HIGH AVIDITY HPV T-CELL RECEPTORS

Abstract

The present invention pertains to novel high avidity antigen recognizing constructs against Human Papilloma Virus antigens. The invention provides novel T cell receptor (TCR) based molecules which are selective and specific for HPV 16/18 proteins E5, E6 and E7. The TCR of the invention, and HPV antigen-binding fragments derived therefrom, are of use for the diagnosis, treatment and prevention of HPV infection, as well as for the diagnosis, treatment and prevention of HPV infection mediated secondary diseases as HPV infection caused cancers, such as cervical, nasopharyngeal or head and neck cancer. Further provided are nucleic acids encoding the proteins of the invention, and recombinant cells expressing the same.

Claims

1. An antigen recognizing construct comprising a complementary determining region 3 (CDR3) having at least 80% sequence identity to an amino acid sequence selected from SEQ ID Nos. 9 to 28.

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

3. The antigen recognizing construct according to claim 1, which is a T cell receptor, and comprises in its alpha chain a CDR3 having at least 80% sequence identity to an amino acid sequence selected from SEQ ID Nos. 9, 10, 12, 14, 16, 18, 21, 23, 25, 27, and/or comprises in its beta chain an CDR3 having at least 80% sequence identity to an amino acid sequence selected from SEQ ID Nos.11, 13, 15, 17, 19, 20, 22, 24, 26, 28.

4. The antigen recognizing construct according to claim 1, which is a TCR, or a fragment thereof, composed of a TCR and a TCR chain, wherein said TCR comprises the CDR1 to 3 sequences as comprised in SEQ ID NO: 29 and 31, or 30 and 31; or 32 and 33; or 34 and 35; or 36 and 37; or 38 and 39 ; or 38 and 40; or 41 and 42; or 43 and 44; or 45 and 46; or 47 and 48.

5. The antigen recognizing construct according to claim 1, which is a TCR, or a fragment thereof, composed of a TCR and a TCR chain, wherein said TCR comprises the variable sequences according to SEQ ID NO: 29 and 31, or 30 and 31; or 32 and 33; or 34 and 35; or 36 and 37; or 38 and 39; or 38 and 40; or 41 and 42; or 43 and 44; or 45 and 46; or 47 and 48.

6. A nucleic acid encoding for an antigen recognizing construct according to claim 1.

7. A vector comprising a nucleic acid according to claim 6.

8. A host cell comprising an antigen recognizing construct according to claim 1.

9. The host cell according to claim 8, which is a lymphocyte, preferably a T lymphocyte, such as a CD4 or CD8 positive T-cell.

10. The antigen recognizing construct according to claim 1 for use in medicine.

11. The antigen recognizing construct according to claim 10, for use in the diagnosis, prevention, and/or treatment of an infectious disease or a proliferative disease, such as a malignant or benign tumor disease.

12. The antigen recognizing construct according to claim 10, wherein the use in medicine is a use in immune therapy, preferably in adoptive T cell therapy.

13. An in vitro method for the manufacturing of an HPV specific antigen recognizing construct (ARC) expressing cell line, comprising a. Providing a suitable host cell, b. Providing a genetic construct encoding for an ARC, wherein said ARC comprises a CDR3 having an amino acid sequence with at least 80% sequence identity to an amino acid sequence selected from SEQ ID Nos. 9 to 28, c. Introducing into said suitable host cell said genetic construct, d. Expressing said genetic construct by said suitable host cell.

14. The method according to claim 13, wherein said ARC is an ARC comprising a complementary determining region 3 (CDR3) having at least 80% sequence identity to an amino acid sequence selected from SEQ ID Nos. 9 to 28.

15. The method according to claim 13, wherein said cell is a mammalian, in particular a human, T-cell.

16. The method according to claim 13, further comprising the purification of the ARC from the cell and, optionally, the reconstitution of the translated ARC-fragments in a T-cell.

17. A host cell comprising a nucleic acid according to claim 6.

18. A host cell comprising a vector according to claim 7.

Description

[0085] 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:

[0086] FIG. 1: shows examples for the specific CD8+ T cell response against HLA-A*02.01 restricted HPV16 E6 epitope TIHDIILECV in ABabDII mice. ABabDII mice were either immunized i.p. with E6/7 expressing adenovirus followed by s.c. administration of 50 g of E6 peptide together with 50 g of CpG ODN 1826 emulsified in IFA, or with peptide/CpG/IFA mixture alone. The presence of HPV-specific CD8+ T cells in the peripheral blood of immunized animals was assessed by intracellular IFN staining 7 days after each boost. Stimulation with phorbol myristate acetate (PMA) and ionomycin was used as a positive control (+ctrl).

