIGL (T2 + irrel. ) or left alone (Jurkat only). (A) After 24h of incubation, cells were stained with anti-HLA-A2-APC and anti-CD3- phycoerythrin-cyanine7 (PE-Cy7) antibody and acquired at flow cytometry. Percentages of eGFP+ Jurkat cells and eGFP MFI for conditions with rel. and irrel. peptide are marked in bold (B) In a separate experiment, cells without additional staining were analyzed. MFI of eGFP channel was plotted. Figure 5 Reactivity of the selected clone to the naturally processed and presented peptide. 8-9 and WT were co-cultured with tumor cell line (SK-MEL23-human melanoma) in the 1 :1 ratio. After 24h, eGFP signal on Jurkat-ieGFP Biosensors was measured with flow cytometry. Graphs show geometric mean fluorescence intensity (gMFI) of eGFP channel.

Figure 6 TCR expression of reconstituted 8-9 TCR and WT-TCR on the surface of Jurkat-ieGFP Biosensor and CD8+ T cells from a healthy donor. Cells were stained with anti-CD3-APC antibody, an antibody recognizing variable (31 chain of the TCR (anti-TCRBV9-PE), and live/dead marker 7-Amino-Actinomycin D (7AAD) and acquired at flow cytometer. Graph presenting the level of TCR expression as a gMFI of TCRBV9-PE signal on BFP ⁺ cells is shown.

Figure 7 Comparison of functional avidity between reconstituted 8-9 and WT-TCRs. (A) 8-9 TCR or WT-TCR-transduced Jurkat-ieGFP Biosensor cells (effectors) were cocultured with T2 cells (targets) loaded with titrated concentrations of VLD peptide ranging from 1 O’ ¹⁰ to 10’ ⁵ M. Effector to target ratio was 2:1 (50000:25000 cells). After 20h of co-culture, cells were collected and stained with anti-HLA-A2-APC antibody to visualize target cells. Percentage of eGFP positive cells and gMFI of eGFP signal on the HLA-A2 BFP ⁺ cells (transduced effector cells) was measured with flow cytometry and plotted on the graph at different co-culture conditions. (B) 8-9 TCR-, WT-TCR- or mock-transduced CD8 ⁺ T cells from a healthy donor were co-cultured with T2 cells loaded with titrated VLD peptide concentrations ranging from 10’ ¹⁰ to 10’ ⁵ M. After 24h, supernatants were collected and IFNy ELISA was performed. IFNy levels at different co-culture conditions are indicated on the graph.

Figure 8 Testing TCR specificity of selected clones. (A-D) Several clones (clone names given below x-axis) and WT were co-cultured with T2 cells loaded with either relevant VLDGLDVLL peptide (T2 + rel.), irrelevant peptide SLLQHLIGL (T2 + irrel.) or left alone (T cells only). After 24h of co-culture, cells were collected and percentage of eGFP positive cells and gMFI of eGFP signal on the transduced effector cells was measured with flow cytometry, gMFI of eGFP channel was plotted. (E-F) Expression of precison paired TCRs in TCR-deficient Jurkat-76 CD+ ieGFP cells. Transduced Jurkat-76 cells were analyzed for TCR expression by staining with anti TCR Vf3>1 (TRBV9) antibody [PE, clone BL37.2, Beckman Coulter] antibody and subsequent flow cytometric analysis. DETAILED DESCRIPTION OF THE INVENTION

Before the invention is described in detail with respect to some of its preferred embodiments, the following general definitions are provided.

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

The present invention will be described with respect to particular embodiments and with reference to certain figures, but the invention is not limited thereto but only by the claims.

Where the term “comprising” is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term “consisting of” is considered to be a preferred embodiment of the term “comprising of”. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which preferably consists only of these embodiments.

Where an indefinite or definite article is used when referring to a singular noun, e.g. “a”, “an” or “the”, this includes a plural of that noun unless something else is specifically stated.

Technical terms are used by their common sense. If a specific meaning is conveyed to certain terms, definitions of terms will be given in the following in the context of which the terms are used.

A first aspect of the invention relates to a library of synthetic polynucleotides encoding TCR alpha chains and TCR beta chains of a TCR capable of binding to a PRAME peptide having the sequence VLDGLDVLL (SEQ ID NO: 3), comprising

- Polynucleotides encoding TCR alpha chains comprising: a) TCR alpha chain constant regions which are selected from the group of TCR alpha constant regions which are at least about 80% identical to the amino acid sequence as set out in SEQ ID NO 1 , b) TCR alpha chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5 and a CDR3 having the amino acid sequence of SEQ ID NO: 6;

- Polynucleotides encoding TCR beta chains comprising a) TCR beta chain constant regions which are selected from the group of TCR beta constant regions which are at least about 80% identical to the amino acid sequence as set out in SEQ ID NO 2 _i or TCR beta chain constant regions which are selected from the group of TCR beta constant regions which are at least about 80% identical to the amino acid sequence as set out in SEQ ID NO 15. b) TCR beta chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 7, a CDR2 having the amino acid sequence of SEQ ID NO: 8 and a CDR3 having the amino acid sequence of SEQ ID NO: 9.

Another aspect refers to a library of TCR alpha chains and TCR beta chains, comprising

- TCR alpha chains comprising: a) TCR alpha chain constant regions which are selected from the group of TCR alpha constant regions which are at least about 80% identical to the amino acid sequence as set out in SEQ ID NO 1 ; b) a TCR alpha chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5 and a CDR3 having the amino acid sequence of SEQ ID NO: 6;

- TCR beta chains comprising a) TCR beta chain constant regions comprising constant regions which are selected from the group of TCR beta constant regions which are at least about 80% identical to the amino acid sequence as set out in SEQ ID NO 15; or

TCR beta chain constant regions comprising constant regions which are selected from the group of TCR beta constant regions which are at least about 80% identical to the amino acid sequence as set out in SEQ ID NO 16. b) TCR beta chain constant region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 7, a CDR2 having the amino acid sequence of SEQ ID NO: 8 and a CDR3 having the amino acid sequence of SEQ ID NO: 9. SEQ ID NO: 1 defines TCR alpha chain constant regions which are set out in following amino acid sequence:

IQNPDPAVYQLRDXKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLD MRSMDFKSNSAVAWSXKSDFACANAFNNSIIPEDTFFPSPEXXCDVKLVE KSFETDXNLNFQNLXVIXXRILLLKVAGFNLLMTLRLWSS wherein

X at position 14 is selected from the group consisting of S, V, W, A, C, F, G, I, L, M and P;

X at position 66 is selected from the group consisting of N, Q, S, T and Y;

X at position 92 is selected from the group consisting of S, V, W, A C, F, G, I, L, M and P;

X at position 93 is selected from the group consisting of S, V, W, A C, F, G, I, L, M and P;

X at position 107 is selected from the group consisting of T, V, W, A, C, F, G, I, L, M and P;

X at positionl 15 is selected from the group consisting of S, V, W, A, C, F, G, I, L, M and P;

X at position 118 is selected from the group consisting of G, I, L, M, P, V, W, A, C and F; and

X at position 119 is selected from the group consisting of F, G, I, L, M, P, V, W, A and C;

The skilled person understands that in SEQ ID NO: 1 X at position 14 corresponds to X01 , X at position 66 corresponds to X02, X at position 92 corresponds to X03, X at position 93 corresponds to X04, X at position 107 corresponds to X05, X at position 115 corresponds to X06, X at position 118 corresponds to X07, X at position 119 corresponds to position X08 in Figure 1A.

SEQ ID NO: 2 defines TCR beta chain constant regions (including both Cbetal and Cbeta2 versions) which are set out in following amino acid sequence:

EDLXXVFPPEVAVFEPSXAEIXHTQKATLVCLAXGFXPDHVELSWWVNGKEVHSGV STDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWT QDRAKPVTQIVSAEAWGRADCGFTSXSYQQGVLSATILYEILLGKAXLYAVLVXALV LMAMVKRKDXXX Wherein

X at position 4 is selected from the group consisting of K, N;

X at position 5 is selected from the group consisting of N, R, D, E, H and K;

X at position 18 is selected from the group consisting of E, H, K, R and D;

X at position 22 is selected from the group consisting of S, V, W, A, C, F, G, I, L, M and P;

X at position 34 is selected from the group consisting of T, D, E, H, K, N and R;

X at position 37 is selected from the group consisting of Y, F;

X at position 136 is selected from the group consisting of E, V;

X at position 157 is selected from the group consisting of T, V, W, A, C, F, G, I, L, M and P; and

X at position 164 is selected from the group consisting of S, V, W, A, C, F, G, I, L, M and P;

X at position 177 is selected from the group consisting of S, F;

X at position 178 is R or deleted;

X at position 179 is G or deleted.

The skilled person understands that in SEQ ID NO 2, X at position 5 corresponds to X09, X at position 18 corresponds to X10, X at position 22 corresponds to X11 , X at position 34 corresponds to X12, X at position 157 corresponds to X13, X at position 164 corresponds to X14 in Figure 1 B.

X at position 4, 37, 136, 177, 178, 179 covers both variations for Cbeta 1 and Cbeta2.

