NOVEL T CELL RECEPTORS AND IMMUNE THERAPY USING THE SAME FOR THE TREATMENT OF CANCER AND INFECTIOUS DISEASES

Abstract

The present invention pertains to antigen recognizing constructs against antigens of the Merkel cell polyomavirus (MCV). The invention in particular provides novel T cell receptor (TCR) based molecules which are selective and specific for the infected host cells and tumor cell expressed MCV derived antigens. The TCR of the invention, and antigen binding fragments derived therefrom, are of use for the diagnosis, treatment and prevention of 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. An antigen recognizing construct comprising at least one complementary determining region (CDR) 3 having at least 80% sequence identity to an amino acid sequence selected from SEQ ID NOs. 3, 9, 15, 21, 27 and 33.

2. The antigen recognizing construct according to claim 1, wherein said antigen recognizing construct is capable of specifically and/or selectively binding to a Merkel Cell Polyoma Virus (MCV) associated antigen, such as an ST or LT protein, preferably such as a peptide according to SEQ ID NO: 37 or 38, optionally wherein the antigen is in a complex with MHC class I, preferably HLA-A*02.

3. The antigen recognizing construct according to claim 1 or 2, wherein the antigen recognizing construct is an antibody, or derivative or fragment thereof, or a T cell receptor (TCR), or a derivative or fragment thereof.

4. The antigen recognizing construct according to any one of claims 1 to 3, comprising a TCR or chain; and/or a TCR or chain; wherein the TCR or chain comprises a CDR3 having at least 80% sequence identity to an amino acid sequence selected from SEQ ID Nos. 3, 15, and 27, and/or wherein the TCR or chain comprises a CDR3 having at least 80% sequence identity to an amino acid sequence selected from SEQ ID Nos. 9, 21, and 33.

5. The antigen recognizing construct according to claim 4, wherein the TCR or chain further comprises a CDR1 having at least 80% sequence identity to an amino acid sequence selected from SEQ ID Nos. 1, 13, and 25; and/or a CDR2 having at least 80% sequence identity to an amino acid sequence selected from SEQ ID Nos. 2, 14, and 26.

6. The antigen recognizing construct according to claim 4 or 5, wherein the TCR or chain further comprises a CDR1 having at least 80% sequence identity to an amino acid sequence selected from SEQ ID Nos. 7, 19, and 31; and/or a CDR2 having at least 80% sequence identity to an amino acid sequence selected from SEQ ID Nos. 8, 20, and 32.

7. The antigen recognizing construct according to any of claims 1 to 6, comprising a TCR variable chain region having at least 90% sequence identity to an amino acid sequence selected from SEQ ID Nos. 4, 10, 16, 22, 28, and 34.

8. The antigen recognizing construct according to any of claims 1 to 7, comprising a binding fragment of a TCR, and wherein said binding fragment comprises CDR1 to CDR3 optionally selected from the CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID Nos. 1, 2, 3, or 7, 8, 9 or 13, 14, 15, or 19, 20, 21, or 25, 26, 27 or 31, 32, 33, in each case independently with not more than 3, preferably 2, most preferably not more than 1 amino acid substitution, deletion, addition or insertion.

9. A nucleic acid encoding for an antigen recognizing construct according to any one of claims 1 to 8.

10. A vector comprising a nucleic acid according to claim 9.

11. A host cell comprising an antigen recognizing construct according to any one of claims 1 to 8, or a nucleic acid according to claim 9, or a vector according to claim to, optionally the host cell is a lymphocyte, preferably a T lymphocyte or T lymphocyte progenitor, more preferably a CD4 or CD8 positive T-cell.

12. A pharmaceutical composition comprising the antigen recognizing construct according to any of claims 1 to 8, or the nucleic acid according to claim 9, or the vector according to claim to, or the host cell according to claim 11, and a pharmaceutical acceptable carrier, stabilizer and/or excipient.

13. The antigen recognizing construct according to any one of claims 1 to 8, or a nucleic acid according to claim 9, or a vector according to claim to, or a host cell according to claim 11, or the pharmaceutical composition according to claim 12, for use in medicine, optionally for use in the diagnosis, prevention, and/or treatment of an infectious or proliferative disease, preferably wherein the proliferative disease is Merkel Cell Carcinoma (MCC).

14. A method of manufacturing a Merkel Cell Polyoma Virus (MCV) specific antigen recognizing construct, comprising a. providing a suitable host cell, b. providing a genetic construct comprising a coding sequence encoding the antigen recognizing construct according to any of claims 1 to 8, c. introducing into said suitable host cell said genetic construct, d. expressing said genetic construct by said suitable host cell.

