ANTI-TIRC7 ANTIGEN BINDING PROTEINS

Abstract

The present disclosure provides human T-cell immune response cDNA 7 (TIRC7) antibodies, having improved TIRC7 binding affinity, and/or activity. The TIRC7 antibodies of the invention are generated by mutation of a parent TIRC7 antibody reports most of the humanized candidates, position 43 was Q (Gln, amide). But in the best humanized VH (cAb1466-VH) mutation of position 43 was Q (Gln, amide) to K (Lys, basic) appears to important for successful affinity/stability over the next best humanized VH.

Claims

1. An Antigen Binding Protein (ABP) capable of binding to T-cell immune response cDNA 7 (TIRC7), comprising: (i) one, preferably two, heavy chain variable domain(s) comprising the CDRH1 region set forth in SEQ ID NO: 01 (GYTFTTYV), the CDRH2 region set forth in SEQ ID NO: 02 (INPYNDGT), and the CDRH3 region set forth in SEQ ID NO: 03 (AEFITKTVGGSNWYLDV), or wherein in each case independently the CDRH1, CDRH2 and/or CDRH3 comprise a sequence having no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to SEQ ID NO: 01, SEQ ID NO: 02, or SEQ ID NO: 03, respectively; (ii) one, preferably two, light chain variable domain(s) comprising the CDRL1 region set forth in SEQ ID NO: 05 (SSISY), the CDRL2 region set forth in SEQ ID NO: 06 (DTS), and the CDRL3 region set forth in SEQ ID NO: 07 (HQRSSYTWT) or wherein in each case independently CDRL1, CDRL2 and/or CDRL3 comprise a sequence having no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to SEQ ID NO: 05, SEQ ID NO: 06, or SEQ ID NO: 07, respectively; characterized in that, said heavy chain variable domain(s) comprise(s) the sequence set forth in SEQ ID NO: 29, with no more than ten amino acid substitution(s), insertion(s) or deletion(s) compared to this sequence.

2. The ABP according to claim 1, wherein said one, preferably two, heavy chain variable domain(s) and said one, preferably two, light chain variable domain(s), each comprise an antibody framework having at least a portion of a human antibody consensus framework sequence.

3. The ABP according to claim 1, wherein within the heavy chain variable domain(s) said no more than ten amino acid substitution(s), insertion(s) or deletion(s) are not within the framework (FR) 3 region, or preferably wherein the heavy chain variable domain(s) FR3 is identical to the FR3 shown in SEQ ID NO: 29.

4. The ABP according to claim 1, wherein the one, preferably two, heavy chain variable domain(s) comprise(s) a CDR3 having the sequence shown in SEQ ID NO: 03 (AEFITKTVGGSNWYLDV).

5. The ABP according to claim 1, which when contacted with an immune cell suppresses immune cell activation and/or proliferation, preferably wherein suppressing or reducing T-cell-activation, and/or -proliferation, and/or wherein said ABP when contacted with a T-cell reduces T-cell mediated Interferon-y release.

6. The ABP according claim 1, which comprises an effector group and/or which is labelled.

7. The ABP according to claim 1, which is Fc receptor binding attenuated.

8. The ABP according to claim 1, which is isolated and/or substantially pure.

9. The ABP according to claim 1, which is an antibody, such as a monoclonal antibody; or which is a fragment of an antibody, such as a fragment of a monoclonal antibody.

10. The ABP according to claim 1, wherein said ABP is an IgG, IgE, IgD, IgA, or IgM immunoglobulin; preferably an IgG immunoglobulin.

11. The ABP according to claim 1, which is an antibody fragment selected from the list consisting of: Fab, Fab′-SH, Fv, scFv and F(ab′)2.

12. The ABP according to claim 1, which is a chimeric antigen receptor (CAR).

13. The ABP according to claim 1, which comprises one or more additional antigen binding domain(s) that bind(s) to antigen(s) other than said TIRC7; such as antigen(s) present on a mammalian T-cell, and most preferably human CD3 or a human T cell receptor (TCR).

14. The ABP according to claim 13, which is bispecific, and, preferably, which comprises one or two binding sites binding to TIRC7 and one or two binding sites binding to an antigen other than said TIRC7, such as antigen(s) present on a mammalian T-cell, and most preferably to human CD3 or human TCR.

15. The ABP according to claim 1, which binds to an extracellular domain of TIRC7 with an KD of less than 100 pM, preferably of less than 50 pM, as measured by plasmon surface resonance.

16-27. (canceled)

28. A method of modulating a cell-mediated immune response in a human cell that expresses human TIRC7 in a subject, comprising contacting said cell or administering to said subject a therapeutically effective amount of the ABP of claim 1, thereby modulating, preferably inhibiting, the cell-mediated immune response.

29. The method of claim 28, for the prevention and/or treatment of a disease associated with a pathological immune response in the subject, and wherein the disorder associated with a pathological immune response is characterized by an expression and/or activity of TIRC7 in cells involved with the pathological immune response.

30. The method of claim 29, wherein the pathological immune response is a cell-mediated immune response, preferably a T-cell mediated immune response.