The constant region of the TCR beta chain occurs naturally either in the Cbetal version or in the Cbeta2 version, which are typically equally distributed in a population of natural unstimulated T cells. The skilled person understands that the invention refers to both versions of the constant region. Thus, the TCR beta chain constant regions may be a Cbetal version as set out in SEQ ID NO: 15 or a Cbeta2 version as set out in SEQ ID NO: 16. Thus, TCR beta chain constant regions comprising constant regions which are selected from the group of TCR beta constant regions which are at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 2. cover both Cbetal and Cbeta2 versions.

SEQ ID NO: 15 defines TCR beta chain constant regions (Cbetal ) which are set out in following amino acid sequence: EDLNXVFPPEVAVFEPSXAEIXHTQKATLVCLAXGFFPDHVELSWWVNG KEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQ VQ FYG LS E N D E WTQ D RAKP VTQ I VS AE AWG RAD C G FTS VS YQ Q G VLS A TILYEILLGKAXLYAVLVXALVLMAMVKRKDF wherein

X at position 5 is selected from the group consisting of N, R, D, E, H and K; X at position 18 is selected from the group consisting of E, H, K, R and D;

X at position 22 is selected from the group consisting of S, V, W, A, C, F, G, I, L, M and P;

X at position 34 is selected from the group consisting of T, D, E, H, K, N and R;

X at position 157 is selected from the group consisting of T, V, W, A, C, F, G, I, L, M and P; and

X at position 164 is selected from the group consisting of S, V, W, A, C, F, G, I, L, M and P;

The skilled person understands that for SEQ ID NO: 15 X09 to X14 are the identical positions as set out in Figure 1 B. Thus, In SEQ ID NO: 15, X at position 5 corresponds to X09, X at position 18 corresponds to X10, X at position 22 corresponds to X1 1 , X at position 34 corresponds to X12, X at position 157 corresponds to X13, X at position 164 corresponds to X14 in Figure 1 B.

Where a sequence is defined as being to a particular percentage identical to an amino acid sequence as set out herein, the amino acids at the specific variable positions identified by the invention (e.g. denotated as X01 etc.) can only be selected from the amino acids as defined herein. In other words, at the variable position according to the invention, only the indicated alternative amino acid sequences can be selected.

SEQ ID NO: 16 defines TCR beta chain constant regions (Cbeta2) which are set out in following amino acid sequence:

EDLKXVFPPEVAVFEPSXAEIXHTQKATLVCLAXGFYPDHVELSWWVNG KEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQ VQ FYG LS E N D E WTQ D RAKP VTQ I VS AE AWG RAD C G FTS E S YQ Q G VLS A TILYEILLGKAXLYAVLVXALVLMAMVKRKDSRG wherein X at position 5 is selected from the group consisting of N, R, D, E, H and K; X at position 18 is selected from the group consisting of E, H, K, R and D; X at position 22 is selected from the group consisting of S, V, W, A, C, F, G, I, L, M and P;

X at position 34 is selected from the group consisting of T, D, E, H, K, N and R;

X at position 157 is selected from the group consisting of T, V, W, A, C, F, G, I, L, M and P; and

X at position 164 is selected from the group consisting of S, V, W, A, C, F, G, I, L, M and P;

Applying to all aspects of the invention, in some embodiments, the TCR alpha chain constant regions are selected from the group of TCR alpha constant regions which are at least about 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1 and the TCR beta chain constant regions are selected from the group of TCR beta constant regions which are at least about 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2.

More specifically, in some embodiments the polynucleotides encoding TCR alpha chain constant regions are selected from the group of TCR alpha constant regions which are at least about 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1 .

In some embodiments the polynucleotides encoding TCR beta chain constant regions are selected from the group of TCR beta constant regions which are at least about 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2.

In some embodiments the polynucleotides encoding TCR beta chain constant regions are selected from the group of TCR beta constant regions which are at least about 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 15.

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

In some embodiments the polynucleotides encoding TCR alpha chains comprising constant regions are selected from the group of TCR alpha constant regions which are at least about 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1.

In some embodiments the polynucleotides encoding TCR beta chains comprising constant regions are selected from the group of TCR beta constant regions which are at least about 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2.

In some embodiments the polynucleotides encoding TCR beta chains comprising constant regions are selected from the group of TCR beta constant regions which are at least about 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 15.

Accordingly, in specific embodiments, the invention also relates to a library of synthetic polynucleotides encoding TCR alpha chains and TCR beta chains of a TCR capable of binding to a PRAME peptide having the sequence VLDGLDVLL (SEQ ID NO: 3), comprising

- Polynucleotides encoding TCR alpha chains comprising: a) TCR alpha chain constant regions which are selected from the group of TCR alpha constant regions comprising the amino acid sequence as set out in SEQ ID NO: 1 ; b) TCR alpha chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5 and a CDR3 having the amino acid sequence of SEQ ID NO: 6;

- Polynucleotides encoding TCR beta chains comprising a) TCR beta chain constant regions which are selected from the group of TCR beta constant regions comprising the amino acid sequence as set out in SEQ ID NO 15; or

TCR beta chain constant regions which are selected from the group of TCR beta constant regions comprising the amino acid sequence as set out in SEQ ID NO 16; b) TCR beta chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 7, a CDR2 having the amino acid sequence of SEQ ID NO: 8 and a CDR3 having the amino acid sequence of SEQ ID NO: 9.

Further embodiments relate to a library of TCR alpha chains and TCR beta chains, comprising

- TCR alpha chains comprising: a) TCR alpha chain constant regions which are selected from the group of TCR alpha constant regions comprising the amino acid sequence as set out in SEQ ID NO 1 ; b) TCR alpha chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5 and a CDR3 having the amino acid sequence of SEQ ID NO: 6;

- TCR beta chains comprising a) TCR beta chain constant regions comprising constant regions which are selected from the group of TCR beta constant regions comprising the amino acid sequence as set out in SEQ ID NO: 15;

TCR beta chain constant regions comprising constant regions which are selected from the group of TCR beta constant regions comprising the amino acid sequence as set out in SEQ ID NO: 16; b) TCR beta chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 7, a CDR2 having the amino acid sequence of SEQ ID NO: 8 and a CDR3 having the amino acid sequence of SEQ ID NO: 9. “Nucleic acid" generally means a polymer of DNA or RNA, which can be singlestranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroam idate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide. Preferably, the nucleic acids described herein are recombinant. As used herein, the term "recombinant" refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above. For purposes herein, the replication can be in vitro replication or in vivo replication. The nucleic acids can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art or commercially available (e.g. from GeneArt, Thermo Fisher and similar companies). See, for example Sambrook et al., a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides). The nucleic acid can comprise any nucleotide sequence which encodes any of the recombinant TCRs, polypeptides, or proteins, or functional portions or functional variants thereof.

The selection marker may be any useful selection marker, such as antibiotic resistance, fluourescent marker, binding epitope. The selection marker may be thus selected from, but not limited to, CD20 or Her2/neu markers, or other conventional tags such as a myc-tag, FLAG-tag, T7-tag, HA (hemagglutinin)-tag, His-tag, S-tag, GST-tag, myc, T7, GST, or fluorescent marker, such as GFP, BFP, mTagBFP.

A TCR is composed of two different and separate protein chains, namely the TCR alpha (a) and the TCR beta (P) chain. The TCR a chain comprises variable region (encoded by the variable (V), joining (J) genomic segments) and constant region (encoded by the constant genomic segment). The TCR p chain comprises the variable region (encoded by the variable (V), diversity (D), joining (J) genomic segments) and constant region (encoded by the constant (C) genomic segment). The rearranged V(D)J regions of both the TCR a and the TCR p chain contain hypervariable regions (CDR, complementarity determining regions), among which the CDR3 region determines the specific epitope recognition. At the C-terminal region both TCR a chain and TCR [3 chain contain a hydrophobic transmembrane domain and end in a short cytoplasmic tail.

Typically, the library contains TCR alpha chains, which contain only variations in their constant region within one library. The remaining part, i.e. the variable region of the TCR is identical within one library. Accordingly, the library contains TCR beta chains, which contain only variations in their constant region within one library. The remaining part, i.e. the variable region of the TCR is identical within one library.

In one embodiment the library comprises polynucleotides encoding a TCR alpha chain and a TCR beta chain, each polynucleotide comprising:

- Polynucleotide encoding TCR alpha chains as defined herein and

- Polynucleotide encoding TCR beta chains as defined herein;

- Optionally a selection marker

- Optionally one or more ribosomal entry sites or self-cleaving peptides of the 2A family.

The polynucleotide may contain one or more internal ribosomal entry sites (IRES) sequence or self-cleaving peptides of the 2A family, comprising the 2A peptide sequence derived from a porcine tsechovirus (P2A), derived from other species like Thosea asigna virus 2A peptide (T2A), derived from foot and mouth disease virus 2A peptide (F2A) (as described in Szymczak et al.: Development of 2A peptide-based strategies in the design of multicistronic vectors) and E2A, resulting in the expression a single messenger RNA (mRNA) molecule under the control of the viral promoter within the transduced cell.

The term “library” refers to a collection of distinct molecules comprising typically more than 10 ³ , more than 10 ⁴ , more than 10 ⁵ , more than 10 ⁶ , more than 10 ⁷ , more than 10 ⁸ , more than 10 ⁹ or even more than 10” members. A library in the context of the present invention is a mixture of heterogeneous polypeptides or nucleic acids. The library is composed of members, each of which has a single polypeptide or nucleic acid sequence. Sequence differences between library members are responsible for the diversity present in the library. The library may take the form of a simple mixture of polypeptides or nucleic acids, or may be in the form of cells containing the library of nucleic acids. Preferably, a cell contains only one or a limited number of library members. Advantageously, the nucleic acids are incorporated into expression vectors, in order to allow expression of the polypeptides encoded by the nucleic acids.