15. The method according to claim 14, further comprising the isolation and purification of the antigen recognizing construct from the suitable host cell and, optionally, reconstitution of the antigen recognizing construct in a T-cell.

Description

[0158] 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:

[0159] FIG. 1: MCV trLT and sT antigens. Grey filled areas illustrate common region encoded by exon 1; open boxes show unique regions. Numbers indicate amino acid positions. Colored boxes depict positions of the respective epitope. Amino acid sequences and predicted (by IEDB) MHC affinities are also indicated.

[0160] FIG. 2: A) Pairing TCR and chains were linked via 2A self-cleaving peptide sequence from Porcine teschovirus-1 (P2A) in the fashion TCR-P2A-TCR. The human TCR constant regions were replaced by their murine counterparts to improve the pairing between the chains of the introduced TCR and avoid mispairing with endogenous TCR and chains. The transgene cassettes were codon-optimized for human expression and cloned into pMP71-PRE retroviral vector. B) FACS analysis of human peripheral blood lymphocytes transduced with TCRs as indicated. Cells were stained with antibodies recognizing human CD8 and constant region of murine TCR . Percentages of CD8+ T cells expressing the transgene are also indicated.

[0161] FIG. 3: IFN release assay after co-culture of TCR-transduced PBLs with TAP-deficient T2 cells loaded with titrated amounts of SMF (A) or KLL (B) peptides.

[0162] FIG. 4: Detection of MCV peptides by the TCR of the invention.

[0163] FIG. 5: Cytolytic activity in T cells expressing MCV-specific TCR.

[0164] FIG. 6: In Vive activity of TCR transduced T cells against tumor growth in mice. Each line represents one mouse bearing one tumor. KLL-85: n=9, T1367 (irrelevant TCR): n=2; no treatment: n=3. The arrow indicates the time point of adopted T cell transfer (ATT).

[0165]