31. The method of claim 29, wherein the disease is an autoimmune disease, such as an autoimmune disease selected from of psoriatic arthritis, graft versus host disease, autoimmune hepatitis, primar sclerosing cholangitis, primary biliary cirrhosis, IgG4 related autoimmune disease, fibrotic diseases e.g. pulmonary fibrosis, Sjörgen syndrome, systemic lupus erythematosis, Grave's disease, uveitis or uveitis with tubulointestinal nephritis, vasculitis, chronic fatigue syndrome and systemic scleroderma.

32. A humanized anti-TIRC7 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein (1) the heavy chain variable region comprises complementarity determining regions (CDRs): CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and (2) the light chain variable region comprises CDRs: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6, and (3) the heavy chain variable region and light chain variable region have amino acid sequences comprising the amino acid sequences of: SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22; SEQ ID NO: 23 and SEQ ID NO: 24; SEQ ID NO: 25 and SEQ ID NO: 26; SEQ ID NO: 27 and SEQ ID NO: 28; SEQ ID NO: 29 and SEQ ID NO: 30; SEQ ID NO: 31 and SEQ ID NO: 32; SEQ ID NO: 33 and SEQ ID NO: 34; SEQ ID NO: 35 and SEQ ID NO: 36; SEQ ID NO: 37 and SEQ ID NO: 38; SEQ ID NO: 39 and SEQ ID NO: 40; SEQ ID NO: 41 and SEQ ID NO: 42; SEQ ID NO: 43 and SEQ ID NO: 44; SEQ ID NO: 45 and SEQ ID NO: 46; SEQ ID NO: 47 and SEQ ID NO: 48; SEQ ID NO: 49 and SEQ ID NO: 50; SEQ ID NO: 51 and SEQ ID NO: 52; SEQ ID NO: 53 and SEQ ID NO: 54; SEQ ID NO: 55 and SEQ ID NO: 56; SEQ ID NO: 57 and SEQ ID NO: 58; SEQ ID NO: 59 and SEQ ID NO: 60; SEQ ID NO: 61 and SEQ ID NO: 62; SEQ ID NO: 63 and SEQ ID NO: 64; SEQ ID NO: 65 and SEQ ID NO: 66; SEQ ID NO: 67 and SEQ ID NO: 68; SEQ ID NO: 69 and SEQ ID NO: 70; SEQ ID NO: 71 and SEQ ID NO: 72; SEQ ID NO: 73 and SEQ ID NO: 74; SEQ ID NO: 75 and SEQ ID NO: 76; SEQ ID NO: 77 and SEQ ID NO: 78; SEQ ID NO: 79 and SEQ ID NO: 80; SEQ ID NO: 81 and SEQ ID NO: 82; SEQ ID NO: 83 and SEQ ID NO: 84; or SEQ ID NO: 85 and SEQ ID NO: 86.

Description

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCES

[0172] The figures show:

[0173] FIG. 1 shows an alignment and annotation of VH (A) and VL (B) sequences for 17-20 (parent mouse antibody) with the closest matching germline sequence.

[0174] FIG. 2 shows a model of the 17-20 (parent antibody) VH and VL domains. CDR regions of the heavy and light chain are indicated.

[0175] FIG. 3 shows a Model of the 17-20 VH and VL domains. CDR regions are indicated. The differences between the chimeric antibody cAb1457-10.0 and the humanized antibody cAb1459-10.0 are shown with amino acid side chains represented as “sticks”. The majority of humanized sites appear to have no influence on the CDRs but positions H-80, H-82 and H-85 are very close to CDRs H1 and H2, potentially having an impact on their folding and orientation.

[0176] FIG. 4 shows an alignment of humanized VH sequences with the original parental sequence. CDRs are indicated supporting amino acids are in additional boxes.

[0177] FIG. 5 shows an Alignment of humanized VL sequences with the original parental sequence. CDRs are indicated supporting amino acids are in additional boxes.

[0178] FIG. 6 shows a model of the 17-20 (parent) VH and VL domains. CDRs of heavy and light chains are indicated.

[0179] FIG. 7 shows a comparison of antibodies of the invention sorted by protein yield. Further indicated is purified protein (%), and monomer content (%).

[0180] FIG. 8 shows a comparison of the biological activity of antibodies of the invention sorted by protein yield. Amount of inhibition of IFNg production, induction of TIRC7+ cells, induction of TIRC7+ TREGs and Induction of TREGs is shown with the following coding: *=weak effect.fwdarw.*****=very strong effect.

[0181] FIG. 9 shows an VH chain humanization alignment—ranked by production and function (FIGS. 7 and 8). Potential interesting sites are highlighted. In these diagrams, the numbering and CDR positioning is according to Chothia et al. (1992) J. Mol. Biol., 227, 776-798, Tomlinson et al. (1995) EMBO J., 14, 4628-4638 and Williams et al. (1996) J. Mol. Biol., 264, 220-232 (Example given in VBase. https://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#JHEX). The chain “Mu 17-20-VK denotes the murine parent sequence also referred to as cAB1457 herein). CDR regions are underlined.

[0182] FIG. 10 shows an VL chain humanization alignment—ranked by production and function (FIGS. 9 and 10). In these diagrams, the numbering and CDR positioning is according to Chothia et al. (1992) J. Mol. Biol., 227, 776-798, Tomlinson et al. (1995) EMBO J., 14, 4628-4638 and Williams et al. (1996) J. Mol. Biol., 264, 220-232 (Example given in VBase. https://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#JHEX). The chain “Mu 17-20-VK denotes the murine parent sequence also referred to as cAB1457 herein). CDR regions are underlined.

