NOVEL ENGINEERED T CELL RECEPTORS AND IMMUNE THERAPY USING THE SAME

Abstract

The present invention pertains to antigen recognizing constructs against COL6A3 antigens. The invention in particular provides novel engineered T cell receptor (TCR) based molecules which are selective and specific for the tumor expressing antigen COL6A3. The TCR of the invention, and COL6A3 antigen binding fragments derived therefrom, are of use for the diagnosis, treatment and prevention of COL6A3 expressing cancerous diseases. Further provided are nucleic acids encoding the antigen recognizing constructs of the invention, vectors comprising these nucleic acids, recombinant cells expressing the antigen recognizing constructs and pharmaceutical compositions comprising the compounds of the invention.

Claims

1. A method of treating a patient who has cancer, comprising administering to the patient a population of transformed T cells expressing at least one vector encoding a T cell receptor (TCR), wherein the TCR comprises SEQ ID NOs: 6, 7, 14, and 15, wherein each of SEQ ID NOs: 6, 7, 14, and 15 comprises at most one conservative amino acid substitution, wherein the TCR binds to a peptide consisting of the amino acid sequence of FLLDGSANV (SEQ ID NO: 1) in a complex with an MHC class I molecule, and wherein the cancer is selected from acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, cancer of the oropharynx, ovarian cancer, cancer of the penis, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, cancer of the uterus, ureter cancer, gastric cancer, and urinary bladder cancer.

2. The method of claim 1, wherein the population of transformed cells are produced by a method comprising isolating a T cell from a subject, transforming the cell with at least one vector encoding the TCR to produce a transformed cell, and expanding the transformed cell to produce the population of transformed cells.

3. The method of claim 2, wherein the subject is the patient.

4. The method of claim 1, wherein said T cells are CD8+ T cells.

5. The method of claim 1, wherein the TCR comprises an chain comprising the amino acid sequence of SEQ ID NO: 2 and a chain comprising the amino acid sequence of SEQ ID NO: 10, or SEQ ID NO: 22, or SEQ ID NO: 24, or SEQ ID NO: 25, or SEQ ID NO: 27.

6. The method of claim 1, wherein the MHC class I molecule is HLA-A*02.

7. The method of claim 1, wherein the population of transformed cells are administered in the form of a pharmaceutical composition.

8. The method of claim 7, wherein the pharmaceutical composition comprises a chemotherapeutic agent selected from the group consisting of asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and vincristine.

9. The method of claim 1, wherein the TCR comprises: a CDR1 chain comprising the amino acid sequence of SEQ ID NO: 5 or 26 a CDR2 chain comprising the amino acid sequence of SEQ ID NO: 6, a CDR3 chain comprising the amino acid sequence of SEQ ID NO: 7, a CDR1 chain comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 13 and 37-44, a CDR2 chain comprising the amino acid sequence of SEQ ID NO: 14, and a CDR3 chain comprising the amino acid sequence of SEQ ID NO: 15, and wherein each of SEQ ID NOs: 5, 26, 13, and 37-44 comprises at most one conservative amino acid substitution.

10. The method of claim 9, wherein the CDR1 chain comprises the amino acid sequence of SEQ ID NO: 5 and the CDR1 chain comprises the amino acid sequences of SEQ ID NO: 13, wherein each of SEQ ID NOs: 5 and 13 comprises at most one conservative amino acid substitution.

11. The method of claim 9, wherein the CDR1 chain comprises the amino acid sequence of SEQ ID NO: 5 and the CDR1 chain comprises the amino acid sequences of SEQ ID NO: 37, wherein each of SEQ ID NOs: 5 and 37 comprises at most one conservative amino acid substitution.

12. The method of claim 9, wherein the CDR1 chain comprises the amino acid sequence of SEQ ID NO: 5 and the CDR1 chain comprises the amino acid sequences of SEQ ID NO: 38, wherein each of SEQ ID NOs: 5 and 38 comprises at most one conservative amino acid substitution.

13. The method of claim 9, wherein the CDR1 chain comprises the amino acid sequence of SEQ ID NO: 5 and the CDR1 chain comprises the amino acid sequences of SEQ ID NO: 39, wherein each of SEQ ID NOs: 5 and 39 comprises at most one conservative amino acid substitution.

14. The method of claim 9, wherein the CDR1 chain comprises the amino acid sequence of SEQ ID NO: 5 and the CDR1 chain comprises the amino acid sequences of SEQ ID NO: 40, wherein each of SEQ ID NOs: 5 and 40 comprises at most one conservative amino acid substitution.

15. The method of claim 9, wherein the CDR1 chain comprises the amino acid sequence of SEQ ID NO: 5 and the CDR1 chain comprises the amino acid sequences of SEQ ID NO: 42, wherein each of SEQ ID NOs: 5 and 42 comprises at most one conservative amino acid substitution.

16. The method of claim 9, wherein the CDR1 chain comprises the amino acid sequence of SEQ ID NO: 5 and the CDR1 chain comprises the amino acid sequences of SEQ ID NO: 43, wherein each of SEQ ID NOs: 5 and 43 comprises at most one conservative amino acid substitution.

17. The method of claim 9, wherein the CDR1 chain comprises the amino acid sequence of SEQ ID NO: 26 and the CDR1 chain comprises the amino acid sequences of SEQ ID NO: 38, wherein each of SEQ ID NOs: 26 and 38 comprises at most one conservative amino acid substitution.

18. The method of claim 9, wherein the CDR1 chain comprises the amino acid sequence of SEQ ID NO: 26 and the CDR1 chain comprises the amino acid sequences of SEQ ID NO: 39, wherein each of SEQ ID NOs: 26 and 39 comprises at most one conservative amino acid substitution.

19. The method of claim 9, wherein the CDR1 chain comprises the amino acid sequence of SEQ ID NO: 26 and the CDR1 chain comprises the amino acid sequences of SEQ ID NO: 40, wherein each of SEQ ID NOs: 26 and 40 comprises at most one conservative amino acid substitution.