TABLE-US-00001 TABLE1 TCRsequencesoftheinvention SEQ ID NO: TCR Chain Region Sequence 1 SMF-48 alpha CDR1 TSESNYY 2 SMF-48 alpha CDR2 QEAYKQQN 3 SMF-48 alpha CDR3 CAFMKRGNRDDKIIF 4 SMF-48 alpha variabledomain MTRVSLLWAVVVSTCLESGMAQTVTQS QPEMSVQEAETVTLSCTYDTSESNYYLF WYKQPPSRQMILVIRQEAYKQQNATEN RFSVNFQKAAKSFSLKISDSQLGDTAMY FCAFMK 5 SMF-48 alpha constantdomain NIQNPEPAVYQLKDPRSQDSTLCLFTDF DSQINVPKTMESGTFITDKCVLDMKAM DSKSNGAIAWSNQTSFTCQDIFKETNA TYPSSDVPCDATLTEKSFETDMNLNFQ NLSVMGLRILLLKVAGFNLLMTLRLWS S 6 SMF-48 alpha full-length MTRVSLLWAVVVSTCLESGMAQTVTQS QPEMSVQEAETVTLSCTYDTSESNYYLF WYKQPPSRQMILVIRQEAYKQQNATEN RFSVNFQKAAKSFSLKISDSQLGDTAMY FCAFMKRGNRDDKIIFGKGTRLHILPNI QNPEPAVYQLKDPRSQDSTLCLFTDFD SQINVPKTMESGTFITDKCVLDMKAMD SKSNGAIAWSNQTSFTCQDIFKETNATY PSSDVPCDATLTEKSFETDMNLNFQNL SVMGLRILLLKVAGFNLLMTLRLWSS 7 SMF-48 beta CDR1 DFQATT 8 SMF-48 beta CDR2 SNEGSKA 9 SMF-48 beta CDR3 CSASPTSGRRTQYF 10 SMF-48 beta variabledomain MLLLLLLLGPGSGLGAVVSQHPSWVIC KSGTSVKIECRSLDFQATTMFWYRQFP KQSLMLMATSNEGSKATYEQGVEKDK FLINHASLTLSTLTVTSAHPEDSSFYICS A 11 SMF-48 beta constantdomain EDLRNVTPPKVSLFEPSKAEIANKQKAT LVCLARGFFPDHVELSWWVNGKEVHS GVCTDPQAYKESNYSYCLSSRLRVSATF WHNPRNHFRCQVQFHGLSEEDKWPE GSPKPVTQNISAEAWGRADCGITSASY HQGVLSATILYEILLGKATLYAVLVSGL VLMAMVKKKNS 12 SMF-48 beta full-length MLLLLLLLGPGSGLGAVVSQHPSWVIC KSGTSVKIECRSLDFQATTMFWYRQFP KQSLMLMATSNEGSKATYEQGVEKDK FLINHASLTLSTLTVTSAHPEDSSFYICS ASPTSGRRTQYFGPGTRLTVLEDLRNV TPPKVSLFEPSKAEIANKQKATLVCLAR GFFPDHVELSWWVNGKEVHSGVCTDP QAYKESNYSYCLSSRLRVSATFWHNPR NHFRCQVQFHGLSEEDKWPEGSPKPV TQNISAEAWGRADCGITSASYHQGVLS ATILYEILLGKATLYAVLVSGLVLMAMV KKKNS 13 SMF-72 alpha CDR1 TSESDYY 14 SMF-72 alpha CDR2 QEAYKQQN 15 SMF-72 alpha CDR3 CAYRGTSGTYKYIF 16 SMF-72 alpha variabledomain MACPGFLWALVISTCLEFSMAQTVTQS QPEMSVQEAETVTLSCTYDTSESDYYLF WYKQPPSRQMILVIRQEAYKQQNATEN RFSVNFQKAAKSFSLKISDSQLGDAAM YFCAYR 17 SMF-72 alpha constantdomain NIQNPEPAVYQLKDPRSQDSTLCLFTDF DSQINVPKTMESGTFITDKCVLDMKAM DSKSNGAIAWSNQTSFTCQDIFKETNA TYPSSDVPCDATLTEKSFETDMNLNFQ NLSVMGLRILLLKVAGFNLLMTLRLWS S 18 SMF-72 alpha full-length MACPGFLWALVISTCLEFSMAQTVTQS QPEMSVQEAETVTLSCTYDTSESDYYLF WYKQPPSRQMILVIRQEAYKQQNATEN RFSVNFQKAAKSFSLKISDSQLGDAAM YFCAYRGTSGTYKYIFGTGTRLKVLANI QNPEPAVYQLKDPRSQDSTLCLFTDFD SQINVPKTMESGTFITDKCVLDMKAMD SKSNGAIAWSNQTSFTCQDIFKETNATY PSSDVPCDATLTEKSFETDMNLNFQNL