[0087] FIG. 2: shows an example for the specific CD8+ T cell response against HLA-A201 restricted HPV16 E7 epitope TLGIVCPI in ABabDII mice. ABabDII mice were immunized s.c. at the tail base with 50 g of E7 peptide together with 50 g of CpG ODN 1826 emulsified in IFA. The presence of HPV-specific CD8+ T cells in the peripheral blood of immunized animals was assessed by intracellular IFN staining 7 days after each boost. Stimulation with phorbol myristate acetate (PMA) and ionomycin was used as a positive control (+ctrl).

[0088] FIG. 3: shows FACSort strategy (IFN capture assay) for the specific CD8+ T cells against HLA-A*02.01 restricted HPV16 E5, E6 and E7 epitopes. Spleen cells from responsive mice were depleted for CD4+ T cells and restimulated for 10 days with 10.sup.8 mol of the respective peptide. For 5RACE PCR HPV-specific CD8+ T cells were sorted directly into RNA purification buffer using IFN capture assay.

[0089] FIG. 4: shows HPV16 E6 and E7 TCR transduction of freshly prepared human PBLs. 110.sup.6 freshly isolated or frozen hPBMCs were stimulated with anti-CD3 and anti-CD28-coated plates in the presence of 200 U/ml recombinant human interleukin 2. Transductions were done 48 and 72 h after stimulation by addition of retrovirus containing supernatant and protamine sulfate followed by spinoculation (1.sup.st transduction) or preloading of virus onto retronectin (Takara)-coated plates and spinoculation (2.sup.nd transduction). Transduction efficacy was assessed by staining with anti-CD8 and anti-mouse constant TCR mAbs.

[0090] FIG. 5: shows the HPV16 E6/E7 peptide-specific IFN- release of TCR transduced human PBMCs; 5859 TCR corresponds to Seq ID No: 34 & 35, 5843B14 TCR corresponds to Seq ID No: 36 & 37, 14800 TCR corresponds to Seq ID NO: 43&44. IFN production was measured by enzyme-linked immunosorbent assay after 16 h coculture of 110.sup.4 TCR-transduced T cells with 110.sup.4 peptide-loaded T2 cells. Stimulation with phorbol myristate acetate (PMA) and ionomycin was used as a positive control (max); ut=untransduced PBMCs.

[0091] FIG. 6: shows the HPV16 E6 IFN- release of TCR transduced human PBMCs on titrated E6-specific peptide and CASKI cell line. Left panel shows the TCR of SEQ ID NO 14/15 (T3), right panel shows TCR of SEQ ID NO 16/17 (T4). IFN production was measured by enzyme-linked immunosorbent assay after 16 h coculture of 110.sup.4 TCR-transduced T cells with 110.sup.4 peptide-loaded T2 cells or 110.sup.4 cells of CASKI cell line. Stimulation with phorbol myristate acetate (PMA) and ionomycin was used as a positive control (P+I). IFN release comparable to recognition of CASKI cells was also seen for HLA-transduced cervical cancer cell lines SiHa cell line and head and neck cancer cell lines SCC-090 and SCC-152 (not shown).

[0092] FIG. 7: shows the HPV16 E5 transduction of freshly prepared human PBLs and HPV16 E5 peptide-specific IFN- release of TCR transduced human PBMCs. TCR used was the TCR of SEQ ID NO 12/13 (T2). 110.sup.6 freshly isolated or frozen hPBMCs were stimulated with anti-CD3 and anti-CD28-coated plates in the presence of 200 U/ml recombinant human interleukin 2. Transductions were done 48 and 72 h after stimulation by addition of retrovirus containing supernatant and protamine sulfate followed by spinoculation (1.sup.st transduction) or preloading of virus onto retronectin (Takara)-coated plates and spinoculation (2.sup.nd transduction). Transduction efficacy was assessed by staining with anti-CD8 and anti-mouse constant TCR mAbs. IFN production was measured by enzyme-linked immunosorbent assay after 16 h coculture of 110.sup.4 TCR-transduced T cells with 110.sup.4 peptide-loaded T2 cells. Stimulation with phorbol myristate acetate (PMA) and ionomycin was used as a positive control (P+I).

[0093] SEQ ID No 1 to 8: show the HPV 16 and 18 epitopes that are bound by the TCR of the invention.

[0094] SEQ ID No 9 to 28: show alpha and beta chain CDR3 sequences of the TCR of the invention.

[0095] SEQ ID No 29 to 48: show the full variable sequences of and chains of the TCR of the present invention.