20. The method of claim 9, wherein the CDR1 chain comprises the amino acid sequence of SEQ ID NO: 26 and the CDR1 chain comprises the amino acid sequences of SEQ ID NO: 44, wherein each of SEQ ID NOs: 26 and 44 comprises at most one conservative amino acid substitution.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0099] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0100] The present invention will now be further described in the following examples with reference to the accompanying figures and sequences, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties. In the Figures and Sequences:

[0101] FIG. 1 shows the conversion of a TCR into stabilized V/V single-chain TCR (scTv) via yeast surface display. ScTv molecules displayed on the surface of transformed Saccharomyces cerevisiae EBY100 were stained with FITC-labeled anti-Vbeta1 antibody and PE-labeled HLA-A*02/COL6A3-002 tetramer. The unmodified scTv R4P3F9 (left panel, SEQ ID NO: 22) is compared to an scTv clone bearing single point mutations to stabilize the scTv scaffold (right panel), which was derived from the selection of a random mutation scTv library.

[0102] FIG. 2 shows scTv affinity maturation via yeast surface display. Stabilized scTv molecules with and without affinity maturated CDR1 beta were stained with HLA-A*02 tetramers containing COL6A3-002 (SEQ ID NO: 1) and counterstained with a mix of HLA-A*02 tetramers containing 9 peptides (SEQ ID NO: 28 to 36) with high sequence similarity to COL6A3-002. Stabilized scTv (SEQ ID NO 27) with non-maturated beta chain CDR1 sequence RSGDLS (SEQ ID NO: 13) is compared with scTv clones bearing the affinity maturated beta chain CDR1 sequences AMDHPY (SEQ ID NO: 40) and ARWHRN (SEQ ID NO: 39).

[0103] FIG. 3 shows size exclusion chromatography elution profiles of anti-CD3 Fab-scTv R4P3F9S fusion variants 75-1 to 75-25.

[0104] FIG. 4 shows the HLA-A*02/COL6A3-002 binding kinetics of anti-CD3 Fab-scTv R4P3F9S fusion variants 75-1 to 75-25 as measured by biolayer interferometry. Analyzed concentrations of HLA-A*02/COL6A3-002 are indicated.

[0105] FIG. 5 shows binding analysis of anti-CD3 Fab-scTv R4P3F9S fusion variants 75-1 to 75-25 as measured via biolayer interferometry (BLI). 1 M of HLA-A*02 in complex with the indicated similar peptides was analyzed.

[0106] FIG. 6 shows a comparison of HLA-A*02/COL6A3-002 and HLA-A*02/COL6A1-001 (SEQ ID NO: 30) binding kinetics of different anti-CD3 Fab-scTv R4P3F9S fusion variants. Analyzed concentrations of Fab-scTv molecules are indicated.

[0107] FIG. 7 shows staining of maturated R4P3F9 TCR variant expressing human CD8.sup.+ T cells with PE-labeled HLA-A*02/COL6A3-002 tetramers. For control purpose, no TCR (Mock) or the 1G4 TCR specific for NYESO1-001 was expressed and staining with PE-labeled HLA-A*02/NYESO1-001 tetramers was used.

[0108] FIG. 8 shows IFN-gamma release of maturated R4P3F9 TCR variant expressing human CD8.sup.+ T cells in response to COL6A3-002. For control purpose, no TCR (mock) or the 1G4 TCR specific for NYESO1-001 was expressed. IFN-gamma release was determined by ELISA after co-culture of electroporated CD8.sup.+ T cells with T2 cells loaded with a serial dilution of COL6A3-002.

[0109] FIG. 9 shows IFN-gamma release of maturated R4P3F9 TCR variant expressing human CD8.sup.+ T cells in response to COL6A3-002 and different similar peptides. For control purpose, no TCR (mock) or the 1G4 TCR specific for NYESO1-001 was expressed. IFN-gamma release was determined by ELISA after co-culture of electroporated CD8.sup.+ T cells with T2 cells loaded with 10 M of COL6A3-002 or similar peptides.

[0110] FIG. 10 shows staining of maturated R4P3F9 TCR variant expressing human CD8.sup.+ T cells with PE-labeled peptide-HLA-A*02 tetramers. For control purpose, no TCR (Mock) or the 1G4 TCR specific for NYESO1-001 was expressed and staining with PE-labeled HLA-A*02/NYESO1-001 tetramers was used.

[0111] FIG. 11 shows IFN-gamma release of maturated R4P3F9 TCR variant expressing human CD8.sup.+ T cells in response to COL6A3-002 or COL6A1-001. For control purpose, no TCR (mock) or the 1G4 TCR specific for NYESO1-001 was expressed. IFN-gamma release was determined by ELISA after co-culture of electroporated CD8.sup.+ T cells with T2 cells loaded with a serial dilution of COL6A3-002 or COL6A1-001.

[0112] FIG. 12 shows IFN-gamma release of primary human CD8.sup.+ T cells expressing R4P3F9 TCR variants upon co-culture with different tumor cell lines. SF539, SW982 and Hs840.T cells present the target peptide at different levels. MCF-7 cells do not present the target peptide. As controls, effector cells without exogenous TCR were analyzed along with cells with TCRs of interest. IFN-gamma release was determined by ELISA. * marks a data point that is out of scale.