SVMGLRILLLKVAGFNLLMTLRLWSS 19 SMF-72 beta CDR1 SEHNR 20 SMF-72 beta CDR2 FQNEAQ 21 SMF-72 beta CDR3 CASSKLAGKNIQYF 22 SMF-72 beta variabledomain MGTSLLCWMALCLLGADHADTGVSQD PRHKITKRGQNVTFRCDPISEHNRLYW YRQTLGQGPEFLTYFQNEAQLEKSRLLS DRFSAERPKGSFSTLEIQRTEQGDSAM YLCASS 23 SMF-72 beta constantdomain EDLRNVTPPKVSLFEPSKAEIANKQKAT LVCLARGFFPDHVELSWWVNGKEVHS GVCTDPQAYKESNYSYCLSSRLRVSATF WHNPRNHFRCQVQFHGLSEEDKWPE GSPKPVTQNISAEAWGRADCGITSASY HQGVLSATILYEILLGKATLYAVLVSGL VLMAMVKKKNS 24 SMF-72 beta full-length MGTSLLCWMALCLLGADHADTGVSQD PRHKITKRGQNVTFRCDPISEHNRLYW YRQTLGQGPEFLTYFQNEAQLEKSRLLS DRFSAERPKGSFSTLEIQRTEQGDSAM YLCASSKLAGKNIQYFGAGTRLSVLEDL RNVTPPKVSLFEPSKAEIANKQKATLVC LARGFFPDHVELSWWVNGKEVHSGVC TDPQAYKESNYSYCLSSRLRVSATFWH NPRNHFRCQVQFHGLSEEDKWPEGSP KPVTQNISAEAWGRADCGITSASYHQG VLSATILYEILLGKATLYAVLVSGLVLMA MVKKKNS 25 KLL-85 alpha CDR1 SIFNT 26 KLL-85 alpha CDR2 LYKAGEL 27 KLL-85 alpha CDR3 CAGQLNQGAQKLVF 28 KLL-85 alpha variabledomain MLLEHLLIILWMQLTWVSGQQLNQSP QSMFIQEGEDVSMNCTSSSIFNTWLWY KQDPGEGPVLLIALYKAGELTSNGRLTA QFGITRKDSFLNISASIPSDVGIYFCAGQ 29 KLL-85 alpha constantdomain NIQNPEPAVYQLKDPRSQDSTLCLFTDF DSQINVPKTMESGTFITDKTVLDMKAM DSKSNGAIAWSNQTSFTCQDIFKETNA TYPSSDVPCDATLTEKSFETDMNLNFQ NLSVMGLRILLLKVAGFNLLMTLRLWS S 30 KLL-85 alpha full-length MLLEHLLIILWMQLTWVSGQQLNQSP QSMFIQEGEDVSMNCTSSSIFNTWLWY KQDPGEGPVLLIALYKAGELTSNGRLTA QFGITRKDSFLNISASIPSDVGIYFCAGQ LNQGAQKLVFGQGTRLTINPNIQNPEP AVYQLKDPRSQDSTLCLFTDFDSQINVP KTMESGTFITDKTVLDMKAMDSKSNG AIAWSNQTSFTCQDIFKETNATYPSSDV PCDATLTEKSFETDMNLNFQNLSVMGL RILLLKVAGFNLLMTLRLWSS 31 KLL-85 beta CDR1 SGHNS 32 KLL-85 beta CDR2 FNNNVP 33 KLL-85 beta CDR3 CASSFYSADTQYF 34 KLL-85 beta variabledomain MDSWTFCCVSLCILVAKHTDAGVIQSP RHEVTEMGQEVTLRCKPISGHNSLFWY RQTMMRGLELLIYFNNNVPIDDSGMP EDRFSAKMPNASFSTLKIQPSEPRDSAV YFCASS 35 KLL-85 beta constantdomain EDLRNVTPPKVSLFEPSKAEIANKQKAT LVCLARGFFPDHVELSWWVNGKEVHS GVSTDPQAYKESNYSYCLSSRLRVSATF WHNPRNHFRCQVQFHGLSEEDKWPE GSPKPVTQNISAEAWGRADCGITSASY HQGVLSATILYEILLGKATLYAVLVSGL VLMAMVKKKNS 36 KLL-85 beta full-length MDSWTFCCVSLCILVAKHTDAGVIQSP RHEVTEMGQEVTLRCKPISGHNSLFWY RQTMMRGLELLIYFNNNVPIDDSGMP EDRFSAKMPNASFSTLKIQPSEPRDSAV YFCASSFYSADTQYFGPGTRLTVLEDLR NVTPPKVSLFEPSKAEIANKQKATLVCL ARGFFPDHVELSWWVNGKEVHSGVST DPQAYKESNYSYCLSSRLRVSATFWHN PRNHFRCQVQFHGLSEEDKWPEGSPK PVTQNISAEAWGRADCGITSASYHQGV LSATILYEILLGKATLYAVLVSGLVLMA MVKKKNS