[0183] FIG. 11 shows a proliferation assay of mixed lymphocytes treated with antibody variants of the invention after allo-stimulation. Top panel shows an experiment with 20 μg/ml of tested antibody, lower panel with 40 μg/ml of tested antibody. Controls are no antibody treatment, IgG control and the parent chimeric antibody. Antibody numbering is as indicated in table 1. Patent Control pertains to the use of the parent mouse antibody (cAb1457). Shown are mean values of 3 independent experiments.

[0184] FIG. 12. shows a proliferation assay of lymphocytes treated with antibody variants of the invention after Phytohaemagglutinin (PHA)-stimulation mitogen. Top panel shows an experiment with 20 μg/ml of tested antibody, lower panel with 40 μg/ml of tested antibody. Controls are no antibody treatment, IgG control and the parent chimeric antibody. Antibody numbering is as indicated in table 1. Patent Control pertains to the use of the parent mouse antibody (cAb1457). Shown are mean values of 3 independent experiments.

[0185] The ABP of the invention are all described in the sequence listing and the following table 1:

TABLE-US-00003 SEQ ID Antibody NO: Name Description Sequence 1 cAb1457 CDRH1 GYTFTTYV 2 cAb1457 CDRH2 INPYNDGT 3 cAb1457 CDRH3 AEFITKTVGGSNWYLDV 4 cAb1457 HCV QVQLKQSGPELVKPGASVKMSCKASGYTFTTYV MHWVKQKPGQGLEWIGYINPYNDGTNYNEKF KGKATLTSDKSSSTAYMELSTLTSEDSAVYYCAE FITKTVGGSNWYLDVWGAGTTVTVSS 5 cAb1457 CDRL1 SSISY 6 cAb1457 CDRL2 DTS 7 cAb1457 CDRL3 HQRSSYTWT 8 cAb1457 LCV DIVLTQSPAIMSASPGEKVTMTCSASSSISYIHW FQQKPGTSPKRWIYDTSKLPSGVPARFSGSGSG TSYSLTISSMEAEDAATYYCHQRSSYTWTFGGG TKLEIK 9 cAb1458 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTMTEDTSTDTAYMELSSLRSEDTAVYYC AEFITKTVGGSNWYLDVWGQGTTVTVSS 10 cAb1458 LCV DIQLTQSPSFLSASVGDRVTITCSASSSISYIHWY QQKPGKAPKRLIYDTSKLPSGVPSRFSGSGSGTE FTLTISSLQPEDFATYYCHQRSSYTWTFGGGTKL EIK 11 cAb1459 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTMTEDTSTDTAYMELSSLRSEDTAVYYC AEFITKTVGGSNWYLDVWGQGTTVTVSS 12 cAb1459 LCV DIQLTQSPSFLSASVGDRVTITCSASSSISYIHWF QQKPGKAPKRWIYDTSKLPSGVPSRFSGSGSGT EYTLTISSLQPEDFATYYCHQRSSYTWTFGGGT KLEIK 13 cAb1460 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTMTEDTSTDTAYMELSSLRSEDTAVYYC AEFITKTVGGSNWYLDVWGQGTTVTVSS 14 cAb1460 LCV EIVLTQSPATLSLSPGERATLSCSASSSISYIHWY QQKPGQAPRRLIYDTSKLPSGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCHQRSSYTWTFGGGTKL EIK 15 cAb1461 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTMTEDTSTDTAYMELSSLRSEDTAVYYC AEFITKTVGGSNWYLDVWGQGTTVTVSS 16 cAb1461 LCV EIVLTQSPATLSLSPGERATLSCSASSSISYIHWF QQKPGQAPKRWIYDTSKLPSGVPARFSGSGSGT DYTLTISSLEPEDFAVYYCHQRSSYTWTFGGGT KLEIK 17 cAb1462 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTMTSDKSTSTAYMELSSLRSEDTAVYYC AEFITKTVGGSNWYLDVWGQGTTVTVSS 18 cAb1462 LCV DIQLTQSPSFLSASVGDRVTITCSASSSISYIHWY QQKPGKAPKRLIYDTSKLPSGVPSRFSGSGSGTE FTLTISSLQPEDFATYYCHQRSSYTWTFGGGTKL EIK 19 cAb1463 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTMTSDKSTSTAYMELSSLRSEDTAVYYC