[0113]

TABLE-US-00001 TABLE1 Peptidesequencesoftheinvention (positionsareaccordingtoIMGTnumbering:(Franois Ehrenmann,PatriceDuroux,ChantalGinestoux;Proteindisplays: human(Homosapiens)TRAV;IMGTRepertoire.IMGTthe internationalImMunoGeneticsinformationsystemwww.imgt.org.; Created:16/03/2011.Version:03/06/2016;FranoisEhrenmann, PatriceDuroux,ChantalGinestoux;Proteindisplays:human (Homosapiens)TRBV;IMGTRepertoire.IMGT,theinternational ImMunoGeneticsinformationsystemwww.imgt.org.; Created:16/03/2011.Version:03/06/2016.) SEQ ID NO: Name Description Sequence 1 COL6A3-002 FLLDGSANV 2 R4P3F9 R4P3F9TCR MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIA alpha alpha SLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDG chain- RFTAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGAGSYQ full LTFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTD length FDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSN KSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETD TNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 3 R4P3F9 R4P3F9TCR MKSLRVLLVILWLQLSWVWSQ alpha alpha leader chain- leader peptide 4 R4P3F9 R4P3F9TCR QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYS alpha alpha GKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQ variable chain- PSDSATYLCAAYSGAGSYQLTFGKGTKLSVIP variable domain 5 R4P3F9 R4P3F9TCR DRGSQS CDRa1 alpha chain- CDR1 6 R4P3F9 R4P3F9TCR IYSNGD CDRa2 alpha chain- CDR2 7 R4P3F9 R4P3F9TCR CAAYSGAGSYQLT CDRa3 alpha chain- CDR3 8 R4P3F9- R4P3F9TCR NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDS alpha alpha DVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNS constant chain- IIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGF constant RILLLKVAGFNLLMTLRLWSS domain 9 R4P3F9- R4P3F9TCR NIQN alpha alpha constant chain- start constant domain start 10 R4P3F9 R4P3F9TCR MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTL beta beta RCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNIL chain- ERFSAQQFPDLHSELNLSSLELGDSALYFCASSVESSYGY full TFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATL length VCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPAL NDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEW TQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILY EILLGKATLYAVLVSALVLMAMVKRKDF 11 R4P3F9 R4P3F9TCR MGFRLLCCVAFCLLGAGPV beta beta leader chain- leader peptide 12 R4P3F9 R4P3F9TCR DSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLD beta beta QGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSS variable chain- LELGDSALYFCASSVESSYGYTFGSGTRLTVV variable domain 13 R4P3F9 R4P3F9TCR RSGDLS CDRb1 beta chain- CDR1 14 R4P3F9 R4P3F9TCR YYNGEE CDRb2 beta chain- CDR2 15 R4P3F9 R4P3F9TCR CASSVESSYGYT CDRb3 beta chain- CDR3 16 R4P3F9 R4P3F9TCR EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDH beta beta VELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRL constant chain- RVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQI constant VSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYA domain VLVSALVLMAMVKRKDF 17 R4P3F9 R4P3F9TCR EDLNK beta betachain- constant constant start1 domain start1 18 R4P3F9 R4P3F9TCR EDLKN beta betachain- constant constant start2 domain start2 19 Aga2p- Aga2p MQLLRCFSIFSVIASVLAQELTTICEQIPSPTLESTPYSL R4P3F9 fusion STTTILANGKAMQGVFEYYKSVTFVSNCGSHPSTTSKGSP protein INTQYVFGGGGSDYKDDDDKGGGASQKEVEQNSGPLSVPE withscTv GAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGD R4P3F9and KEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGA tags GSYQLTFGKGTKLSVIPNIQNGGGGSGGGGSGGGGSGGGG SGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQ GLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSL ELGDSALYFCASSVESSYGYTFGSGTRLTVVEDLNKAAAG GSGGEQKLISEEDL 20 Aga2p Leader MQLLRCFSIFSVIASVLAQELTTICEQIPSPTLESTPYSL sequence STTTILANGKAMQGVFEYYKSVTFVSNCGSHPSTTSKGSP andAga2p INTQYVF 21 FLAGtag FLAGtag GGGGSDYKDDDDKGGGAS plus linkers 22 scTv Single QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYS R4P3F9 chain GKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQ variable PSDSATYLCAAYSGAGSYQLTFGKGTKLSVIPNIQNGGGG domainsof SGGGGSGGGGSGGGGSGVTQTPKHLITATGQRVTLRCSPR R4P3F9 SGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSA with QQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTFGSG linker; TRLTVVEDLNK aF55Sin alpha variable domain 23 Myctag Linkerand AAAGGSGGEQKLISEEDL Myctag 24 scTv scTv QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYS R4P3F9- R4P3F9 GKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQ bQ43K with PSDSATYLCAAYSGAGSYQLTFGKGTKLSVIPNIQNGGGG stabilizing SGGGGSGGGGSGGGGSGVTQTPKHLITATGQRVTLRCSPR mutation SGDLSVYWYKQSLDQGLQFLIQYYNGEERAKGNILERFSA bQ43Kin QQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTFGSG beta TRLTVVEDLNK variable domain 25 scTv scTv QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYS R4P3F9- R4P3F9 GKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQ bL72S with PSDSATYLCAAYSGAGSYQLTFGKGTKLSVIPNIQNGGGG stabilizing SGGGGSGGGGSGGGGSGVTQTPKHLITATGQRVTLRCSPR mutation SGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNISERFSA bL72Sin QQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTFGSG beta TRLTVVEDLNK variable domain 26 CDRa1 aG29R DRRSQS mutant1 mutation 27 scTv Stabilized QKEVEQNSGPLSVPEGAIASLNCTYSDRRSQSFFWYRQYS R4P3F9S versionof GKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQ scTv PSDSATYLCAAYSGAGSYQLTFGKGTKLSVIPGGGGSGGG R4P3F9 GSGGGGSGGGGSGGGGSGVTQTPKHLITATGQRVTLRCSP RSGDLSVYWYKQSLDQGLQFLIQYYNGEERAKGNISERFS AQQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTFGS GTRLTVV 28 AGRN-001 Similar ALLDGRVQL peptides 29 CLASP1-001 Similar RLLDGAFKL peptides 30 COL6A1-001 Similar ILLDGSASV peptides 31 COL6A2-001 Similar FLLDGSERL peptides 32 COL6A3-006 Similar FLFDGSANLV peptides 33 COL6A3-008 Similar FLFDGSANL peptides 34 COL6A3-014 Similar FLLDGSEGV peptides 35 VWA2-001 Similar FLLDGSNSV peptides 36 VWF-001 Similar FLLDGSSRL peptides 37 CDRb1 Betachain- ARWHNN mutant1 CDR1 variant1 38 CDRb1 Betachain- AKDHLN mutant2 CDR1 variant2 39 CDRb1 Betachain- ARWHRN mutant3 CDR1 variant3 40 CDRb1 Betachain- AMDHPY mutant4 CDR1 variant4 41 CDRb1 Betachain- ATDHYN mutant5 CDR1 variant5 42 CDRb1 Betachain- ARYHTN mutant6 CDR1 variant6 43 CDRb1 Betachain- APYHLN mutant7 CDR1 variant7 44 CDRb1 Betachain- AKDHTN mutant8 CDR1 variant8 45 CDRb1 Betachain- ARYHRN mutant9 CDR1 variant9 46 CDRb1 Betachain- ARWHSN mutant10 CDR1 variant10 47 CDRb1 Betachain- ATDHYN mutant11 CDR1 