Typically, the library comprises at least 1 x 10 ⁴ , at least 1 x 10 ⁵ , at least 1 x 10 ⁶ , at least 1 x 10 ⁷ , at least 1 x 10 ⁸ , at least 1 x 10 ⁹ , preferably, 1 x 10 ¹⁰ , more preferably 2.1 x 10 ¹¹ , such as 2.25 x 10 ¹³ unique molecules. Preferably, the library has 1 x 10 ⁶ to 1 x 10 ¹⁵ , more preferably 1x 1O ¹⁰ to 1x10 ¹⁴ unique molecules. Comprising a number of unique molecules means that each of these molecules of this number differs to each of the other molecules of this number. In one embodiment “unique molecules” means that these molecules exist only once in the library. It should be understood that there is likely to be more than one copy of many unique molecules in a particular physical realization.

The data shows that different constant regions selected from the library show improved expression on the cell surface:

Hence the constant regions as set out in SEQ ID NOs: 12, 13, and 17 to 32 and any combination of alpha and beta constant regions thereof are also embodiments of the invention.

Thus, in some embodiments, a TCR alpha chain constant region may comprise an amino acid sequence which is at least about 80% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 12, 17, 19, 21 , 23, 25,

27, 29 and 31 wherein the amino acids at position 14, 66, 92, 93, 107, 115, 118 and 119 are not changed. Accordingly, a TCR beta chain constant region may comprise an amino acid sequence which is at least about 80% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 13, 18, 20, 22, 24, 26,

28, 30 and 32 wherein the amino acids at position 5, 18, 22, 34, 157 and 164 are not changed.

The TCR alpha chain constant regions and the TCR beta chain constant region are selected from the following: a) TCR alpha chain constant region comprising an amino acid sequence which is at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 12 wherein the amino acids at position 14, 66, 92, 93, 107, 115, 118 and 119 are not changed and a TCR beta chain constant region comprising an amino acid sequence which is at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 13, wherein the amino acids at position 5, 18, 22, 34, 157 and 164 are not changed; b) TCR alpha chain constant region comprising an amino acid sequence which is at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 17 wherein the amino acids at position 14, 66, 92, 93, 107, 115, 118 and 119 are not changed and a TCR beta chain constant region comprising an amino acid sequence which is at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 18, wherein the amino acids at position 5, 18, 22, 34, 157 and 164 are not changed; c) TCR alpha chain constant region comprising an amino acid sequence which is at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 19 wherein the amino acids at position 14, 66, 92, 93, 107, 115, 118 and 119 are not changed and a TCR beta chain constant region comprising an amino acid sequence which is at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 20, wherein the amino acids at position 5, 18, 22, 34, 157 and 164 are not changed; d) TCR alpha chain constant region comprising an amino acid sequence which is at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 21 wherein the amino acids at position 14, 66, 92, 93, 107, 115, 118 and 119 are not changed and a TCR beta chain constant region comprising an amino acid sequence which is at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 22, wherein the amino acids at position 5, 18, 22, 34, 157 and 164 are not changed; e) TCR alpha chain constant region comprising an amino acid sequence which is at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 23 wherein the amino acids at position 14, 66, 92, 93, 107, 115, 118 and 119 are not changed and a TCR beta chain constant region comprising an amino acid sequence which is at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 24, wherein the amino acids at position 5, 18, 22, 34, 157 and 164 are not changed; f) TCR alpha chain constant region comprising an amino acid sequence which is at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 25 wherein the amino acids at position 14, 66, 92, 93, 107, 115, 118 and 119 are not changed and a TCR beta chain constant region comprising an amino acid sequence which is at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 26, wherein the amino acids at position 5, 18, 22, 34, 157 and 164 are not changed; g) TCR alpha chain constant region comprising an amino acid sequence which is at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 27 wherein the amino acids at position 14, 66, 92, 93, 107, 115, 118 and 119 are not changed and a TCR beta chain constant region comprising an amino acid sequence which is at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 28, wherein the amino acids at position 5, 18, 22, 34, 157 and 164 are not changed; h) TCR alpha chain constant region comprising an amino acid sequence which is at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 29 wherein the amino acids at position 14, 66, 92, 93, 107, 115, 118 and 119 are not changed and a TCR beta chain constant region comprising an amino acid sequence which is at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 30, wherein the amino acids at position 5, 18, 22, 34, 157 and 164 are not changed; i) TCR alpha chain constant region comprising an amino acid sequence which is at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 32 wherein the amino acids at position 14, 66, 92, 93, 107, 115, 118 and 119 are not changed and a TCR beta chain constant region comprising an amino acid sequence which is at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 32, wherein the amino acids at position 5, 18, 22, 34, 157 and 164 are not changed;

In specific embodiments the TCR alpha chain constant regions and the TCR beta chain constant region are selected from the following: a) TCR alpha chain constant region comprising the amino acid sequence as set out in SEQ ID NO: 12 and a TCR beta chain constant region comprising the amino acid sequence as set out in SEQ ID NO: 13; b) TCR alpha chain constant region comprising the amino acid sequence as set out in SEQ ID NO: 17 and a TCR beta chain constant region comprising the amino acid sequence as set out in SEQ ID NO: 18; c) TCR alpha chain constant region comprising the amino acid sequence as set out in SEQ ID NO: 19 and a TCR beta chain constant region comprising the amino acid sequence as set out in SEQ ID NO: 20; d) TCR alpha chain constant region comprising the amino acid sequence as set out in SEQ ID NO: 21 and a TCR beta chain constant region comprising the amino acid sequence as set out in SEQ ID NO: 22; e) TCR alpha chain constant region comprising the amino acid sequence as set out in SEQ ID NO: 23 and a TCR beta chain constant region comprising the amino acid sequence as set out in SEQ ID NO: 24; f) TCR alpha chain constant region comprising the amino acid sequence as set out in SEQ ID NO: 25 and a TCR beta chain constant region comprising the amino acid sequence as set out in SEQ ID NO: 26; g) TCR alpha chain constant region comprising the amino acid sequence as set out in SEQ ID NO: 27 and a TCR beta chain constant region comprising the amino acid sequence as set out in SEQ ID NO: 28; h) TCR alpha chain constant region comprising the amino acid sequence as set out in SEQ ID NO: 29 and a TCR beta chain constant region comprising the amino acid sequence as set out in SEQ ID NO: 30; i) TCR alpha chain constant region comprising the amino acid sequence as set out in SEQ ID NO: 31 and a TCR beta chain constant region comprising the amino acid sequence as set out in SEQ ID NO: 32;

Also encompassed is a library of vectors comprising the polynucleotide library as defined herein.

A “vector” is any molecule or composition that has the ability to carry a nucleic acid sequence into a suitable host cell where synthesis of the encoded polypeptide can take place. Typically, and preferably, a vector is a nucleic acid that has been engineered, using recombinant DNA techniques that are known in the art, to incorporate a desired nucleic acid sequence (e.g. a nucleic acid of the invention). The vector may comprise DNA or RNA and/or comprise liposomes. The vector may be a plasmid, shuttle vector, phagemide, cosmid, expression vector, retroviral vector, lentiviral vector, adenoviral vector or particle. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known to those of ordinary skill in the art. A vector preferably is an expression vector that includes a nucleic acid according to the present invention operably linked to sequences allowing for the expression of said nucleic acid.

Preferably, the vector is a retroviral particle. Further encompassed is a library of cells, comprising said library of vectors. Moreover, also a library of cells expressing the polynucleotides as defined herein is encompassed.

Thus, in the library of cells the above described vector comprising a nucleic acid sequence coding for the above described TCR may be introduced or the nucleic acid may be introduced by other means, e.g. In vitro transcribed RNA (ivtRNA) coding for said TCR may be introduced.

The cell may be a peripheral blood lymphocyte such as a T cell. The method of cloning and exogenous expression of the TCR is for example described in Engels et al. (Relapse or eradication of cancer is predicted by peptide-major histocompatibility complex affinity. Cancer Cell, 23(4), 516-26. 2013). The transduction of primary human T cells with a lentiviral vector is, for example, described in Cribbs “simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells” BMC Biotechnol. 2013; 13: 98.

The term “transduction” refers to the process by which an exogenous nucleic acid sequence is introduced into a host cell, e.g. into a T cell. It is noted that introduction or transfer of nucleic acid sequences is not limited to the mentioned methods but can be achieved by any number of means including electroporation, microinjection, gene gun delivery, lipofection, superfection, infection by retroviruses or other suitable viruses for transduction or transfection.

Preferably the cell may be a T cell. The T cell may express endogenous TCR chains. The T cell may be a CD4+ or a CD8+ T cell. Preferably, the T cell is a CD8+ T cell. In one embodiment the cell may be a Jurkat-ieGFP TCR ^A CD8+ cell.

A further aspect relates to a library of polypeptides encoded by the library of synthetic polynucleotides as defined herein. Another aspect refers to the use of the library as described herein for identifying a TCR receptor with enhanced TCR alpha and TCR beta chain pairing.