EXAMPLES

[0096] HPV Epitopes used for Immunization of Animals:

TABLE-US-00001 TABLE1 HPV SEQ Gene Protein HLAA2.01Epitope IDNO: 16-E5 Humanpapillomavirustype16E5protein YIIFVYIPL(63-71) 1 16-E6 Humanpapillomavirustype16E6protein KLPQLCTEL(11-19) 2 16-E6 Humanpapillomavirustype16E6protein TIHDIILECV(29-38) 3 16-E7 Humanpapillomavirustype16E7protein YMLDLQPET(86-93) 4 16-E7 Humanpapillomavirustype16E7protein YMLDLQPETT(11-20) 5 16-E7 Humanpapillomavirustype16E7protein TLGIVCPI(86-93) 6 18-E6 Humanpapillomavirustype18E6protein KCIDFYSRI(67-75) 7 18-E7 Humanpapillomavirustype18E7protein FQQLFLNTL(86-94) 8

[0097] Peptide Epitopes with CTL reactivity in ABabDII mice

Example 1

T-Cell Receptor T1 Recognizing HPV 16-E5 Epitope YIIFVYIPL

[0098]

TABLE-US-00002 Immunogenic CDR3 peptide/ SEQ TCR Antigen purification TCRsequence IDNO: T1* E5 YIIFVYIPL TRAV13-1*01-CAASSYNQGGKLF-TRAJ23*-1 9 IFN- TRAV26-2*01-CILRDVNAGGTSYGKLTF-TRAJ52*01 10 CAPTURE TRBV12-3*01-CASSLLSSYNEQFF-TRBD2*01-TRBJ2-3*01 11

[0099] CDR1 sequences are bold

[0100] CDR2 sequences are double underlined

[0101] CDR3 sequences are underlined

[0102] Full length variable chain sequences (CDR3 sequences are underlined):

TABLE-US-00003 TRAV13-1*01-CAASSYNQGGKLIF-TRAJ23*01 (SEQIDNO:29) MTSIRAVFIFLWLQLDLVNGENVEQHPSTLSVQEGDSAVIKCTYSDSASNYFPWYKQELGKGPQLIID IRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAASSYNQGGKLIFGQGTELSVKPN TRAV26-2*01-CILRDVNAGGTSYGKLTF-TRAJ52*01 (SEQIDNO:30) MKLVTSITVLLSLGIMGDAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQLPS QGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILRDVNAGGTSYGKLTFGQGTIL TVHPN TRBV12-3*01-CASSLLSSYNEQFF-TRBD2*01-TRBJ2-3*01 (SEQIDNO:31) MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQTMMRGLELLI YFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSLLSSYNEQFFGPGTRLTVL

Example 2

T-Cell Receptor T2 Recognizing HPV 16-E5 Epitope YIIFVYIPL

[0103]

TABLE-US-00004 Immunogenic CDR3 peptide/ SEQ TCR Antigen purification TCRsequence IDNO: T2 E5 YIIFVYIPL TRAV12-2*02-CAVNVDFNKFYF-TRAJ21*01 12 IFN- TRBV4-1*01-CASSQDWNNEQFF-TRBD1*01-TRBJ2-1*01 13 CAPTURE

[0104] Full length variable chain sequences (CDR3 sequences are underlined):

TABLE-US-00005 TRAV12-2*02-CAVNVDFNKFYF-TRAJ21*01 (SEQIDNO):32: MMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAI ASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKED GRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNVDFNKF YFGSGTKLNVKPN TRBV4-1*01-CASSQDWNNEQFF-TRBD1*01-TRBJ2-1*01 SEQIDNO:33: MGCRLLCCAVLCLLGAVPIDTEVTQTPKHLVMGMTN KKSLKCEQHMGHRAMYWYKQKAKKPPELMFVYSYEKLSIN ESVPSRFSPECPNSSLLNLHLHALQPEDSALYLCASSQDW NNEQFFGPGTRLTVL

Example 3

T-Cell Receptor T3 Recognizing HPV 16-E6 Epitope TIHDIILECV

[0105]

TABLE-US-00006 Immunogenic CDR3 peptide/ SEQ TCR Antigen purification TCRsequence IDNO: T3 E6 TIHDIILECV TRAV20*02-CA 14 VQANRGSTLGRL YF-TRAJ18*01 IFN- TRBV28*01-CA 15 CAPTURE SSLWGRLAKNIQ YF-TRBD1*01- TRBJ2-4*01

[0106] Full length variable chain sequences (CDR3 sequences are underlined):