variant11 48 CDRb1 Betachain- RWGDLN mutant12 CDR1 variant12 49 CDRb1 Betachain- ARDHLN mutant13 CDR1 variant13 50 75-1 Fabheavy MKWVTFISLLFLFSSAYSEVQLVESGGGLVQPGGSLRLSC chainwith AASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQ stabilized KFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYY scTv GDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG R4P3F9S GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTSPPSPAPPVAGQKEVEQNSGPLSVPEGAIASL NCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRF TAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGAGSYQLT FGKGTKLSVIPNIQNGGGGSGGGGSGGGGSGGGGSGGGGS GVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYKQSLDQG LQFLIQYYNGEERAKGNISERFSAQQFPDLHSELNLSSLE LGDSALYFCASSVESSYGYTFGSGTRLTVVEDLKN 51 75- Fabheavy MKWVTFISLLFLFSSAYSEVQLVESGGGLVQPGGSLRLSC Fab chain AASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQ heavy KFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYY chain GDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTSPPSPAPPVAG 52 75- Fablight MKWVTFISLLFLFSSAYSDIQMTQSPSSLSASVGDRVTIT Fab chain CRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRF light SGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGT chain KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 53 1G4alpha 1G4TCR METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVL alpha NCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGR chain- LNASLDKSSGRSTLYIAASQPGDSATYLCAVRPTSGGSYI full PTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTD length FDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSN KSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETD TNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 54 1G4alpha 1G4TCR METLLGLLILWLQLQWVSSK leader alpha chain- leader peptide 55 1G4alpha 1G4TCR QEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPG variable alpha KGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQ chain- PGDSATYLCAVRPTSGGSYIPTFGRGTSLIVHP variable domain 56 1G4alpha 1G4TCR YIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDS constant alpha DVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNS chain- IIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGF constant RILLLKVAGFNLLMTLRLWSS domain 57 1G4beta 1G4TCR MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTL betachain- QCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVP full NGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGE length LFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKAT LVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPA LNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDE WTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATIL YEILLGKATLYAVLVSALVLMAMVKRKDSRG 58 1G4beta 1G4TCR MSIGLLCCAALSLLWAGPVNA leader betachain- leader peptide 59 1G4beta 1G4TCR GVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMG variable betachain- LRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAA variable PSQTSVYFCASSYVGNTGELFFGEGSRLTVL domain 60 1G4beta Betachain- EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDH constant constant VELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRL domain RVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQI VSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYA VLVSALVLMAMVKRKDSRG 61 NYES01-001 Control SLLMWITQV peptide 62 C-14 C-14;C-5 MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTL beta;C-5 TCRfull RCSPAMDHPYVYWYQQSLDQGLQFLIQYYNGEERAKGNIL beta length ERFSAQQFPDLHSELNLSSLELGDSALYFCASSVESSYGY betachain TFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATL withCDRb1 VCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPAL mutant4 NDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEW TQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILY EILLGKATLYAVLVSALVLMAMVKRKDF 63 C-14 C-14TCR MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIA alpha full SLNCTYSDRRSQSFFWYRQYSGKSPELIMFIYSNGDKEDG length RFTAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGAGSYQ alpha LTFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTD chainwith FDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSN CDRa1 KSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETD mutant1 TNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 64 75-5 Fabheavy MKWVTFISLLFLFSSAYSEVQLVESGGGLVQPGGSLRLSC chainwith AASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQ stabilized KFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYY scTv GDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG R4P3F9S GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS andCDRb1 GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP mutant4 KSCDKTHTSPPSPAPPVAGQKEVEQNSGPLSVPEGAIASL NCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRF TAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGAGSYQLT FGKGTKLSVIPNIQNGGGGSGGGGSGGGGSGGGGSGGGGS GVTQTPKHLITATGQRVTLRCSPAMDHPYVYWYKQSLDQG LQFLIQYYNGEERAKGNISERFSAQQFPDLHSELNLSSLE LGDSALYFCASSVESSYGYTFGSGTRLTVVEDLKN 65 75-14 Fabheavy MKWVTFISLLFLFSSAYSEVQLVESGGGLVQPGGSLRLSC chainwith AASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQ stabilized KFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYY scTv GDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG R4P3F9S, GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS CDRa1 GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP mutant1 KSCDKTHTSPPSPAPPVAGQKEVEQNSGPLSVPEGAIASL andCDRb1 NCTYSDRRSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRF mutant4 TAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGAGSYQLT FGKGTKLSVIPNIQNGGGGSGGGGSGGGGSGGGGSGGGGS GVTQTPKHLITATGQRVTLRCSPAMDHPYVYWYKQSLDQG LQFLIQYYNGEERAKGNISERFSAQQFPDLHSELNLSSLE LGDSALYFCASSVESSYGYTFGSGTRLTVVEDLKN 66 75-25 Fabheavy MKWVTFISLLFLFSSAYSEVQLVESGGGLVQPGGSLRLSC chainwith AASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQ stabilized KFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYY scTv GDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG R4P3F9Sin GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS beta/alpha GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP orientation, KSCDKTHTSPPSPAPPVAGGVTQTPKHLITATGQRVTLRC CDRa1 SPRSGDLSVYWYKQSLDQGLQFLIQYYNGEERAKGNISER mutant1 FSAQQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTF GSGTRLTVVEDLKNGGGGSGGGGSGGGGSGGGGSGGGGSQ KEVEQNSGPLSVPEGAIASLNCTYSDRRSQSFFWYRQYSG KSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQP SDSATYLCAAYSGAGSYQLTFGKGTKLSVIPNIQN