A further aspect refers to a method for isolating a TCR with enhanced TCR alpha and beta chain pairing, comprising the steps:

- Co-culturing the library of cells as described herein with antigen presenting cells presenting the HLA bound form of the target antigen of the TCR;

And at least one of the steps

- Selecting cells showing elevated expression levels of the heterologous TCR compared to wildtype,

- Selecting cells showing elevated activation levels compared to wild type.

Wildtype with regard to “compared to wildtype” means in particular a TCR which is identical in the amino acid sequence to the TCR it is compared except that it contains in all positions X01 to X14 the wildtype amino acid. The skilled person is aware that the wild type amino acids for XOl to X014 regarding the Cbeta2 chain are as follows: X01 = S; X02 = N; X03 = S; X04 = S; X05 = T; X06 = S; X07 = G; X08 = F; X09 = N;

X10 = E; X11 = S; X12 = T; X13 = T and X14 = S. The skilled person is aware that the wild type amino acids for X01 to X014 regarding the Cbetal chain are as follows: X01 = S; X02 = N; X03 = S; X04 = S; X05 = T; X06 = S; X07 = G; X08 = F; X09 = K;

X10 = E; X11 = S; X12 = T; X13 = T and X14 = S.

The expression levels can be measured using flow cytometry as described herein. The avidity may be measured e.g. by IFNy ELISA as described herein.

Accordingly, also a TCR isolated from the TCR library as described herein is encompassed.

Preferably, the TCR isolated from the TCR library shows elevated expression levels of the heterologous TCR compared to wild type and/or elevated activation levels compared to wild type.

In a specific embodiment, the invention relates to a TCR capable of binding to a PRAME peptide having the sequence VLDGLDVLL (SEQ ID NO: 3), wherein the TCR comprises: - A TCR alpha chain comprising a variable TCR alpha region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5 and a CDR3 having the amino acid sequence of SEQ ID NO: 6; and

- A constant TCR alpha region comprising the sequence SEQ ID NO: 12; and

- A TCR beta chain comprising

- a variable TCR beta chain region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 7, a CDR2 having the amino acid sequence of SEQ ID NO: 8 and a CDR3 having the amino acid sequence of SEQ ID NO: 9; and

- A constant TCR beta region comprising the sequence SEQ ID NO: 13.

In some embodiments the TCR alpha chains comprise constant regions are selected from the group of TCR alpha constant regions which are at least about 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1 and the TCR beta chains comprise constant regions are selected from the group of TCR beta constant regions which are at least about 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2.

The TCRs according to the invention may show elevated expression levels of the heterologous TCR compared to wild type. Alternatively, or in addition, the TCRs according to the invention may show elevated activation levels compared to wild type. Preferably the TCRs according to the invention show elevated expression levels of the heterologous TCR compared to wild type and show elevated activation levels compared to wild type.

In one embodiment, in at least one of the variable positions, i.e. X01 to X14, the amino acid is not the amino acid which occurs naturally in wild type. That means that preferably in at least two, more preferably at least three, even more preferably at least four, such as at least five, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, such as in all 14 positions of X01 to X14, the amino acid is not the amino acid which occurs naturally in wild type. The term “not the amino acid which occurs naturally in wild type” means in the context of this application that it is substituted by an amino acid as defined for the variable positions X1 to X14, which is not the wild type amino acid for this position. The skilled person is aware that the wild type amino acids for XOl to X014 regarding the Cbeta2 chain are as follows: X01 = S; X02 = N; X03 = S; X04 = S; X05 = T; X06 = S; X07 = G; X08 = F; X09 = N; X10 = E; X11 = S; X12 = T; X13 = T and X14 = S. The skilled person is aware that the wild type amino acids for XOl to X014 regarding the Cbetal chain are as follows: X01 = S; X02 = N; X03 = S; X04 = S; X05 = T; X06 = S; X07 = G; X08 = F; X09 = K; X10 = E; X11 = S; X12 = T; X13 = T and X14 = S.

Also encompassed is a method of preparing a library of synthetic polynucleotides encoding a plurality of TOR alpha chain constant regions and TCR beta chain constant regions, the method comprising:

- Providing sequences of polynucleotides a) encoding TCR alpha chain constant regions and TCR beta chain constant regions as defined herein; or b) encoding the TCR alpha chain and TCR beta chain as defined herein ;

- Assembling the synthetic polynucleotide sequences to produce a library of synthetic polynucleotides.

A further aspect refers to a kit comprising one or more of the libraries described herein.

Another aspect of the invention refers to nucleotide sequences of the synthetic polynucleotides defined herein in computer readable format. Accordingly, the invention also encompasses the amino acid sequences as defined herein in computer readable format.

“Nucleic acid" generally means a polymer of DNA or RNA, which can be singlestranded or double-stranded, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroam idate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide. The nucleic acid may be made synthetically, e.g. using art-recognized nucleic acid chemistry or enzymatically using, e.g. a polymerase. Preferably, the nucleic acids described herein are recombinant. As used herein, the term "recombinant" refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above. For purposes herein, the replication can be in vitro replication or in vivo replication. The nucleic acids can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art or commercially available (e.g. from Genscript, Thermo Fisher and similar companies). See, for example Sambrook et al., a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides). The nucleic acid can comprise any nucleotide sequence which encodes any of the recombinant TCRs, polypeptides, or proteins, or functional portions or functional variants thereof.

The nucleic acid encoding the TOR may be modified. Useful modifications in the overall nucleic acid sequence may be codon optimization. Alterations may be made which lead to conservative substitutions within the expressed amino acid sequence. These variations can be made in complementarity determining and non-complementarity determining regions of the amino acid sequence of the TCR chain that do not affect function. Usually, additions and deletions should not be performed in the CDR3 region. Moreover, as modifications in the constant regions that might impact the pairing of the TCR chains, changes beyond the variations indicated herein for the specific positions 14, 66, 92, 93, 107, 115, 118 and 119 (X01 -X08 of Figure 1 A) of the TCR alpha chain constant region of SEQ ID NO: 1 and positions 5, 18, 22, 34, 157 and 164 (X09 to X14 of Figure 1 B) TCR beta chain constant region of SEQ ID NO: 2 (and the corresponding positions in SEQ ID NO: 15 and 16) should be avoided when modifying.

The terms “recombinant” and “recombinant TCR” as used in the present application refers to TCRs that have been introduced by any of the genetic engineering techniques into the T cells. The “recombinant TCR” also termed “exogenous TCR” may be engineered or may be a naturally occurring TCR which has a desired antigen specificity and which was isolated. Typically, these recombinant TCRs which were not endogenous to the T cell population, i.e. were not naturally expressed in the T cell population, i.e. were not expressed in the T cell population before transfer of the recombinant TCR. According to some embodiments of the invention the amino acid sequence of the TCR is modified to comprise a detectable label, a therapeutic agent or pharmacokinetic modifying moiety.

Non-limiting examples for detectable labels are radiolabels, fluorescent labels, nucleic acid probes, enzymes and contrast reagents. Therapeutic agents which may be associated with the TCRs include radioactive compounds, immune-modulators, enzymes or chemotherapeutic agents. The therapeutic agents could be enclosed by a liposome linked to TCR so that the compound can be released slowly at the target site. This will avoid damage during the transport in the body and ensure that the therapeutic agent, e.g. toxin, has maximum effect after binding of the TCR to the relevant antigen presenting cells. Other examples for therapeutic agents are: peptide cytotoxins, i.e. proteins or peptides with the ability to kill mammalian cells, such as ricin, diphtheria toxin, pseudomonas bacterial exotoxin A, DNase and RNase. Small molecule cytotoxic agents, i.e. compounds with the ability to kill mammalian cells having a molecular weight of less than 700 Daltons. Such compounds could contain toxic metals capable of having a cytotoxic effect. Furthermore, it is to be understood that these small molecule cytotoxic agents also include pro-drugs, i.e. compounds that decay or are converted under physiological conditions to release cytotoxic agents. Such agents may for example include docetaxel, gemcitabine, cisplatin, maytansine derivatives, rachelmycin, calicheamicin, etoposide, ifosfamide, irinotecan, porfimer sodium photofrin II, temozolomide, topotecan, trimetrexate glucoronate, mitoxantrone, auristatin E, vincristine and doxorubicin; radionuclides, such as, iodine 131 , rhenium 186, indium 111 , yttrium 90. bismuth 210 and 213, actinium 225 and astatine 213. The association of the radionuclides with the TCRs or derivatives thereof may for example be carried out by chelating agents; immune-stimulators, also known as immunostimulants, i.e. immune effector molecules which stimulate immune response. Exemplary immune-stimulators are cytokines such as IL-2 and IFN-y, antibodies or fragments thereof, including anti-T cell or NK cell determinant antibodies (e.g anti-CD3, anti-CD28 or anti-CD16); alternative protein scaffolds with antibody like binding characteristics; Superantigens, i.e. antigens that cause non-specific activation of T- cells resulting in polyclonal T cell activation and massive cytokine release, and mutants thereof; chemokines such as IL-8, platelet factor 4, melanoma growth stimulatory protein, etc. complement activators; xenogeneic protein domains, allogeneic protein domains, viral/bacterial protein domains, viral/bacterial peptides. The antigen receptor molecules (T cell receptor molecules) on human T lymphocytes are non-covalently associated with the CD3 (T3) molecular complex on the cell surface. Perturbation of this complex with anti-CD3 monoclonal antibodies induces T cell activation. Thus, some embodiments refer to a TCR as described herein associated (usually by fusion to an N-or C-terminus of the alpha or beta chain) with an anti-CD3 antibody, or a functional fragment or variant of said anti-CD3 antibody. Antibody fragments and variants/analogues which are suitable for use in the compositions and methods described herein include minibodies, Fab fragments, F(ab<'>)2fragments, dsFv and scFv fragments, Nanobodies™ (Ablynx (Belgium), molecules comprising synthetic single immunoglobulin variable heavy chain domain derived from a camelid (e.g. camel or llama) antibody) and Domain Antibodies (comprising an affinity matured single immunoglobulin variable heavy chain domain or immunoglobulin variable light chain domain (Domantis (Belgium)) or alternative protein scaffolds that exhibit antibody-like binding characteristics such as Affibodies (comprising engineered protein A scaffold Affibody (Sweden)) or Anticalins (comprising engineered anticalins Pieris (German)).