TABLE-US-00007 TRAV20*02-CAVQANRGSTLGRLYF-TRAJ18*01 SEQIDNO:34 MEKMLECAFIVLWLQLGWLSGEDQVTQSPEALRLQEG ESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEE KEKERLKATLTKKESFLHITAPKPEDSATYLCAVQANRGS TLGRLYFGRGTQLTVWPD TRBV28*01-CASSLWGRLAKNIQYF-TRBD1*01-TRBJ2-4*01 SEQIDNO:35 MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGE KVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEK GDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSLWG RLAKNIQYFGAGTRLSVL

Example 4

T-Cell Receptor T4 Recognizing HPV 16-E6 Epitope TIHDIILECV

[0107]

TABLE-US-00008 Immunogenic CDR3 peptide/ SEQ TCR Antigen purification TCRsequence IDNO: T4 E6 TIHDIILECV TRAV21*02-CA 16 VRETSGSRLTF- TRAJ58*01 IFN- TRBV28*01-CA 17 CAPTURE SSFWGRSTDTQY F-TRBD1*01- TRBJ2-3*01

[0108] Full length variable chain sequences (CDR3 sequences are underlined):

TABLE-US-00009 TRAV21*02-CAVRETSGSRLTF-TRAJ58*01 SEQIDNO:36 METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENL VLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTS GRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRETSGSR LTFGEGTQLTVNPD TRBV28*01-CASSFWGRSTDTQYF-TRBD1*01-TRBJ2-3*01 SEQIDNO:37 MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGE KVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEK GDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSFWG RSTDTQYFGPGTRLTVL

Example 5

T-Cell Receptor T5 Recognizing HPV 16-E6 Epitope TIHDIILECV

[0109]

TABLE-US-00010 Immunogenic CDR3 peptide/ SEQ TCR Antigen purification TCRsequence IDNO: T5* E6 TIHDIILECV TRAV17*01-CA 18 TVSTDSWGKKLQ F-TRAJ24*02 IFN- TRBV10-3*02- 19 CAPTURE CAISDSNGINIQ YF-TRBD2*02- TRBJ2-4*01 TRBV28*01-CA 20 SSLWGRAGKDTQ YF-TRBD2*02- TRBJ2-3*01

[0110] Full length variable chain sequences (CDR3 sequences are underlined):

TABLE-US-00011 TRAV17*01-CATVSTDSWGKKLQF-TRAJ24*02 SEQIDNO:38 METLLGVSLVILWLQLARVNSQQGEEDPQAL SIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRS NEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCAT VSTDSWGKLQFGAGTQVVVTPD TRBV10-3*02-CAISDSNGINIQYF-TRBD2*02-TRBJ2-4*01 SEQIDNO:39 MRSWPGPEMGTRLFFYVALCLLWTGHMDAGITQSPR HKVTETGTPVTLRCHQTENHRYMYWYRQDPGHGLRLIHYS YGVKDTDKGEVSDGYSVSRSKTEDFLLTLESATSSQTSVY FCAISDSNGINIQYFGAGTRLSVL TRBV28*01-CASSLWGRAGKDTQYF-TRBD2*02-TRBJ2-3*01 SEQIDNO:40 MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGE KVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEK GDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSLWG RAGKDTQYFGPGTRLTVL

Example 6

T-Cell Receptor T6 Recognizing HPV 16-E6 Epitope KLPQLCTEL

[0111]

TABLE-US-00012 Immunogenic CDR3 peptide/ SEQ TCR Antigen purification TCRsequence IDNO: T6 E6 KLPQLCTEL TRAV8-6*01-C 21 AVSLNSGNTPLV F-TRAJ29*01 IFN- TRBV20-1*01- 22 CAPTURE CSARDLAGNTGE LFF-TRBD2*01- TRBJ2-2*01

[0112] Full length variable chain sequences (CDR3 sequences are underlined):

TABLE-US-00013 TRAV8-6*01-CAVSLNSGNTPLVF-TRAJ29*01 SEQIDNO:41 MLLLLVPAFQVIFTLGGTRAQSVTQLDSQVPV FEEAPVELRCNYSSSVSVYLFWYVQYPNQGLQLLLKYLSG STLVESINGFEAEFNKSQTSFHLRKPSVHISDTAEYFCAV SLNSGNTPLVFGKGTRLSVIAN TRBV20-1*01-CSARDLAGNTGELFF-TRBD2*01-TRBJ2-2*01 SEQIDNO:42 MLLLLLLLGPGSGLGAVVSQHPSWVICKSGTSVKIECRSL DFQATTMFWYRQFPKQSLMLMATSNEGSKA TYEQGVEKDK FLINHASLTLSTLTVTSAHPEDSSFYICSARDLAGNTGEL FFGEGSRLTVL