EXAMPLES

[0114] Native T cell receptors (TCRs) against cancer antigens are often of lower affinity when compared to TCRs targeting viral antigens, and this may be one possible explanation for tumor immune escape (Aleksic et al. 2012). Therefore, it is desirable to have higher affinity TCR variants designed for the use as antigen recognizing constructs in an adoptive cell therapy, or as recognition module of a soluble approach, i.e., using bispecific molecules (Hickman et al. 2016). This invention thus relates to the modification and optimization of the naturally occurring T cell receptor R4P3F9 (SEQ ID NOs: 2 and 10) targeting the tumor associated peptide COL6A3-002 (SEQ ID NO: 1) with an affinity of about 60 M (DE102016115246).

Example 1: Generation of Stable scTv

[0115] For the present invention, the previously investigated TCR R4P3F9 (SEQ ID NOs: 2 and 10) was converted into a single chain TCR construct (scTv, SEQ ID NO: 22) for maturation via yeast surface display by combination of the variable alpha (SEQ ID NO: 4) and beta (SEQ ID NO: 12) domain with appendages of the respective constant domains (SEQ ID NOs: 9 and 17) and an appropriate glycine-serine linker sequence. The DNA of the corresponding sequence was synthesized and transformed into Saccharomyces cerevisiae EB Y100 (MATa AGA1::GAL1AGA1::URA3 ura352 trp1 leu2delta200 his3delta200 pep4::HIS3 prbd1.6R can1 GAL) (ATCC MYA 4941) together with a yeast display vector containing a leader sequence and the Aga2p yeast mating protein (SEQ ID NO: 20), based on pCT302 (Boder et al. 2000). The resulting fusion protein after homologous recombination in the yeast (SEQ ID NO: 19) contains a leader peptide at the N-terminus of the Aga2p protein, responsible for the display of the protein of interest (Boder et al. 1997), short peptide tags including linker sequences (SEQ ID NOs: 21 and 23) for expression controls and the protein of interest, namely the scTv R4P3F9 (SEQ ID NO: 22) or its variants. The transformation was performed as described in DE102016121899 and resulted in up to 10.sup.9 yeast clones per library. The libraries were generated via a random mutation PCR approach spanning the whole gene sequence of the scTv R4P3F9.

[0116] The selection process for the yeast clones bearing the best expressing scTv that is selectively binding to COL6A3-002 in context of HLA-A*02 was essentially performed as described in Smith et al 2015. In order to ascertain high expression and correct conformation of the scTv R4P3F9 variant, displayed on the yeast surface, an anti-Vbeta1 (Beckman Coulter, clone BL37.2) antibody was used, together with HLA-A*02/COL6A3-002 tetramer (FIG. 1). The scTv conversion by yeast surface display revealed two crucial stabilizing mutations in the framework region together with the original CDR sequences for the proper presentation of the scTv on the cell surface, namely bQ43K (SEQ ID NO: 24) and bL72S (SEQ ID NO: 25), both located on the beta chain. Furthermore, during stability maturation position 29 in the CDR1 of the alpha chain (SEQ ID NO: 5) was converted from glycine to arginine (CDRa1 mutant 1, SEQ ID NO: 26), which resulted in improved tetramer binding.

Example 2: Affinity Maturation of Stabilized scTv

[0117] To generate scTv molecules with higher binding affinity towards HLA-A*02/COL6A3-002, the CDRb1 (SEQ ID NO: 13) was degenerated using the previously identified stabilized scTv R4P3F9S scaffold (SEQ ID NO: 27) expressing the stabilizing mutations aG29R, bQ43K and bL72S. The CDRb1 residues were randomized by using degenerate DNA oligo primers essentially as described previously (Smith et al. 2015). The resulting DNA library was transformed as described in example 1. To retain scTv binding selectivity, negative selection was employed against HLA-A*02 tetramers comprising peptides derived from normal tissues (SEQ ID NOs: 28 to 36), which show high sequence similarity to COL6A3-002 peptide.

[0118] For the selection of affinity enhanced and selective scTv R4P3F9S variants a decreasing concentration of HLA-A*02/COL6A3-002 tetramer was used for each sorting round. After three selection rounds, single scTv clones were isolated and sequenced, resulting in a variety of affinity maturated CDRb1 sequences (SEQ ID NOs: 37 to 49). For scTv with maturated CDRb1 sequences a strong improvement in COL6A3-002 binding could be demonstrated while the selectivity of COL6A3-002 binding was retained as no binding of 9 similar peptides was observed (FIG. 2).