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

The pharmacokinetic modifying moiety may be for example at least one polyethylene glycol repeating unit, at least one glycol group, at least one sialyl group or a combination thereof. The association of at least one polyethylene glycol repeating unit, at least one glycol group, at least one sialyl group may be caused in a number of ways known to those skilled in the art. In a preferred embodiment the units are covalently linked to the TCR. The TCRs according to the invention can be modified by one or several pharmacokinetic modifying moieties. In particular, the soluble form of the TCR is modified by one or several pharmacokinetic modifying moieties. The pharmacokinetic modifying moiety may achieve beneficial changes to the pharamacokinetic profile of the therapeutic, for example improved plasma half-life, reduced or enhanced immunogenicity, and improved solubility. llse functional moieties and modifications include “suicide” or “safety switches” that can be used to shut off effector host cells carrying an inventive TCR in a patient’s body. An example is the inducible Caspase 9 (iCasp9) “safety switch” described by Gargett and Brown Front Pharmacol. 2014; 5: 235. Briefly, effector host cells are modified by well-known methods to express a Caspase 9 domain whose dimerization depends on a small molecule dimerizer drug such as AP1903/CIP, and results in rapid induction of apoptosis in the modified effector cells. The system is for instance described in EP2173869 (A2). Examples for other “suicide” “safety switches” are known in the art, e.g. Herpes Simplex Virus thymidine kinase (HSV-TK), expression of CD20 and subsequent depletion using anti-CD20 antibody or myc tags (Kieback et al, Proc Natl Acad Sci U S A. 2008 Jan 15;105(2):623-8).

TCRs with an altered glycosylation pattern are also envisaged herein. As is known in the art, glycosylation patterns can depend on the amino acid sequence (e.g., the presence or absence of particular glycosylation amino acid residues, discussed below) and/or the host cell or organism in which the protein is produced. Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. Addition of N- linked glycosylation sites to the binding molecule is conveniently accomplished by altering the amino acid sequence such that it contains one or more tri-peptide sequences selected from asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline). O-linked glycosylation sites may be introduced by the addition of or substitution by, one or more serine or threonine residues to the starting sequence.

Another means of glycosylation of TCRs is by chemical or enzymatic coupling of glycosides to the protein. Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. Similarly, deglycosylation (i.e., removal of carbohydrate moieties present on the binding molecule) may be accomplished chemically, e.g. by exposing the TCRs to trifluoromethanesulfonic acid, or enzymatically by employing endo- and exo-glycosidases.

It is also conceivable to add a drug such as a small molecule compound to the TCR, in particular a soluble form of the inventive TCR. Linkage can be achieved via covalent bonds, or non-covalent interactions such as through electrostatic forces. Various linkers, known in the art, can be employed in order to form the drug conjugates.

The TCR, in particular a soluble form of the inventive TCR can additionally be modified to introduce additional domains which aid in identification, tracking, purification and/or isolation of the respective molecule (tags). Thus, in some embodiments, the TCR a chain or the TCR [3 chain may be modified to comprise an epitope tag.

Epitope tags are useful examples of tags that can be incorporated into the TCR of the invention. Epitope tags are short stretches of amino acids that allow for binding of a specific antibody and therefore enable identification and tracking of the binding and movement of soluble TCRs or host cells within the patient’s body or cultivated (host) cells. Detection of the epitope tag, and hence, the tagged TCR, can be achieved using a number of different techniques.

Tags can further be employed for stimulation and expansion of host cells carrying an inventive TCR by cultivating the cells in the presence of binding molecules (antibodies) specific for said tag.

In general, the TCR can be modified in some instances with various mutations that modify the affinity and the off-rate of the TCR with the target antigen. In particular, the mutations may increase the affinity and/or reduce the off-rate. Thus, the TCR may be mutated in at least one CDR and the variable domain framework region thereof.

However, in a preferred embodiment the CDR regions of the TCR are not modified or in vitro affinity maturated such as for the TCR receptors in the examples. This means that the CDR regions have naturally occurring sequences. This can be advantageous, since in vitro affinity maturation may lead to immunogenicity to the TCR molecule. This may lead to the production of anti-drug antibodies decreasing or inactivating the therapeutic effect and the treatment and /or induce adverse effects.

The mutation may be one or more substitution(s), deletion(s) or insertions(s). These mutations may be introduced by any suitable method known in the art, such as polymerase chain reaction, restriction enzyme-based cloning, ligation independent cloning procedures, which are described for Example in Sambrook, Molecular Cloning - 4 ^th Edition (2012) Cold Spring Harbor Laboratory Press.

Another aspect of the invention refers to pharmaceutical composition comprising the TCR as described herein, the nucleic acid encoding the TCR, the vector comprising said nucleic acid and/or the cell comprising said TCR. Those active components of the present invention are preferably used in such a pharmaceutical composition, in doses mixed with an acceptable carrier or carrier material, that the disease can be treated or at least alleviated. Such a composition can (in addition to the active component and the carrier) include filling material, salts, buffer, stabilizers, solubilizers and other materials, which are known state of the art.

The term "pharmaceutically acceptable" defines a non-toxic material, which does not interfere with effectiveness of the biological activity of the active component. The choice of the carrier is dependent on the application.

The pharmaceutical composition may contain additional components which enhance the activity of the active component or which supplement the treatment. Such additional components and/or factors can be part of the pharmaceutical composition to achieve synergistic effects or to minimize adverse or unwanted effects.

Techniques for the formulation or preparation and application/medication of active components of the present invention are published in "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA, latest edition. An appropriate application is a parenteral application, for example intramuscular, subcutaneous, intramedular injections as well as intrathecal, direct intraventricular, intravenous, intranodal, intraperitoneal or intratumoral injections. The intravenous injection is the preferred treatment of a patient.

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

An injectable composition is a pharmaceutically acceptable fluid composition comprising at least one active ingredient, e.g. an expanded T cell population (for example autologous or allogenic to the patient to be treated) expressing a TCR. The active ingredient is usually dissolved or suspended in a physiologically acceptable carrier, and the composition can additionally comprise minor amounts of one or more non-toxic auxiliary substances, such as emulsifying agents, preservatives, and pH buffering agents and the like. Such injectable compositions that are useful for use with the fusion proteins of this disclosure are conventional; appropriate formulations are well known to those of ordinary skill in the art. Typically, the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier.

Accordingly, another aspect of the invention refers to the TCR as described herein, the polypeptide comprising a functional portion of said TCR, the multivalent TCR complex according as described herein, the nucleic acid encoding said TCR, the vector comprising said nucleic acid, the cell comprising said TCR, or the antibody specifically binding to a portion of the TCR as described herein for use as a medicament.

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

In one embodiment the cancer is a hematological cancer or a solid tumor.

Hematological cancers also called blood cancers which do not form solid tumors and therefore are dispersed in the body. Examples of hematological cancers are leukemia, lymphoma or multiple myeloma. There are two major types of solid tumors, sarcomas and carcinomas. Sarcomas are for example tumors of the blood vessel, bone, fat tissue, ligament, lymph vessel, muscle or tendon.

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

In some embodiments the cancer is a hematological cancer. Preferably the hematological cancer may be acute myeloid leukemia, myelodysplastic syndromes and multiple myeloma. Also contemplated herein are pharmaceutical compositions and kits containing one or more of (i) an isolated TCR as described herein; (ii) viral particles comprising a nucleic acid encoding a recombinant TCR; (iii) immune cells, such as T cells or NK cells, modified to express a recombinant TCR as described herein; (iv) nucleic acids encoding a recombinant TCR as described herein. In some embodiments, the present disclosure provides compositions comprising lentiviral vector particles comprising a nucleotide sequence encoding a recombinant TCR described herein (or T cells that have been modified using the vector particles described herein to express a recombinant TCR). Such compositions can be administered to subjects in the methods of the present disclosure as described further herein.

Compositions comprising the modified T cells as described herein can be utilized in methods and compositions for adoptive immunotherapy in accordance with known techniques, or variations thereof that will be apparent to those skilled in the art based on the instant disclosure.