AEFITKTVGGSNWYLDVWGQGTTVTVSS 20 cAb1463 LCV DIQLTQSPSFLSASVGDRVTITCSASSSISYIHWF QQKPGKAPKRWIYDTSKLPSGVPSRFSGSGSGT EYTLTISSLQPEDFATYYCHQRSSYTWTFGGGT KLEIK 21 cAb1464 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTMTSDKSTSTAYMELSSLRSEDTAVYYC AEFITKTVGGSNWYLDVWGQGTTVTVSS 22 cAb1464 LCV EIVLTQSPATLSLSPGERATLSCSASSSISYIHWY QQKPGQAPRRLIYDTSKLPSGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCHQRSSYTWTFGGGTKL EIK 23 cAb1465 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTMTSDKSTSTAYMELSSLRSEDTAVYYC AEFITKTVGGSNWYLDVWGQGTTVTVSS 24 cAb1465 LCV EIVLTQSPATLSLSPGERATLSCSASSSISYIHWF QQKPGQAPKRWIYDTSKLPSGVPARFSGSGSGT DYTLTISSLEPEDFAVYYCHQRSSYTWTFGGGT KLEIK 25 cAb1466 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTLTSDKSTSTAYMELSSLRSEDTAVYYCA EFITKTVGGSNWYLDVWGQGTTVTVSS 26 cAb1466 LCV DIQLTQSPSFLSASVGDRVTITCSASSSISYIHWY QQKPGKAPKRLIYDTSKLPSGVPSRFSGSGSGTE FTLTISSLQPEDFATYYCHQRSSYTWTFGGGTKL EIK 27 cAb1467 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTLTSDKSTSTAYMELSSLRSEDTAVYYCA EFITKTVGGSNWYLDVWGQGTTVTVSS 28 cAb1467 LCV DIQLTQSPSFLSASVGDRVTITCSASSSISYIHWF QQKPGKAPKRWIYDTSKLPSGVPSRFSGSGSGT EYTLTISSLQPEDFATYYCHQRSSYTWTFGGGT KLEIK 29 cAb1468 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTLTSDKSTSTAYMELSSLRSEDTAVYYCA EFITKTVGGSNWYLDVWGQGTTVTVSS 30 cAb1468 LCV EIVLTQSPATLSLSPGERATLSCSASSSISYIHWY QQKPGQAPRRLIYDTSKLPSGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCHQRSSYTWTFGGGTKL EIK 31 cAb1469 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTLTSDKSTSTAYMELSSLRSEDTAVYYCA EFITKTVGGSNWYLDVWGQGTTVTVSS 32 cAb1458 LCV EIVLTQSPATLSLSPGERATLSCSASSSISYIHWF QQKPGQAPKRWIYDTSKLPSGVPARFSGSGSGT DYTLTISSLEPEDFAVYYCHQRSSYTWTFGGGT KLEIK 33 cAb1901 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGQGLEWMGYINPYNDGTNYNEK FQGRVTSTSDKSISTAYMELSRLRSDDTVVYYCA EFITKTVGGSNWYLDVWGQGTTVTVSS 34 cAb1901 LCV DIQLTQSPSFLSASVGDRVTITCSASSSISYIHWF QQKPGKAPKRWIYDTSKLPSGVPSRFSGSGSGT EYTLTISSLQPEDFATYYCHQRSSYTWTFGGGT KLEIK 35 cAb2021 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGQGLEWMGYINPYNDGTSYNEKF QGRVTMTSDKSTSTVYMELSSLRSEDTAVYYCA EFITKTVGGSNWYLDVWGQGTTVTVSS 36 cAb2021 LCV EIVLTQSPDFQSVTPKEKVTITCRASSSISYIHWF QQKPDQSPKRLIYDTSKSFSGVPSRFSGSGSGTD YTLTINSLEAEDAATYYCHQRSSYTWTFGGGTK LEIK 37 cAb2022 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGQGLEWMGYINPYNDGTSYNEKF QGRVTMTSDKSTSTVYMELSSLRSEDTAVYYCA EFITKTVGGSNWYLDVWGQGTTVTVSS 38 cAb2022 LCV DIQLTQSPSAMSASVGDRVTITCRASSSISYIHW FQQKPGKVPKRLIYDTSKLQSGVPSRFSGSGSGT EYTLTISSLQPEDFATYYCHQRSSYTWTFGGGT KLEIK 39 cAb2023 HCV QVQLVQSGSELKKPGASVKVSCKASGYTFTTYV MHWVRQAPGQGLEWMGYINPYNDGTTYNEG FTGRFVFSSDKSVSTAYLQISSLKAEDTAVYYCA EFITKTVGGSNWYLDVWGQGTTVTVSS 40 cAb2023 LCV EIVLTQSPDFQSVTPKEKVTITCRASSSISYIHWF QQKPDQSPKRLIYDTSKSFSGVPSRFSGSGSGTD YTLTINSLEAEDAATYYCHQRSSYTWTFGGGTK LEIK 41 cAb2024 HCV QVQLVQSGSELKKPGASVKVSCKASGYTFTTYV MHWVRQAPGQGLEWMGYINPYNDGTTYNEG FTGRFVFSSDKSVSTAYLQISSLKAEDTAVYYCA EFITKTVGGSNWYLDVWGQGTTVTVSS 42 cAb2024 LCV DIQLTQSPSAMSASVGDRVTITCRASSSISYIHW