Example 3: Production of Bispecific Antibody-scTv Fusion Proteins

[0119] Stabilized and affinity maturated scTv against HLA-A*02/COL6A3-002 can be expressed in fusion with an antibody moiety directed against CD3 allowing tumor-specific retargeting and activation of T cells independent of their natural specificity. The inventors generated bispecific antibody-TCR fusion proteins comprising an anti-CD3 Fab (UCHT1) heavy chain (SEQ ID NO: 51) fused to scTv R4P3F9S variants (SEQ ID NOs: 50, 64, 65 and 66) and an anti-CD3 Fab (UCHT1) light chain (SEQ ID NO: 52). The resulting Fab-scTv fusion proteins have a molecular mass of approximately 75 kDa. Based on different CDR1 sequences of the scTv R4P3F9S alpha (SEQ ID NOs: 5 and 26) and beta chain (SEQ ID NOs: 13 and 37 to 49) different Fab-scTv fusion variants (75-1 to 75-25, Table 2) were expressed in transiently transfected ExpiCHO cells as recommended by the manufacturer. Proteins were purified by protein L and size exclusion chromatography. All fusion variants could be produced with yields ranging from 80 g up to 1 mg (Table 2) and homogeneously formed heterodimers at the expected size as analyzed by size exclusion chromatography (FIG. 3).

TABLE-US-00002 TABLE2 Nomenclatureandyieldsofbispecific Fab-scTvfusionproteins.Themoleculesare basedonSEQIDNOs50and52andthe indicatedCDRa1andCDRb1variants. Yield Variant CDRa1/SEQ CDRb1/SEQ [g] 75-1 DRGSQS RSGDLS 267.9 (SEQIDNO.5) (SEQIDNO.13) 75-2 DRGSQS ARWHNN 78.4 (SEQIDNO.5) (SEQIDNO.37) 75-3 DRGSQS AKDHLN 646.7 (SEQIDNO.5) (SEQIDNO.38) 75-4 DRGSQS ARWHRN 704.3 (SEQIDNO.5) (SEQIDNO.39) 75-5 DRGSQS AMDHPY 397.2 (SEQIDNO.5) (SEQIDNO.40) 75-6 DRGSQS ATDHYN 268.1 (SEQIDNO.5) (SEQIDNO.41) 75-7 DRGSQS ARYHTN 83.2 (SEQIDNO.5) (SEQIDNO.42) 75-8 DRGSQS APYHLN 765.7 (SEQIDNO.5) (SEQIDNO.43) 75-9 DRGSQS AKDHTN 1067.2 (SEQIDNO.5) (SEQIDNO.44) 75-10 DRRSQS RSGDLS 389.6 (SEQIDNO.26) (SEQIDNO.13) 75-11 DRRSQS ARWHNN 270.4 (SEQIDNO.26) (SEQIDNO.37) 75-12 DRRSQS AKDHLN 943.6 (SEQIDNO.26) (SEQIDNO.38) 75-13 DRRSQS ARWHRN 560.3 (SEQIDNO.26) (SEQIDNO.39) 75-14 DRRSQS AMDHPY 360.7 (SEQIDNO.26) (SEQIDNO.40) 75-15 DRRSQS ATDHYN 541.5 (SEQIDNO.26) (SEQIDNO.41) 75-16 DRRSQS ARYHTN 403.6 (SEQIDNO.26) (SEQIDNO.42) 75-17 DRRSQS APYHLN 195.5 (SEQIDNO.26) (SEQIDNO.43) 75-18 DRRSQS AKDHTN 731.3 (SEQIDNO.26) (SEQIDNO.44) 75-19 DRRSQS ARYHRN 794 (SEQIDNO.26) (SEQIDNO.45) 75-20 DRRSQS ARWHSN 85.5 (SEQIDNO.26) (SEQIDNO.46) 75-21 DRRSQS ATDHYN 276 (SEQIDNO.26) (SEQIDNO.47) 75-22 DRRSQS RWGDLN 255 (SEQIDNO.26) (SEQIDNO.48) 75-23 DRRSQS ARDHLN 217 (SEQIDNO.26) (SEQIDNO.49) 75-24.sup.a DRGSQS RSGDLS 166.6 (SEQIDNO:5) (SEQIDNO.13) 75-25.sup.a DRRSQS RSGDLS 267 (SEQIDNO.26) (SEQIDNO.13) .sup.abeta-alpha orientation of scTv

Example 4: Fab-scTv Fusion Protein Binding to COL6A3-002 and Similar Peptides

[0120] Binding affinity of anti-CD3-scTv R4P3F9S fusion proteins towards HLA-A*02 monomers with COL6A3-002 or different similar peptides was measured by biolayer interferometry. Measurements were done on an Octet RED384 system using settings recommended by the manufacturer. Briefly, purified Fab-scTv molecules were loaded onto biosensors (FAB2G) prior to analyzing serial dilutions of HLA-A*02/COL6A3-002. Compared to variants 75-1 and 75-24 comprising wild-type CDRa1 and wild-type CDRb1 increased binding affinities of up to 40-fold were observed for Fab-scTv variants with maturated CDRa1 and/or CDRb1 sequences (Table 3, FIG. 4). In order to evaluate the selectivity of binding to HLA-A*02/COL6A3-002, purified Fab-scTv molecules loaded onto FAB2G biosensors were screened for binding to 1 M similar peptides (SEQ ID NOs: 28 to 36), each in complex with HLA-A*02. Except of HLA-A*02/COL6A1-001 (SEQ ID NO: 30), which was bound by most of the Fab-scTv variants containing maturated CDRa1 (SEQ ID NO: 26), Fab-scTv variants showed no binding to similar peptides (FIG. 5) arguing for high binding selectivity. For some Fab-scTv variants the therapeutic window between HLA-A*02/COL6A3-002 and HLA-A*02/COL6A1-001 binding was investigated by loading biotinylated peptide-HLA complexes onto biosensors (SA) and analyzing dilution series of Fab-scTv variants. While variant 75-10 comprising maturated CDRa1 (SEQ ID NO: 26) and wild-type CDRb1 (SEQ ID NO: 13) sequence showed 8-fold increased binding affinity to HLA-A*02/COL6A3-002 over HLA-A*02/COL6A1-001, an up to 57-fold increased binding affinity was detected for Fab-scTv variant 75-13 comprising a maturated CDRb1 (SEQ ID NO: 39) arguing for an improvement in therapeutic window (Table 4, FIG. 6).