TABLE-US-00002 TABLE2 Peptidesequencesoftheinvention PeptideCode Sequence SEQIDNO: trLT74-82 SMFDEVDEA 37 trLT/ST15-23 KLLEIAPNC 38

EXAMPLES

Example 1: Immunization

[0166] For the immunization of human TCR expressing mice (ABabDII) peptide epitopes SMFDEVDEA (SMF), which is located on trLT at position 74-82, and KLLEIAPNC located at position 15-23 on both, trLT and sT, were used (FIG. 1). Similar to other polyomaviruses, Merkel cell carcinoma virus (MCV) encodes alternatively spliced large T (LT) and small T (ST) antigen transcripts that share exon 1 of the T antigen locus. Sequence analysis demonstrated that in all Merkel cell carcinoma (MCC) cases truncating mutations result in deletion of the C-terminus of LT which is referred to trLT. MCV DNA is integrated in MCC genome only but not in surrounding healthy tissue. Additionally, MCC cells require expression of viral T antigens.

[0167] The following TCRs were recovered ABabDII mice as described before (Obenaus et al. Nat Biotechnol. 2015 April; 33(4):402-7) (CDR1-3 regions are underlined):

Example 2: TCR: SMF-48

[0168]

TABLE-US-00003 Antigen(epitope):MCVtrLT74-82(SMFDEVDEA-SEQ IDNO:36) TRAV38-1*03-CAFMKRGNRDDKIIF-TRAJ30*01 (SEQIDNO:6) MTRVSLLWAVVVSTCLESGMAQTVTQSQPEMSVQEAETVTLSCTYDTSE SNYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLK ISDSQLGDTAMYFCAFMKRGNRDDKIIFGKGTRLHILPNIQNPEPAVYQ LKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSN GAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQ NLSVMGLRILLLKVAGFNLLMTLRLWSS TRBV20-1*01-CSASPTSGRRTQYF-TRBJ2-3*01 (SEQIDNO:12) MLLLLLLLGPGSGLGAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFW YRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSA HPEDSSFYICSASPTSGRRTQYFGPGTRLTVLEDLRNVTPPKVSLFEPS KAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKES NYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQ NISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVL MAMVKKKNS

Example 3: TCR: SMF-72

[0169]

TABLE-US-00004 Antigen(epitope):MCVtrLT74-82(SMFDEVDEA-SEQ IDNO:36) TRAV38-2*01-CAYRGTSGTYKYIF-TRAJ40*01 (SEQIDNO:18) MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSES DYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKIS DSQLGDAAMYFCAYRGTSGTYKYIFGTGTRLKVLANIQNPEPAVYQLKDP RSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAW SNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMG LRILLLKVAGFNLLMTLRLWSS TRBV7-9*03-CASSKLAGKIVIQYF-TRBJ2-4*01 (SEQIDNO:24) MGTSLLCWMALCLLGADHADTGVSQDPRHKITKRGQNVTFRCDPISEHNRL YWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTE QGDSAMYLCASSKLAGKNIQYFGAGTRLSVLEDLRNVTPPKVSLFEPSKAE IANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYC LSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAW GRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS

Example 4: TCR: KLL-85

[0170]

TABLE-US-00005 Antigen(epitope):MCVsT/LT(KLLEIAPNC-SEQID NO:37) TRAV35*01-CAGQLNQGAQKLVF-TRAJ54*01 (SEQIDNO:30) MLLEHLLIILWMQLTWVSGQQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWL WYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDV GIYFCAGQLNQGAQKLVFGQGTRLTINPNIQNPEPAVYQLKDPRSQDSTLC LFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQ DIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAG FNLLMTLRLWSS TRBV12-3*01-CASSFYSADTQYF-TRBJ2-3*01 (SEQIDNO:36) MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSL FWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSE PRDSAVYFCASSFYSADTQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEI ANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCL SSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWG RADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS

Example 5: Functional Tests

[0171] The isolated TCR of the invention could be re-expressed in human peripheral blood lymphocytes (FIG. 2).

[0172] To compare the functional avidity of the isolated TCRs, TCR-transduced PBLs were co-cultured with TAP-deficient T2 cells loaded with titrated amounts of SMF (left diagram) or KLL (right diagram) peptides and measured release of IFN as indicator for activation of T cells. All three TCRs mediated recognition of sub-nanomolar levels of peptide pulsed T2 cells (see FIG. 3).

[0173] The processing and presentation of SMF- and KLL-epitopes as well as recognition by TCR-engineered PBLs were tested in vitro by culturing the PBLs with derivatives of H1299 cells that express HLA-A*02 (H1299-A2) with or without concomitant expression of trLT (H1299-A2-trLT) or ST (H1299-A2-sT). As shown in FIG. 4, all three TCRs recognized H1299-A2-trLT but not the parental cell line H1299-A2. As expected, only KLL-85 expressing T cells reacted to H1299-A2-sT, confirming the specificity of our isolated TCRs.

Example 6: Cytolytic and Therapeutic Potential of TCR Transduced T Cells of the Invention

[0174] The inventors also examined the ability of MCC cell lines to provoke cytolytic activity in T cells expressing MCV-specific TCR. In accordance with the IFN- secretion assay, only HLA-A02-positive cells (WaGa and MKL1-A02) induced CD107 expression (FIG. 5).

[0175] For in vivo analysis, HLA-A02+ HHDxRag/ mice lacking B and T lymphocytes were used to exclude endogenous T cell responses (Leisegang et al., 2016). The inventors transduced a MC703 fibrosarcoma cell line, which was established from HHD mouse, to express KLL epitope (MC703-KLL). MC703-KLL cells were grown in HHDxRag/ mice until large tumors were established (average diameter of 9-100 mm). Tumor bearing mice were treated with HHD-derived T cells which were transduced with KLL85 TCR (see table 1 above). As controls, a group of mice was treated with T cells expressing an irrelevant TCR (T1367) and another didn't receive any treatment. In contrast to control groups, MC703-KLL tumors regressed upon treatment with KLL85-expressing T cells (FIG. 6) underlying effectiveness of the inventive isolated TCR.