FQQKPGKVPKRLIYDTSKLQSGVPSRFSGSGSGT EYTLTISSLQPEDFATYYCHQRSSYTWTFGGGT KLEIK 43 cAb2287 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWIGYINPYNDGTNYNEKF KGRVTMTSDKSTSTAYMELSSLRSEDTAVYYCA EFITKTVGGSNWYLDVWGQGTTVTVSS 44 cAb2287 LCV DIVLTQSPSFLSASVGDRVTITCSASSSISYIHWF QQKPGKAPKRWIYDTSKLPSGVPSRFSGSGSGT EYTLTISSLQPEDFATYYCHQRSSYTWTFGGGT KLEIK 45 cAb2288 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWIGYINPYNDGTNYNEKF KGRVTLTSDKSTSTAYMELSSLRSEDTAVYYCAE FITKTVGGSNWYLDVWGQGTTVTVSS 46 cAb2288 LCV DIVLTQSPSFLSASVGDRVTITCSASSSISYIHWF QQKPGKAPKRWIYDTSKLPSGVPSRFSGSGSGT EYTLTISSLQPEDFATYYCHQRSSYTWTFGGGT KLEIK 47 cAb2712 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGQGLEWIGYINPYNDGTNYNEKF QGRVTLTSDKSSSTAYMELSRLRSDDTVVYYCA EFITKTVGGSNWYLDVWGQGTTVTVSS 48 cAb2712 LCV DIVLTQSPDFQSVTPKEKVTITCRASSSISYIHWF QQKPDQSPKRLIYDTSKLPSGVPSRFSGSGSGTD YTLTINSLEAEDAATYYCHQRSSYTWTFGGGTK LEIK 49 cAb2713 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGQGLEWIGYINPYNDGTNYNEKF QGRVTLTSDKSSSTAYMELSRLRSDDTVVYYCA EFITKTVGGSNWYLDVWGQGTTVTVSS 50 cAb2713 LCV DIQLTQSPSAMSASVGDRVTITCRASSSISYIHW FQQKPGKVPKRLIYDTSKLPSGVPSRFSGSGSGT EYTLTISSLQPEDFATYYCHQRSSYTWTFGGGT KLEIK 51 cAb2714 HCV QVQLVQSGSELKKPGASVKVSCKASGYTFTTYV MHWVRQAPGQGLEWIGYINPYNDGTNYNEGF TGRFVLSSDKSSSTAYLQISSLKAEDTAVYYCAE FITKTVGGSNWYLDVWGQGTTVTVSS 52 cAb2714 LCV DIVLTQSPDFQSVTPKEKVTITCRASSSISYIHWF QQKPDQSPKRLIYDTSKLPSGVPSRFSGSGSGTD YTLTINSLEAEDAATYYCHQRSSYTWTFGGGTK LEIK 53 cAb2715 HCV QVQLVQSGSELKKPGASVKVSCKASGYTFTTYV MHWVRQAPGQGLEWIGYINPYNDGTNYNEGF TGRFVLSSDKSSSTAYLQISSLKAEDTAVYYCAE FITKTVGGSNWYLDVWGQGTTVTVSS 54 cAb2715 LCV DIQLTQSPSAMSASVGDRVTITCRASSSISYIHW FQQKPGKVPKRLIYDTSKLPSGVPSRFSGSGSGT EYTLTISSLQPEDFATYYCHQRSSYTWTFGGGT KLEIK 55 cAb3458 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTLTEDTSTDTAYMELSSLRSEDTAVYYC AEFITKTVGGSNWYLDVWGQGTTVTVSS 56 cAb3458 LCV DIQLTQSPSFLSASVGDRVTITCSASSSISYIHWY QQKPGKAPKRLIYDTSKLPSGVPSRFSGSGSGTE FTLTISSLQPEDFATYYCHQRSSYTWTFGGGTKL EIK 57 cAb3459 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTLTEDTSTDTAYMELSSLRSEDTAVYYC AEFITKTVGGSNWYLDVWGQGTTVTVSS 58 cAb3459 LCV DIQLTQSPSFLSASVGDRVTITCSASSSISYIHWF QQKPGKAPKRWIYDTSKLPSGVPSRFSGSGSGT EYTLTISSLQPEDFATYYCHQRSSYTWTFGGGT KLEIK 59 cAb3460 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTLTEDTSTDTAYMELSSLRSEDTAVYYC AEFITKTVGGSNWYLDVWGQGTTVTVSS 60 cAb3460 LCV EIVLTQSPATLSLSPGERATLSCSASSSISYIHWY QQKPGQAPRRLIYDTSKLPSGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCHQRSSYTWTFGGGTKL EIK 61 cAb3461 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTLTEDTSTDTAYMELSSLRSEDTAVYYC AEFITKTVGGSNWYLDVWGQGTTVTVSS 62 cAb3461 LCV EIVLTQSPATLSLSPGERATLSCSASSSISYIHWF QQKPGQAPKRWIYDTSKLPSGVPARFSGSGSGT DYTLTISSLEPEDFAVYYCHQRSSYTWTFGGGT KLEIK 63 cAb3462 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTMTSDTSTDTAYMELSSLRSEDTAVYYC AEFITKTVGGSNWYLDVWGQGTTVTVSS 64 cAb3462 LCV DIQLTQSPSFLSASVGDRVTITCSASSSISYIHWY QQKPGKAPKRLIYDTSKLPSGVPSRFSGSGSGTE FTLTISSLQPEDFATYYCHQRSSYTWTFGGGTKL EIK 65 cAb3463 