TABLE-US-00003 TABLE 3 Binding affinity of Fab-scTv fusion proteins to HLA-A*02/COL6A3-002. Variant KD (M) kon (1/Ms) koff (1/s) 75-1 8.06E06 1.01E+05 8.17E01 75-2 3.69E06 1.59E+05 5.86E01 75-3 4.92E06 9.71E+04 4.78E01 75-4 5.76E06 9.78E+04 5.63E01 75-5 4.32E04 2.21E+03 9.55E01 75-6 1.13E06 2.06E+05 2.32E01 75-7 1.79E06 1.93E+05 3.44E01 75-8 3.45E06 1.36E+05 4.69E01 75-9 1.41E05 6.02E+04 8.51E01 75-10 1.78E06 1.69E+05 3.01E01 75-11 2.82E07 4.16E+05 1.18E01 75-12 3.74E07 2.67E+05 1.00E01 75-13 4.05E07 3.28E+05 1.33EO1 75-14 3.10E06 8.41E+04 2.61E01 75-15 7.78E07 2.33E+05 1.81E01 75-16 5.87E07 3.37E+05 1.98E01 75-17 2.27E07 3.62E+05 8.20E02 75-18 1.93E06 1.51E+05 2.91E01 75-19 6.00E07 2.96E+05 1.78E01 75-20 5.31E07 6.08E+05 3.23E01 75-21 5.52E07 2.72E+05 1.50E01 75-22 8.22E07 2.48E+05 2.04E01 75-23 3.24E07 3.18E+05 1.03E01 75-24 5.20E06 1.08E+05 5.62E01 75-25 8.33E06 6.23E+04 5.19E01

TABLE-US-00004 TABLE 4 Comparative binding affinity of Fab-scTv fusion proteins to HLA-A*02/COL6A3-002 and HLA-A*02/COL6A1-001. Variant pHLA-A*02 KD (M) KD.sub.COL6A1-001/KD.sub.COL6A3-002 75-10 COL6A3-002 1.37E05 8 COL6A1-001 1.08E04 75-11 COL6A3-002 8.50E07 8 COL6A1-001 6.46E06 75-12 COL6A3-002 7.24E07 12 COL6A1-001 8.98E06 75-13 COL6A3-002 7.39E07 57 COL6A1-001 4.23E05 75-17 COL6A3-002 8.25E07 9 COL6A1-001 7.10E06 75-23 COL6A3-002 1.15E06 22 COL6A1-001 2.55E05

Example 5: Use of Affinity-Maturated TCRs for Cellular Expression

[0121] Modification of T cells to express TCRs recognizing a tumor-specific peptide-HLA is a promising alternative of redirecting T cells to cancer cells. As the usage of maturated CDR1 sequences could improve cell-bound TCRs against HLA-A*02/COL6A3-002, the identified CDRa1 and CDRb1 mutant sequences were grafted onto the parental TCR R4P3F9 (SEQ ID NOs: 2 and 10). The resulting mutant TCR variants (C-1 to C-18, Table 5) were expressed in human CD8.sup.+ T cells after electroporation of respective mRNA generated by in vitro transcription of PCR-amplified DNA constructs. For control purpose, the 1G4 TCR (SEQ ID NOs: 53 and 57) against NYESO1-001 peptide (SEQ ID NO: 61) was expressed. After overnight incubation of RNA-electroporated CD8.sup.+ T cells, expression of introduced TCR variants was analyzed by staining with PE-labeled HLA-A*02/COL6A3-002 tetramers or HLA-A*02/NYESO1-001 tetramers. While the parental TCR R4P3F9 variant C-1 showed only minimal staining with HLA-A*02/COL6A3-002 tetramers, the R4P3F9 TCR variants C-2 to C-18 with maturated CDRa1 and/or CDRb1 showed increased tetramer staining (FIG. 7). Functional activation of CD8.sup.+ T cells (20,000 cells/well) expressing different maturated R4P3F9 TCR variants was investigated by determining levels of released IFN-gamma upon co-culture with T2 cells (20,000 cells/well) loaded with either a dilution series of COL6A3-002 (SEQ ID NO: 1) or 10 M of COL6A3-002 and similar peptides (SEQ ID NOs: 28 to 36). Compared to the parental R4P3F9 TCR variant C-1, maturated TCR variants C-2 to C-18 showed increased IFN-gamma release with maximum levels reached already at lower peptide concentrations (FIG. 8). As expected no IFN-gamma release was observed with T cells expressing no TCR or the 1G4 control TCR specific for NYESO1-001. To analyze the selectivity of COL6A3-002 recognition of the maturated R4P3F9 TCR variants, the IFN-gamma release in response to T2 cells loaded with different similar peptides (SEQ ID NOs: 28 to 36) was analyzed and revealed different selectivity profiles for the maturated R4P3F9 TCR variants. Most interestingly, the TCR variants C-5 (SEQ ID Nos 62 and 2) and C-14 (SEQ ID NOs: 62 and 63) comprising the same maturated CDRb1 (SEQ ID NO: 40) did not show any cross-reactivity towards COL6A1-001 or other similar peptides (FIG. 9) making affinity maturated R4P3F9 TCR variants C-5 and C14 most promising candidates for cellular TCR-based tumor targeting.