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

The number of cells for an effective treatment in the composition is typically greater than 10 cells, and up to 10 ⁶ , up to and including 10 ⁸ or 10 ⁹ cells and can be more than 10 ¹ ° cells. The number of cells will depend upon the ultimate use for which the composition is intended as will the type of cells included therein. For uses provided herein, the cells are generally in a volume of a liter or less, can be 500 ml or less, even 250 ml or 100 ml or less. Hence the density of the desired cells is typically greater than 10 ⁶ cells/ml and generally is greater than 10 ⁷ cells/ml, generally 10 ⁸ cells/ml or greater. The clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 10 ⁹ , 10 ¹ ° or 10 ¹¹ cells. Pharmaceutical compositions provided herein can be in various forms, e.g., in solid, liquid, powder, aqueous, or lyophilized form. Examples of suitable pharmaceutical carriers are known in the art. Such carriers and/or additives can be formulated by conventional methods and can be administered to the subject at a suitable dose. Stabilizing agents such as lipids, nuclease inhibitors, polymers, and chelating agents can preserve the compositions from degradation within the body. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.

The recombinant TCRs as described herein, or the viral vector particles comprising a nucleotide sequence encoding a recombinant TCR provided herein, can be packaged as kits. Kits can optionally include one or more components such as instructions for use, devices, and additional reagents, and components, such as tubes, containers and syringes for practice of the methods. Exemplary kits can include the nucleic acids encoding the recombinant TCRs, the recombinant TCR polypeptides, or viruses provided herein, and can optionally include instructions for use, a device for detecting a virus in a subject, a device for administering the compositions to a subject, and a device for administering the compositions to a subject.

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

Kits contemplated herein also include kits for carrying out the methods for detecting the presence of polynucleotides encoding any one or more of the TCRs disclosed herein. In particular, such diagnostic kits may include sets of appropriate amplification and detection primers and other associated reagents for performing deep sequencing to detect the polynucleotides encoding TCRs disclosed herein. In further embodiments, the kits herein may comprise reagents for detecting the TCRs disclosed herein, such as antibodies or other binding molecules. Diagnostic kits may also contain instructions for determining the presence of the polynucleotides encoding the TCRs disclosed herein or for determining the presence of the TCRs disclosed herein. A kit may also contain instructions. Instructions typically include a tangible expression describing the components included in the kit, and methods for administration, including methods for determining the proper state of the subject, the proper dosage amount, and the proper administration method. Instructions can also include guidance for monitoring the subject over the duration of the treatment time. Kits provided herein also can include a device for administering a composition described herein to a subject. Any of a variety of devices known in the art for administering medications or vaccines can be included in the kits provided herein. Exemplary devices include, but are not limited to, a hypodermic needle, an intravenous needle, a catheter, a needle-less injection device, an inhaler, and a liquid dispenser, such as an eyedropper. Typically, the device for administering a virus of the kit will be compatible with the virus of the kit; for example, a needle-less injection device such as a high pressure injection device can be included in kits with viruses not damaged by high pressure injection, but is typically not included in kits with viruses damaged by high pressure injection.

Kits provided herein also can include a device for administering a compound, such as a T cell activator or stimulator, or a TLR agonist, such as a TLR4 agonist to a subject. Any of a variety of devices known in the art for administering medications to a subject can be included in the kits provided herein. Exemplary devices include a hypodermic needle, an intravenous needle, a catheter, a needle-less injection, but are not limited to, a hypodermic needle, an intravenous needle, a catheter, a needle-less injection device, an inhaler, and a liquid dispenser such as an eyedropper. Typically, the device for administering the compound of the kit will be compatible with the desired method of administration of the compound.

Examples

Library design

“TCR-C-enhancement library” is a DNA library carrying 10 ¹¹ TCR a and [3 constant region variants of the PRAMEVLD-TCR and was generated using TRIM technology. TCR constant regions contain six mutation sites in the TCR C-|3 chain and eight mutation sites in the TCR C-a chain (Figure 1 A and B). Amino acid substitutions were chosen based on a broad literature evaluation for residues that are important for: improving TCR signal transduction through better TCR interaction with CD3 subunits; post-translational modifications that might be detrimental for TCR avidity (e.g N-linked glycosylated positions); binding to lipid rafts resulting in better clustering [Cohen et.al. , Cancer Res. 2007; Kuball et. al. J Exp Med, 2009; Haga-Friedman et. al. J Immunol, 2012; Bethune et. al. eLife 2016;]. Additionally, amino acid positions in murine constant regions that have been proved to be important for TCR avidity and could potentially influence pairing of alpha and beta chains were also chosen for diversification [Sommermayer and llckert J Immunol, 2010], Library diversification was done taking into consideration that some positions might be risky to change thus those positions had a higher frequency of wt-derived amino acid sequences. Also not all 20 amino acids could be used for the diversification at each position as the complexity of the library would be too high and could not be reasonably synthesized.

The sequences coding for alpha and beta chains of PRAMEVLD-TCR are separated with a linker and P2A sequence coding for self-cleaving peptide (Figure 1C). Library contains also a mTag BFP sequence preceded with a linker and P2A sequence. The library was cloned into a retroviral vector that allows transduction of the library into recipient T cells with the aim of finding optimal TCR mutation combinations for increased functional avidity.

Screening procedure

Retroviral supernatants of the library as well as PRAMEVLD-WT-TCR construct, which differed from library PRAMEVLD-TCR amino acid sequence only in regard to the mutation sites in the constant a and [3 chains (Figure 1 A and B), were produced in HEK293FT cells. Viral supernatants were titrated and diluted to achieve multiplicity of infection (MOI) equal or lower than 1 , thus maximizing the number of cells with a single integration of a viral particle. Additionally, a TCR signal reporter system, which allows high-throughput flow cytometry-based screening for exogenously introduced and activated TCRs, was generated. For that purpose, Jurkat 76 T cell line without endogenous expression of TCR, but expressing CD8, was engineered to express eGFP under the control of a nuclear factor of activated T cells (NFAT) response element (Jurkat-ieGFP Biosensors). These cells were used for transduction, followed by bulk fluorescence activated cell sorting for BFP positive cells (transduced cells) (Figure 2). In the next step, transduced cells were co-cultured with VLDGLDVLL (VLD) peptide loaded T2 cells or SK-MEL23 melanoma cell line expressing PRAME antigen, both expressing also the HLA of interest, and after 24h a single cell sort for CD3 ⁺ eGFP ^high cells was performed. CD3 can be only detected on the cell surface if it is associated with an alpha and beta TCR heterodimer, thus cells without a paired alpha beta TCR will be CD3 negative and the amount of CD3 will be directly related to the amount of the alpha-beta paired chains of the TCR. Therefore, CD3 can be used as selection marker. Based on CD3 positivity, Jurkat-ieGFP Biosensor cells successfully expressing TCR were selected. Additionally, through gating on cells with high expression of eGFP, only cells transduced with highly functional TCRs were isolated. Single cell clones were expanded to the numbers allowing further testing for reactivity to T2 cells loaded with 10’ ⁵ M concentration of VLD peptide or tumor cell lines with differential expression of PRAME antigen. PRAMEvLD-WT-TCR-Jurkat-ieGFP Biosensors (WT) were used as a control throughout the whole screening process. Based on the superior functionality compared to the WT-TCR, one promising clone caring TCRs from the library was selected.

Characterization of selected clones

For the further characterization of the selected clone (called 8-9), derived from library screening, the level of TCR expression on the surface of the library clone and WT was analyzed using flow cytometry. Library clone 8-9 showed highly elevated levels of TCR expression compared to the WT (Figure 3 A and B), that may account for its increased functionality observed in the screening procedure.

Additionally, response to the irrelevant peptide derived from the same PRAME antigen was investigated to ensure high specificity of the TCRs expressed on the surface of the selected clone (Figure 4 A and B). For this purpose, T2 cells expressing the HLA of interest were loaded with 10’ ⁵ M concentration of relevant (VLD) or control SLLQHLIGL (SLL; SEQ ID NO: 14) peptide and co-cultured with the library clone 8-9 or the WT cells. After 24h of co-culture, eGFP signal induced upon TCR activation was measured with flow cytometry. The specific activation could only be seen when the library clone or WT were co-cultured with T2 cells loaded with relevant peptide, thereby confirming the peptide specificity of the selected clone.

To test whether the selected library clone can recognize and respond to the antigen naturally processed and presented on the cell surface of tumor cells, its reactivity to tumor cell line SK-MEL23) expressing PRAME antigen (1150.3 reads per kilobase million [RPKM], data not shown) was investigated (Figure 5).

As a result of 24h co-culture with the SK-MEL23 cell line, the library clone showed increased expression of inducible eGFP levels in comparison to WT measured by flow cytometry. The selected library clone was shown to carry highly functional TCRs, which are able to recognize naturally processed and presented peptide more efficiently than WT. Reconstitution of 8-9 clone derived TCR and validation

As a next step, TCR sequence derived from clone 8-9 (called 8-9 TCR) and WT-TCR sequence, which differed from 8-9 TCR nucleotide sequence only in regard to the mutation sites in the constant a and [3 chains introduced through the library, were reconstituted in Jurkat-ieGFP Biosensor cells or in CD8+ T cells derived from three healthy donors. Reconstitution was performed with retroviral transduction at high MOI. With comparable expression of BFP (transduction efficiency marker), TCR expression levels were highly increased in 8-9 TCR transduced Jurkat-ieGFP Biosensor cells or CD8+ T cells from all three healthy donors compared to the same cell types transduced with WT-TCR (Figure 6).