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTMTSDTSTDTAYMELSSLRSEDTAVYYC AEFITKTVGGSNWYLDVWGQGTTVTVSS 66 cAb3463 LCV DIQLTQSPSFLSASVGDRVTITCSASSSISYIHWF QQKPGKAPKRWIYDTSKLPSGVPSRFSGSGSGT EYTLTISSLQPEDFATYYCHQRSSYTWTFGGGT KLEIK 67 cAb3464 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTMTSDTSTDTAYMELSSLRSEDTAVYYC AEFITKTVGGSNWYLDVWGQGTTVTVSS 68 cAb3464 LCV EIVLTQSPATLSLSPGERATLSCSASSSISYIHWY QQKPGQAPRRLIYDTSKLPSGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCHQRSSYTWTFGGGTKL EIK 69 cAb3465 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTMTSDTSTDTAYMELSSLRSEDTAVYYC AEFITKTVGGSNWYLDVWGQGTTVTVSS 70 cAb3465 LCV EIVLTQSPATLSLSPGERATLSCSASSSISYIHWF QQKPGQAPKRWIYDTSKLPSGVPARFSGSGSGT DYTLTISSLEPEDFAVYYCHQRSSYTWTFGGGT KLEIK 71 cAb3466 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTMTEDKSTDTAYMELSSLRSEDTAVYYC AEFITKTVGGSNWYLDVWGQGTTVTVSS 72 cAb3466 LCV DIQLTQSPSFLSASVGDRVTITCSASSSISYIHWY QQKPGKAPKRLIYDTSKLPSGVPSRFSGSGSGTE FTLTISSLQPEDFATYYCHQRSSYTWTFGGGTKL EIK 73 cAb346y HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTMTEDKSTDTAYMELSSLRSEDTAVYYC AEFITKTVGGSNWYLDVWGQGTTVTVSS 74 cAb3467 LCV DIQLTQSPSFLSASVGDRVTITCSASSSISYIHWF QQKPGKAPKRWIYDTSKLPSGVPSRFSGSGSGT EYTLTISSLQPEDFATYYCHQRSSYTWTFGGGT KLEIK 75 cAb3468 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTMTEDKSTDTAYMELSSLRSEDTAVYYC AEFITKTVGGSNWYLDVWGQGTTVTVSS 76 cAb3468 LCV EIVLTQSPATLSLSPGERATLSCSASSSISYIHWY QQKPGQAPRRLIYDTSKLPSGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCHQRSSYTWTFGGGTKL EIK 77 cAb3469 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTMTEDKSTDTAYMELSSLRSEDTAVYYC AEFITKTVGGSNWYLDVWGQGTTVTVSS 78 cAb3469 LCV EIVLTQSPATLSLSPGERATLSCSASSSISYIHWF QQKPGQAPKRWIYDTSKLPSGVPARFSGSGSGT DYTLTISSLEPEDFAVYYCHQRSSYTWTFGGGT KLEIK 79 cAb3470 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTMTEDTSTSTAYMELSSLRSEDTAVYYC AEFITKTVGGSNWYLDVWGQGTTVTVSS 80 cAb3470 LCV DIQLTQSPSFLSASVGDRVTITCSASSSISYIHWY QQKPGKAPKRLIYDTSKLPSGVPSRFSGSGSGTE FTLTISSLQPEDFATYYCHQRSSYTWTFGGGTKL EIK 81 cAb3471 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTMTEDTSTSTAYMELSSLRSEDTAVYYC AEFITKTVGGSNWYLDVWGQGTTVTVSS 82 cAb3471 LCV DIQLTQSPSFLSASVGDRVTITCSASSSISYIHWF QQKPGKAPKRWIYDTSKLPSGVPSRFSGSGSGT EYTLTISSLQPEDFATYYCHQRSSYTWTFGGGT KLEIK 83 cAb3472 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTMTEDTSTSTAYMELSSLRSEDTAVYYC AEFITKTVGGSNWYLDVWGQGTTVTVSS 84 cAb3472 LCV EIVLTQSPATLSLSPGERATLSCSASSSISYIHWY QQKPGQAPRRLIYDTSKLPSGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCHQRSSYTWTFGGGTKL EIK 85 cAb3473 HCV QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYV MHWVRQAPGKGLEWMGYINPYNDGTNYNEK FKGRVTMTEDTSTSTAYMELSSLRSEDTAVYYC AEFITKTVGGSNWYLDVWGQGTTVTVSS 86 cAb3473 LCV EIVLTQSPATLSLSPGERATLSCSASSSISYIHWF QQKPGQAPKRWIYDTSKLPSGVPARFSGSGSGT DYTLTISSLEPEDFAVYYCHQRSSYTWTFGGGT KLEIK CDRH (Complementary Determining Region Heavy Chain); CDRL (Complementary Determining Region Light Chain); HCV (Heavy Chain Variable); LCV (Light Chain Variable)