Example 7

T-Cell Receptor T7 Recognizing HPV 16-E7 Epitope TLGIVCPI

[0113]

TABLE-US-00014 Immunogenic CDR3 peptide/ SEQ TCR Antigen purification TCRsequence IDNO: T7 E7 TLGIVCPI TRAV30*01-CG 23 TGTDSWGKLQF- TRAJ24*02 IFN- TRBV12-4*01- 24 CAPTURE CASSPGLAGGEQ FF-TRBD2*02- TRBJ2-1*01

[0114] Full length variable chain sequences (CDR3 sequences are underlined):

TABLE-US-00015 TRAV30*01-CGTGTDSWGKLQF-TRAJ24*02 SEQIDNO:43 METLLKVLSGTLLWQLTWVRSQQPVQSPQAVILREGED AVINCSSSKALYSVHWYRQKHGEAPVFLMILLKGGEQKGH EKISASFNEKKQQSSLYLTASQLSYSGTYFCGTGTDSWGK LQFGAGTQVVVTPD TRBV12-4*01-CASSPGLAGGEQFF-TRBD2*02-TRBJ2-1*01 SEQIDNO:44 MGSWTLCCVSLCILVAKHTDAGVIQSPRHEVTEMGQ EVTLRCKPISGHDYLFWYRQTMMRGLELLIYFNNNVPIDD SGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSPG LAGGEQFFGPGTRLTVL

Example 8

T-Cell Receptor T8 Recognizing HPV 16-E7 Epitope TLGIVCPI

[0115]

TABLE-US-00016 Immunogenic CDR3 peptide/ SEQ TCR Antigen purification TCRsequence IDNO: T8 E7 TLGIVCPI TRAV22*01-CA 25 VEPNSGNTPLVF- TRAJ29*01 IFN- TRBV7-2*02or 26 CAPTURE 03-CASSLIISY NEQFF-TRBJ2- 1*01

[0116] Full length variable chain sequences (CDR3 sequences are underlined):

TABLE-US-00017 TRAV22*01-CAVEPNSGNTPLVF-TRAJ29*01 SEQIDNO:45 MKRILGALLGLLSAQVCCVRGIQVEQSPPDLILQEGAN STLRCNFSDSVNNLQWFHQNPWGQLINLFYIPSGTKQNGR LSATTVATERYSLLYISSSQTTDSGVYFCAVEPNSGNTPL VFGKGTRLSVIAN TRBV7-2*02or03-CASSLIISYNEQFF-TRBJ2-1*01 SEQIDNO:46 MGTRLLFWVAFCLLGAYHTGAGVSQSPSNKVTEKGK DVELRCDPISGHTALYWYRQRLGQGLEFLIYFQGNSAPDK SGLPSDRFSAERTGESVSTLTIQRTQQEDSAVYLCASSLI ISYNEQFFGPGTRLTVL

Example 9

T-Cell Receptor T9 Recognizing HPV 16-E7 Epitope TLGIVCPI

[0117]

TABLE-US-00018 Immunogenic CDR3 peptide/ SEQ TCR Antigen purification TCRsequence IDNO: T9 E7 TLGIVCPI TRAV38-2/DV- 27 8*01-CAYRSAP YSGAGSYQLTF- TRAJ28*01 IFN- TRBV4-2*01-C 28 CAPTURE ASSQAPGLAGAE QYF-TRBD2*02- TRBJ2-7*01

[0118] Full length variable chain sequences (CDR3 sequences are underlined):

TABLE-US-00019 TRAV38-2/DV-8*01-CAYRSAPYSGAGSYQLTF-TRAJ28*01 SEQIDNO:47 MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAE TVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQ NATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCAYRSA PYSGAGSYQLTFGKGTKLSVIPN TRBV4-2*01-CASSQAPGLAGAEQYF-TRBD2*02-TRBJ2-7*01 SEQIDNO:48 MGCRLLCCAVLCLLGAVPMETGVTQTPRHLVMGMTN KKSLKCEQHLGHNAMYWYKQSAKKPLELMFVYNFKEQTEN NSVPSRFSPECPNSSHLFLHLHTLQPEDSALYLCASSQAP GLAGAEQYFGPGTRLTVT

Example 10

Interferon Release by TCR T2, T3 and T4 Transduced Cell Lines

[0119] FIGS. 6 and 7 show that T-cell lines transduced with the TCR T3 and T4 (FIGS. 6) and T2 (FIG. 7). Results are shown in the figures and figure legend.