TABLE-US-00005 TABLE 5 Nomenclature of cellular TCR variants. The molecules are based on SEQ ID NOs 2 and 10 and the indicated CDRa1 and CDRb1 variants. Variant CDRa1 CDRb1 C-1 DRGSQS (SEQ ID NO. 5) RSGDLS (SEQ ID NO. 13) C-2 DRGSQS (SEQ ID NO. 5) ARWHNN (SEQ ID NO. 37) C-3 DRGSQS (SEQ ID NO. 5) AKDHLN (SEQ ID NO. 38) C-4 DRGSQS (SEQ ID NO. 5) ARWHRN (SEQ ID NO. 39) C-5 DRGSQS (SEQ ID NO. 5) AMDHPY (SEQ ID NO. 40) C-6 DRGSQS (SEQ ID NO. 5) ATDHYN (SEQ ID NO. 41) C-7 DRGSQS (SEQ ID NO. 5) ARYHTN (SEQ ID NO. 42) C-8 DRGSQS (SEQ ID NO. 5) APYHLN (SEQ ID NO. 43) C-9 DRGSQS (SEQ ID NO. 5) AKDHTN (SEQ ID NO. 44) C-10 DRRSQS (SEQ ID NO. 26) RSGDLS (SEQ ID NO. 13) C-11 DRRSQS (SEQ ID NO. 26) ARWHNN (SEQ ID NO. 37) C-12 DRRSQS (SEQ ID NO. 26) AKDHLN (SEQ ID NO. 38) C-13 DRRSQS (SEQ ID NO. 26) ARWHRN (SEQ ID NO. 39) C-14 DRRSQS (SEQ ID NO. 26) AMDHPY (SEQ ID NO. 40) C-15 DRRSQS (SEQ ID NO. 26) ATDHYN (SEQ ID NO. 41) C-16 DRRSQS (SEQ ID NO. 26) ARYHTN (SEQ ID NO. 42) C-17 DRRSQS (SEQ ID NO. 26) APYHLN (SEQ ID NO. 43) C-18 DRRSQS (SEQ ID NO. 26) AKDHTN (SEQ ID NO. 44)

Example 6: Window of COL6A3-002 and COL6A1-001 Recognition of Cellular TCR Variants

[0122] Cellular expression and analysis of R4P3F9 variants was performed as described above. In accordance with previous experiments (FIG. 7), staining of T cells expressing R4P3F9 TCR variants C-2 to C-18 with PE-labeled HLA-A*02/COL6A3-002 tetramers were increased compared to the parental TCR C-1. Additionally, TCR variants C-12 and C-17 showed binding to HLA-A*02/COL6A1-001 (FIG. 10). Expression of all maturated R4P3F9 variants improved functional activation of CD8.sup.+ T cells in response to T2 cells loaded with a dilution series of COL6A3-002 (SEQ ID NO: 1) reaching 5- to 90-fold lower EC.sub.50 values compared to the parental TCR C-1 (FIG. 11, Table 6). The lowest EC.sub.50 value was found for variant C-14. Again, TCR variants C-5 (SEQ ID NOs 62 and 2) and C-14 (SEQ ID NOs: 62 and 63) comprising the same maturated CDRb1 (SEQ ID NO: 40) did not show any cross-reactivity towards COL6A1-001, while other variants showed strong recognition with EC.sub.50 windows (COL6A3-002 vs. COL6A1-001) as low as factor 5.

TABLE-US-00006 TABLE 6 EC.sub.50 values [nM] of IFN- release of T cells expressing R4P3F9 variants after coculture with T2 cells loaded with COL6A3-002 or COL6A1-001. Variant EC.sub.50 COL6A3-002 [nM] EC.sub.50 COL6A1-001 [nM] C-1 2.51 C-2 0.16 C-3 0.14 871.sup.a C-4 0.13 C-5 0.15 C-6 0.48 C-7 0.29 C-8 0.20 350 C-9 0.55 C-10 0.32 1.5 C-11 0.32 8.2 C-12 0.20 1.9 C-13 0.23 9.7 C-14 0.03 C-15 0.31 69 C-16 0.34 78 C-17 0.33 4.1 C-18 0.14 280089.sup.a .sup.aplateau not reached

Example 7: Efficacy of Maturated R4P3F9 Variants C-5 and C-14 on Tumor Cell Lines

[0123] Cellular expression and analysis of R4P3F9 variants was performed as described above.

[0124] Expression of the maturated R4P3F9 variants C-5 (SEQ ID NOs 62 and 2) and C-14 (SEQ ID NOs: 62 and 63) improved functional activation of CD8.sup.+ T cells in response to COL6A3-002 (SEQ ID NO: 1)-presenting tumor cell lines as compared to the parental TCR C-1 (FIG. 12). The tumor cell lines used during this study present different amounts of target peptide. SF539 cells carry 4000 copies of HLA-A*02/COL6A3-002 per cell and SW982 cells carry 460 copies per cell. Whereas the parental TCR C-1 did not mediate strong T-cell activation upon co-culture with target-positive cell lines, TCR variant C-14 showed even stronger improvement of functional activation than TCR variant C-5. These data are in line with EC.sub.50 improvements from TCR C-1 to C-5 and to C-14 (table 6). The target-negative tumor cell line MCF-7 was not recognized by any of these TCRs.

REFERENCES

[0125] Aleksic et al. 2012: Different affinity windows for virus and cancer-specific T-cell receptorsimplications for therapeutic strategies, Eur J Immunol. 2012 December; 42(12):3174-9; [0126] Hickman et al. 2016: Antigen Selection for Enhanced Affinity T-Cell Receptor-Based Cancer Therapies, J Biomol Screen. 2016 September; 21(8):769-85; [0127] Boder and Wittrup 2000: Yeast surface display for directed evolution of protein expression, affinity, and stability, Methods Enzymol. 2000; 328:430-44; [0128] Boder and Wittrup 1997: Yeast surface display for screening combinatorial polypeptide libraries, Nat Biotechnol. 1997 June; 15(6):553-7; [0129] Smith et al. 2015: T Cell Receptor Engineering and Analysis Using the Yeast Display Platform, Methods Mol Biol. 2015; 1319:95-141; [0130] DE102016121899.5 [0131] DE102016115246