EXAMPLES

[0186] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the description, figures and tables set out herein. Such examples of the methods, uses and other aspects of the present invention are representative only, and should not be taken to limit the scope of the present invention to only such representative examples.

[0187] The examples show:

Example 1: Generation of Variant and Humanized Anti-TIRC7 Antibodies

[0188] For the purpose of identifying complementarity determining regions (CDRs) and analyzing the closest matching germline sequences the IMGT Domain Gap Align tool was used: http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi.

[0189] Molecular models were built for VH and VL domains based on homology to previously published antibody crystal structures using in-house software. PDB files can be provided on request for viewing in any molecular visualization software. Images have been generated using PyMol.

[0190] Variable heavy and variable light domains were designed with appropriate restriction sites at the 5′ and 3′ ends to enable cloning into Absolute Antibody® cloning and expression vectors. Variable domains sequences were codon optimized for expression in human cells. Following gene synthesis, the variable domains were cloned into Absolute Antibody® vectors of the appropriate species and type. The correct sequence was verified by Sanger sequencing with raw data analyzed using DNASTAR® Lasergene software. Once confirmed plasmid DNA preps of the appropriate size were performed to generate a sufficient quantity of high quality DNA for transfection.

[0191] HEK 293 (human embryonic kidney 293) mammalian cells were passaged to the optimum stage for transient transfection. Cells were transiently transfected with heavy and light chain expression vectors and cultured for a further 6 days. Cultures were harvested by centrifugation at 4000 rpm and filtered through a 0.22 μM filter. A first step of purification was performed by Protein A affinity chromatography with elution using citrate pH3.0 buffer followed by neutralization with 0.5M Tris, pH 9.0. Eluted protein was then buffer exchanged into PBS using a desalting column. Antibody concentration was determined by UV spectroscopy and the antibodies concentrated as necessary.

[0192] Antibody purity was determined by SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) and HPLC (high performance liquid chromatography). SEC-HPLC was performed on an Agilent 1100 series instrument using an appropriate size exclusion column (SEC). Antibody expression titer was determined by Protein A HPLC.

[0193] The VH and VL sequences for 17-20 were run through the IGMT Gap Align tool to analyse against all known antibody germline sequences (FIG. 1). CDR regions were assigned using the IMGT definition. As expected, the sequence is most closely aligned to mouse, specifically the IGHV1-2, IGHV1-46*01 and IGHV7-4-1*02 for the heavy chain and IGKV1-9, IGKV3-11, IGKV6-21*01 and IGKV1-17*03 for the light chain.

[0194] To enable structure guided humanization models were built for the 17-20 murine VH and VL sequences, as shown in FIG. 2.

[0195] The VH and VL sequences were aligned to with an Absolute Antibody® database of human germline sequences. Table 1 shows the germline sequences that were selected as frameworks for humanization.

[0196] Tables 2 and 3: Heavy and light chain germline sequences selected as humanization frameworks and their percent identity to the original murine VH and VL sequences.

TABLE-US-00004 TABLE 2 Accession Germline ID no. % identity Heavy IGHV1-2 X07448 71.13 chain Light IGKV1-9 Z00013 63.83 chain IGKV3-11 X01668 61.70

TABLE-US-00005 TABLE 3 Germline ID % identity Heavy IGHV1-46*01 70.1 chain IGHV7-4-1*02 61.9 Light IGKV6-21*01 66.7 chain IGKV1-17*03 66.0

[0197] The VH and VL sequences were run through a CDR grafting algorithm to transfer the CDRs from the murine antibody 17-20 onto the selected human germline sequences. Although CDRs are defined as being primarily responsible for binding to an antigen it is possible for amino acids outside of these regions, in what are known as framework regions, to either be involved directly in binding or to play a role in correctly orientating the CDRs. A structure guided approach was used to determine which of the framework amino acids to retain in the as the original mouse amino acid for the sake of retaining binding integrity (FIGS. 3 and 6). Table 1 contains the sequences that were generated.

Example 2: Germline Combination and Non-Intuitive Back Mutations were Necessary to Obtain Functional Humanized Anti-TIRC7 Antibodies

[0198] As described in example 1 humanized heavy chains and humanized light chains were designed. Each of these were synthesized separately and cloned into human IgG1 heavy chain and human kappa light chain expression vectors respectively. At the point of transfection all possible combinations of the humanized sequences were made to create a total of 12 different humanized antibodies. In addition to these the original mouse antibody as well as a chimeric human IgG1 were made as controls.

[0199] Most humanized chain generated either lacked expression, where expressed with reduced monomer content or lost activity. In total five rounds of humanization attempts were necessary to identify some adequate humanized sequences, that in addition to the usual grafting procedure and back mutations close to the CDR regions needed additional non-intuitive back mutations in the heavy chain sequences—which was surprising.

[0200] An alignment of some antibodies shown in table 1 above is provided in FIGS. 4 and 5.

[0201] In one attempt to humanize the heavy chain, the inventors used the IGHV7-4-1 human germline sequence to generate humanized heavy chains. Unfortunately, all of the generated sequences were not expressible at all (see FIG. 7, antibodies having a heavy chain sequence cAB2714-VH or cAB2712-VH).

[0202] In a further attempt, the human germ line sequence IGHV1-46 was used for grafting. The resulting chains are cAB2288-VH and cAB2287-VH, of which cAB2287-VH was not expressible at all, while cAB2288-VH showed some expression. However, antibodies having the cAB2288-VH chain when expressed showed no monomer content (FIG. 7).

[0203] Another attempt IGHV1-2 was used for grafting and resulted in the cAB1901VH chain. Antibodies with this chain were also not expressible at all.

[0204] In two additional rounds of humanization chains based on all three of the above captioned germ line sequences were generated with the introduction of various mutations at selected positions (in particular chains cAB1458-VH, cAB1462-VH, cAB1466-VH, cAB2021-VH and cAB2023-VH) to combine human framework regions from different germ line sequences, which constitutes an usual approach. The generated heavy chain sequences in at least some antibodies were expressible to at least a low degree. However, antibodies comprising the cAB2021-VH or cAB2023-VH chains had low or undetectable monomer content, and were disregarded for the final selection.