The expression of TCR 8-9 compared to the WT-TCR is higher in both, Jurkat-ieGFP cells and CD8+ T cells (Figure 6). The difference between WT-TCR transduced and 8- 9-TCR transduced CD8+ T cells from healthy donors is significantly smaller compared with the difference of WT-TCR transduced and 8-9-TCR transduced ieGFP Jurkat cells. Nevertheless, the reactivity of the 8-9 TCR transduced CD8+ T cells is markedly higher than that of the WT-TCR transduced T cells, comparable to the results seen in ieGFP-Jurkat cells (Figure 7). This supports that the TCRs described herein identified by the method of the invention not only show an increased surface expression but also a reduced mispairing with the endogenous TCR chains compared to WT-TCR.

8-9 TCR or WT-TCR-transduced Jurkat-ieGFP Biosensor cells were co-cultured with T2 cells loaded with titrated concentrations of VLD peptide ranging from 1 O’ ¹⁰ to 10’ ⁵ M. After 20h of co-culture, percentage of eGFP positive cells as well as geometric mean fluorescence intensity of eGFP signal on the BFP positive cells (transduced cells) was assessed with flow cytometry. In both read-outs 8-9 TCR-transduced cells showed higher signals when compared to the WT-TCR-transduced cells (Figure 7A).

In the similar way, 8-9 TCR or WT-TCR-transduced CD8 ⁺ T cells were co-cultured with T2 cells loaded with titrated VLD peptide concentrations. After 24h, supernatants were collected and IFNy ELISA was performed. IFNy levels secreted by CD8 ⁺ T cells transduced with the 8-9 TCR were much higher than for T cells carrying the WT-TCR construct (in the 10’ ⁹ to 10’ ⁵ peptide concentration range) supporting the results derived from Jurkat-ieGFP Biosensor co-cultures (Figure 7B). Further sequences of the library show improved effect

Using the PP library and the already described method, additional sequences were identified that showed an increase activity compared to wt sequence using the Jurkat biosensor system (Fig. 8 A-D).

Several clones and WT were co-cultured with T2 cells loaded with either relevant VLDGLDVLL peptide, irrelevant peptide, or left alone (T cells only). After 24h of coculture, cells were collected and percentage of eGFP positive cells and gMFI of eGFP signal on the transduced effector cells was measured with flow cytometry, gMFI of eGFP channel was plotted.

For analyzing expression of precision paired TCRs in TCR-deficient Jurkat-76 CD+ ieGFP cells, Transduced Jurkat-76 cells were analyzed for TOR expression by staining with anti TCR Vf3>1 (TRBV9) antibody [PE, clone BL37.2, Beckman Coulter] antibody and subsequent flow cytometric analysis. ppTCR clones showed equal or higher expression of the TCR on the cell surface of Jurkat 76 -/- cells compared to wildtype TCR transduce Jurkat 76 -/- cells (Fig. 8E-F). In a co-culture experiment) all identified PP clones showed better reactivity compared to the wildtype clones, depicted by either gMFI or %GFP (Fig. 8 A-D). This shows that the precision pairing library allow identification of several TCR constant chain variants that show superiority over the wildtype TCR constant chain sequences.

The application further comprises the following items:

Item 1 : A library of synthetic polynucleotides encoding TCR alpha chains and TCR beta chains of a TCR capable of binding to a PRAME peptide having the sequence VLDGLDVLL (SEQ ID NO: 3), comprising

- Polynucleotides encoding TCR alpha chains comprising: a) TCR alpha chain constant regions which are selected from the group of TCR alpha constant regions which are at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 1 ; b) TCR alpha chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5 and a CDR3 having the amino acid sequence of SEQ ID NO: 6;

- Polynucleotides encoding TCR beta chains comprising a) - TCR beta chain constant regions which are selected from the group of TCR beta constant regions which are at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 15; or

- TCR beta chain constant regions which are selected from the group of TCR beta constant regions which are at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 16; b) TCR beta chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 7, a CDR2 having the amino acid sequence of SEQ ID NO: 8 and a CDR3 having the amino acid sequence of SEQ ID NO: 9.

Item 2: A library of synthetic polynucleotides encoding TCR alpha chains and TCR beta chains of a TCR capable of binding to a PRAME peptide having the sequence VLDGLDVLL (SEQ ID NO: 3), comprising

- Polynucleotides encoding TCR alpha chains comprising: a) TCR alpha chain constant regions which are selected from the group of TCR alpha constant regions which are at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 1 : wherein the amino acid at position 14 is selected from the group consisting of S, V, W, A, C, F, G, I, L, M and P; the amino acid at position 66 is selected from the group consisting of N, Q, S, T and Y; the amino acid at position 92 is selected from the group consisting of S, V, W, A C, F, G, I, L, M and P; the amino acid at position 93 is selected from the group consisting of S, V, W, A C, F, G, I, L, M and P; the amino acid at position 107 is selected from the group consisting of T, V, W, A, C, F, G, I, L, M and P; the amino acid at positionl 15 is selected from the group consisting of S, V, W, A, C, F, G, I, L, M and P; the amino acid at position 118 is selected from the group consisting of G, I, L, M, P, V, W, A, C and F; and the amino acid at position 119 is selected from the group consisting of F, G, I, L, M, P, V, W, A and C; b) TOR alpha chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5 and a CDR3 having the amino acid sequence of SEQ ID NO: 6;

- Polynucleotides encoding TCR beta chains comprising a) TCR beta chain constant regions which are selected from the group of TCR beta constant regions which are at least about 80% identical to the amino acid sequence as set out in SEQ ID NO: 15 wherein the amino acid at position 5 is selected from the group consisting of N, R,

D, E, H and K; the amino acid at position 18 is selected from the group consisting of E, H, K, R and D; the amino acid at position 22 is selected from the group consisting of S, V, W, A, C, F, G, I, L, M and P; the amino acid at position 34 is selected from the group consisting of T, D,

E, H, K, N and R; the amino acid at position 157 is selected from the group consisting of T,

V, W, A, C, F, G, I, L, M and P; and the amino acid at position 164 is selected from the group consisting of S,

V, W, A, C, F, G, I, L, M and P; or

- TCR beta chain constant regions which are selected from the group of TCR beta constant regions which are at least about 80% identical to the amino acid sequence in SEQ ID NO: 16; wherein the amino acid at position 5 is selected from the group consisting of N, R, D, E, H and K; the amino acid at position 18 is selected from the group consisting of E, H, K, R and D; the amino acid at position 22 is selected from the group consisting of S, V, W, A, C, F, G, I, L, M and P; the amino acid at position 34 is selected from the group consisting of T, D, E, H, K, N and R; the amino acid at position 157 is selected from the group consisting of T,

V, W, A, C, F, G, I, L, M and P; and the amino acid at position 164 is selected from the group consisting of S, V, W, A, C, F, G, I, L, M and P; b) TCR beta chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 7, a CDR2 having the amino acid sequence of SEQ ID NO:8 and a CDR3 having the amino acid sequence of SEQ ID NO: 9.

Item 3: Library of synthetic polynucleotides according to item 1 or 2, wherein the TCR specifically recognizes the amino acid sequence of SEQ ID NO: 3, which is presented by a molecule encoded by an HLA-A*02 gene.

Item 4: Library of synthetic polynucleotides according to items 1 to 3, wherein the library comprises polynucleotides encoding a TCR alpha chain and a TCR beta chain, each polynucleotide comprising:

- Polynucleotide encoding TCR alpha chain as defined in item 1 , and

- Polynucleotide encoding TCR beta chain as defined in item 1 ;

- Optionally a selection marker,

- Optionally one or more ribosomal entry site or self-cleaving peptides of the 2A family.

Item 5: Library of synthetic polynucleotides according to any one of items 1 to 4, wherein the library comprises at least 1 x 10 ⁵ , preferably, 1 x 10 ¹⁰ , more preferably 2.1 x 10 ¹¹ , such as 2.25 x 10 ¹³ unique molecules.

Item 6: A library of vectors comprising the polynucleotide library according to any one of items 1 to 5. Item 7: The library of vectors according to item 6, wherein the vectors are retroviral vectors.

Item 8: A library of cells, comprising the library of vectors according to item 7.

Item 9: A library of cells expressing the polynucleotides defined in items 1 to 4.

Item 10: A library of polypeptides encoded by the library of synthetic polynucleotides according to items 1 to 5.

Item 11 : Use of the library of items 1 to 10 for identifying a TCR receptor with enhanced TCR alpha and TRC beta chain pairing.

Item 12: A method for isolating a library derived TCR with enhanced TCR alpha and beta chain pairing, comprising the steps:

- Co-culturing the library of cells according to items 8 to 9 with antigen presenting cells presenting the HLA bound form of the target antigen of the TCR, and at least one of the steps

- Selecting cells showing elevated expression levels of the heterologous TCR compared to wild type,

- Selecting cells showing elevated activation levels compared to wild type.

Item 13: A TCR isolated from the TCR library as described in item 11 .