[0205] The remaining chains were compared by retained biological activity of antibodies comprising them. The result is shown in FIG. 8. Most surprisingly antibodies comprising a heavy chain into which three non-intuitive back mutations were introduced into framework 3 (FR3) showed astonishing biological activity while having a preferable protein expression and monomer content (chain cAB1466-VH). Taken together, the antibodies denoted as cAB1467-10.0, cAB1468-10.0 and cAB1459-10.0 are the best candidates, with cAB1467-10.0, cAB1468-10.0 (both comprising the cAB1466-VH chain) being the best ones.

[0206] Conclusion:

[0207] Following standard humanization, analysis was done to determine which human germline genes were closest to the original mouse hybridoma sequences and thus could be used as donor frameworks for CDR grafting. In variation from many humanization processes, a combination of framework donors for CDR grafting from 2 different human VH genes was also tried. Extensive experimentation determined that the combination of donors provided better results. A comparison of the sequences with highlighted positions of interest is shown in FIGS. 9 (heavy chain) and 10 (light chain). In these diagrams, the numbering and CDR positioning is according to Chothia et al. (1992) J. Mol. Biol., 227, 776-798, Tomlinson et al. (1995) EMBO J., 14, 4628-4638 and Williams et al. (1996) J. Mol. Biol., 264, 220-232) Example given in VBase. https://www2. mrc-lmb.cam.ac.uk/vbase/alignments2.php#JHEX. [0208] Used IGHV1-2 as donor for FR1 and FR2 (indicated by highlighting) [0209] Used IGHV1-46*01 as donor for FR3 (indicated by highlighting)

[0210] Unusual backmutations to original mouse sequences were needed in framework 3 to create high affinity and stability in the humanized antibodies: [0211] Position 71—human donor R (Arg, basic) backmutation to mouse L (Leu, hydrophobic) [0212] Position 73—human donor T (Thr, nucleophilic), backmutation to mouse K (Lys, basic) [0213] Position 69—human donor M (Met, hydrophobic), backmutation to mouse L (Leu, hydrophobic), small change but appears to make cAb1466-VH better than cAb2021.

[0214] Unusual mutation was also made in framework 2: [0215] In original VH17-20, all proposed human donor VH, and most of the humanized candidates, position 43 was Q (Gln, amide). But in the best humanized VH (cAb1466-VH) mutation of position 43 was Q (Gln, amide) to K (Lys, basic) appears to important for successful affinity/stability over the next best humanized VH (cAb2021-VH).

[0216] It is therefore demonstrated that only a combination of multiple human germ line sequences, and the non-intuitive introduction of murine back mutations could yield a small number of final candidate antibodies having improved expression and biologic activity.

[0217] Light Chain Humanization—the wide diversity of humanized light chains that were able to function well in both affinity and stability of the intact mAb suggest that most of the success of the mAb is due to the heavy chain and the heavy chain is rather promiscuous in its light chain selection.

Example 3: Effect of Improved Anti-TIRC7 Antibodies of the Invention on Human Lymphocytes

[0218] The antibodies of the invention were tested in an immune assay for mixed lymphocyte proliferation and by PHA stimulation of lymphocytes. The results show a surprisingly stronger effect for the improved antibodies of the invention compared to the parent chimeric antibody. The results are shown in FIGS. 11 and 12 respectively.

Example 4: Surface Plasmon Resonance Testing of Antibody Affinity

[0219] To compare target binding of the generated antibodies with the parent molecule, binding constants of the of parent and selected generated humanized versions antibodies were obtained via BiaCore® Surface Plasmon Resonance Testing. The largest TIRC7 extracellular domain which is the known binder for the parent antibody was immobilized on a carboxymethylated dextran surface of a CM5 sensor chip (CM5 ship, research grade). A Biacore T200 instrument was used, 25° C. analysis temperature, and a flow rate of 50 μl/min for quantitative kinetic interaction analyses. The analysis buffer was 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20.

[0220] For each antibody multiple Biacore SPR analysis rows were performed in varying concentrations in order to determine the kinetic constants or absorption effects. The specific constants are very reproducible for the buffer conditions. The parent antibody showed an average K.sub.D of 300+/−100 pM. Measurements for 3 of the humanized antibodies was done in the same setting. Results are provided in table 4. Surprisingly, the results show that AB affinity to TIRC7 of the selected humanized AB variants was at least one order of magnitude higher compared to the parent molecule.

TABLE-US-00006 TABLE 4 Summary of Kinetic Data Kinetic fit 1:1 binding Sample k.sub.a (M.sup.−1s.sup.−1) k.sub.d (s.sup.−1) K.sub.D (pM) parent antibody   .sup. 5.2 ± 1.4E+05   .sup. 1.3 ± 0.7E−04 300 ± 100 cAB1459-10.0 3.56E+06 ± 2.41E+05 2.03E−04 ± 2.22E−05  5.70 ± 0.280 cAB1468-10.0 1.13E+06 ± 5.69E+04 3.16E−04 ± 1.40E−05 27.9 ± 1.04 cAB1467-10.0 1.24E+06 ± 6.66E+04 5.97E−04 ± 2.97E−05 48.0 ± 2.51

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