Item 14: A library of TCR alpha chains and TCR beta chains, comprising

- TCR alpha chains comprising: a) TCR alpha chain constant regions which are selected from the group of TCR alpha constant regions which are at least about 80% identical to the amino acid sequence as set out in SEQ ID NO 1 ; b) TCR alpha chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5 and a CDR3 having the amino acid sequence of SEQ ID NO: 6; - TCR beta chains comprising a) TCR beta chain constant regions comprising constant regions which are selected from the group of TCR beta constant regions which are at least about 80% identical to the amino acid sequence as set out in SEQ ID NO 15; or

TCR beta chain constant regions which are selected from the group of TCR beta constant regions which are at least about 80% identical to the amino acid sequence as set out in SEQ ID NO 16; b) TCR beta chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 7, a CDR2 having the amino acid sequence of SEQ ID NO: 8 and a CDR3 having the amino acid sequence of SEQ ID NO: 9.

Item 15: A library of TCR alpha chains and TCR beta chains, comprising

- TCR alpha chains comprising: a) TCR alpha chain constant regions which are selected from the group of TCR alpha constant regions which are at least about 80% identical to the amino acid sequence as set out in SEQ ID NO 1 wherein

X at position 14 is selected from the group consisting of S, V, W, A, C, F, G, I, L, M and P;

X at position 66 is selected from the group consisting of N, Q, S, T and Y;

X at position 92 is selected from the group consisting of S, V, W, A C, F, G, I, L, M and P;

X at position 93 is selected from the group consisting of S, V, W, A C, F, G, I, L, M and P;

X at position 107 is selected from the group consisting of T, V, W, A, C, F, G, I, L, M and P;

X at positionl 15 is selected from the group consisting of S, V, W, A, C, F, G, I, L, M and P;

X at position 118 is selected from the group consisting of G, I, L, M, P, V, W, A, C and F; and X at position 119 is selected from the group consisting of F, G, I, L, M, P, V, W, A and C; b) TOR alpha chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5 and a CDR3 having the amino acid sequence of SEQ ID NO: 6;

-TCR beta chains comprising a) TCR beta chain constant regions comprising constant regions which are selected from the group of TCR beta constant regions which are at least about 80% identical to the amino acid sequence as set in SEQ ID NO: 2, wherein the amino acid at position 4 is selected from the group consisting of K, N; the amino acid at position 5 is selected from the group consisting of N, R, D, E, H and K; the amino acid at position 18 is selected from the group consisting of E, H, K, R and D; the amino acid at position 22 is selected from the group consisting of

S, V, W, A, C, F, G, I, L, M and P; the amino acid at position 34 is selected from the group consisting of

T, D, E, H, K, N and R; the amino acid at position 37 is selected from the group consisting of Y, F; the amino acid at position 136 is selected from the group consisting of E, V; the amino acid at position 157 is selected from the group consisting of T, V, W, A, C, F, G, I, L, M and P; and the amino acid at position 164 is selected from the group consisting of S, V, W, A, C, F, G, I, L, M and P; the amino acid at position 177 is selected from the group consisting of S, F; the amino acid at position 178 is R or deleted; the amino acid at position 179 is G or deleted.; and

X at position 164 is selected from the group consisting of S, V, W, A, C, F, G, I, L, M and P; b) TOR beta chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 7, a CDR2 having the amino acid sequence of SEQ ID NO: 8 and a CDR3 having the amino acid sequence of SEQ ID NO: 9.

Item 16: A method of preparing a library of synthetic polynucleotides encoding a plurality of encoding TCR alpha chain constant regions and TCR beta chain constant regions, the method comprising:

- Providing the sequences of the polynucleotides encoding the TCR alpha chains and TCR beta chains as defined in items 1 to 4;

- Assembling the synthetic polynucleotide to produce a library of synthetic polynucleotides.

Item 17: A kit comprising the library as defined in items 1 to 9, 14 or 15.

Item 18: The nucleotide sequences of the synthetic polynucleotides defined in items 1 to 4 in computer readable format.

Item 19: The amino acid sequences defined in items 1 to 4 in computer readable format.

Item 20: TCR capable of binding to a PRAME peptide having the sequence VLDGLDVLL (SEQ ID NO: 3), wherein the TCR comprises:

- a TCR alpha chain comprising a) TCR alpha chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5 and a CDR3 having the amino acid sequence of SEQ ID NO: 6; b) a constant TCR alpha region comprising the sequence selected from the group of constant TCR alpha regions represented by the sequence as set in SEQ ID NO 1 :

- a TCR beta chain comprising a) TCR beta chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 7, a CDR2 having the amino acid sequence of SEQ ID NO: 8 and a CDR3 having the amino acid sequence of SEQ ID NO: 9; b) a constant TCR beta region comprising the sequence selected from the group of constant TCR beta regions represented by the sequence as set in SEQ ID NO: 15, or a constant TCR beta region comprising the sequence selected from the group of constant TCR beta regions represented by the sequence as set in SEQ ID NO: 16.

Item 21 : TCR according to item 20, wherein the TCR shows elevated expression levels of the heterologous TCR compared to wild type.

Item 22: TCR according to items 20 and 21 , wherein the TCR shows elevated activation levels compared to wild type.

Item 23: TCR according to items 20 to 22, wherein at in least one of X01 to X14 the amino acid is not the amino acid which occurs naturally in wild type at the respective position.

Item 24: The TCR according to items 20 to 23, wherein in at least two, at least three, at least four, at least five, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, such as in 14 positions of X01 to X14, the amino acid is not the amino acid which occurs naturally in wild type at the respective position.

Item 25. TCR capable of binding to a PRAME peptide having the sequence VLDGLDVLL (SEQ ID NO: 3), wherein the TCR comprises:

- A TCR alpha chain comprising a) a variable TCR alpha chain region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5 and a CDR3 having the amino acid sequence of SEQ ID NO: 6; b) A constant TCR alpha region comprising the sequence SEQ ID NO: 12

- A TCR beta chain comprising a) a variable TCR beta chain region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 7, a CDR2 having the amino acid sequence of SEQ ID NO: 8 and a CDR3 having the amino acid sequence of SEQ ID NO: 9; b) a constant TCR beta region comprising the sequence SEQ ID NO: 13; Item 26. TCR according to items 19 to 25 , wherein the TCR specifically recognizes the amino acid sequence of SEQ ID NO: 3, which is presented by a molecule encoded by an HLA-A*02 gene.

Item 27. TCR according to any one of items 19 to 23, wherein the TCR comprises a) a variable TCR a region having an amino acid sequence which is at least 80% identical to SEQ ID NO: 10 and b) a variable TCR [3 region having an amino acid sequence which is at least 80% identical to SEQ ID NO: 11 .

Item 28: TCR according to any one of items 19 to 24, wherein the TCR comprises a variable TCR a region having the amino acid sequence of SEQ ID NO: 10 and a variable TCR [3 region having the amino acid sequence of SEQ ID NO: 11 .

Item 29: Nucleic acid encoding a TCR according to any one of items 19 to 28.

Item 30: Vector comprising the nucleic acid of item 29.

Item 31 : Vector according to item 30, wherein the vector is an expression vector.

Item 32: Vector according to item 30 or 31 , wherein the vector is a retroviral vector.

Item 33: Vector according to item 30 or 31 , wherein the vector is a lentiviral vector.

Item 34: Cell expressing the TCR according to items 19 to 28, the nucleic acid according to item 27, the vector according to items 30 to 33.

Item 35: Cell according to item 34, wherein the cell is isolated or non-naturally occurring.

Item 36: Cell according to items 34 or 35, wherein the cell comprises the nucleic acid according to item 29 or the vector according to items 30 to 33.

Item 37: Cell according to items 34 to 36, wherein the cell comprises: a) an expression vector which comprises at least one nucleic acid as embodied in item 29, or b) a first expression vector which comprises a nucleic acid encoding the alpha chain of the TCR as embodied in any one of the items 20 to 28, and a second expression vector which comprises a nucleic acid encoding the beta chain of a TCR as embodied in any one of the items 20 to 28. Item 38: Cell according to any one of items 34 to 37, wherein the cell is a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC).

Item 39: Cell according to any one of items 34 to 38, wherein the cell is a T cell.

Item 40: Pharmaceutical composition comprising the TCR according to items 20 to 28, the nucleic acid according to item 29, the vector according to items 30 to 33, the cell according to any one of items 34 to 39.

Item 41 : Pharmaceutical composition according to item 40 wherein the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier.

Item 42: The TCR according to items 20 to 28, the nucleic acid according to item 29, the vector according to items 30 to 33, the cell according to any one of items 34 to 39 for use as a medicament.

Item 43: The TCR according to items 20 to 28, the nucleic acid according to item 27, the vector according to items 30 to 33, the cell according to any one of items 34 to 39 for use in the treatment of cancer.

Item 44: The TCR, the polypeptide, the multivalent TCR complex, the nucleic acid, the vector or the cell for use according to item 43, wherein the cancer is a hematological cancer or a solid tumor.

Item 45: The TCR, the polypeptide, the multivalent TCR complex, the nucleic acid, the vector or the cell for use according to item 44, wherein the cancer is a hematological cancer.

Item 46: The TCR, the polypeptide, the multivalent TCR complex, the nucleic acid, the vector or the cell for use according to item 45, Wherein the hematological cancer is Acute myeloid leukemia (AML), myelodysplastic syndromes (MDS) and multiple myeloma (MM).

Item 47: The TCR, the polypeptide, the multivalent TCR complex, the nucleic acid, the vector or the cell for use according to item 43, wherein the cancer is a solid tumor.