Dual antigen-induced bipartite functional complementation

Abstract

The present invention relates to a set of polypeptides and its uses. In particular, the present invention relates to a set of polypeptides whereby this set comprises two polypeptides each of which comprises a targeting moiety “T” binding to an antigen “A” and a fragment of “F” of a functional domain, wherein said two polypeptides are not associated with each other in absence of a substrate that has “A” at (on) its surface and wherein, upon dimerization of “F”, the resulting dimer becomes functional. Furthermore, medical and diagnostic uses of said set are described. Moreover, the present invention relates to nucleic acid molecule(s) encoding said set of polypeptides. The present invention also relates to a vector comprising the nucleotide sequence of nucleic acid molecule(s) encoding said set of polypeptides. Furthermore, the present invention relates to pharmaceutical compositions comprising said set of polypeptides. Moreover, the present invention relates to a kit comprising said set of polypeptides.

1 . A set of polypeptides comprising:
 a first polypeptide P1 comprising
 (i) a targeting moiety Ti,
 wherein said targeting moiety T1 specifically binds to an antigen A1, and 
 
 (ii) a fragment F1 of a functional domain F, 
   wherein neither said fragment F1 by itself nor said polypeptide P1 by itself is functional with respect to the function of said domain F,   and   a second polypeptide P2 comprising
 (i) a targeting moiety T2,
 wherein said targeting moiety T2 specifically binds to an antigen A2, and 
 
 (ii) a fragment F2 of said functional domain F, 
   wherein neither said fragment F2 by itself nor said polypeptide P2 by itself is functional with respect to the function of said domain F,   wherein said antigen A1 is different from said antigen A2,   wherein said polypeptide P1 and said polypeptide P2 are not associated with each other in the absence of a substrate that has both antigens A1 and A2 at its surface, and   wherein, upon dimerization of said fragment F1 of said polypeptide P1 with said fragment F2 of said polypeptide P2, the resulting dimer is functional with respect to the function of said domain F.   
     
     
         2 . The set of polypeptides according to  claim 1 , wherein said polypeptide P1 and said polypeptide P2 are not associated with each other in the absence of a cell that carries both antigens A1 and A2 at its cell surface. 
     
     
         3 . The set of polypeptides according to  claim 1 , wherein a cell carrying both antigens A1 and A2 at its cell surface induces dimerization of the fragment F1 of said polypeptide P1 with the fragment F2 of said polypeptide P2, whereas a cell which does not carry both antigens A1 and A2 at its cell surface does not induce dimerization of the fragment F1 of said polypeptide P1 with the fragment F2 of said polypeptide P2. 
     
     
         4 . The set of polypeptides according to  claim 1 , wherein said polypeptides P1 and P2 have, in the absence of said substrate or cell, with each other a dissociation constant K D  in the range of 10 −8  M to 10 −2  M, in the range of 10 −7  M to 10 −3  M or in the range of 10 −6  M to 10 −3  M; and/or said polypeptides P1 and P2 have, in the presence of said substrate or cell, with each other a dissociation constant K D  below 10 −6  M, below 10 −7  M below 10 −8  M or below 10 −9  M. 
     
     
         5 . The set of polypeptides according to  claim 1 , wherein said antigen A1 and/or said antigen A2 is an antigen expressed on the surface of cells of a tumour or on the surface of progenitor/precursor cells of a tumour. 
     
     
         6 . (canceled) 
     
     
         7 . The set of polypeptides according to  claim 1 , wherein the combination of antigen A1 and antigen A2 is only found on cancerous cells, and not on cells that are not cancerous. 
     
     
         8 . The set of polypeptides according to  claim 7 , wherein the combination of antigen A1 and antigen A2 is specific for cancerous cells of a certain type of cancer. 
     
     
         9 . The set of polypeptides according to  claim 1 , wherein said antigen A1 is an MHC antigen being an allelic variant selected from the group consisting of:
 HLA-A2, HLA-Cw6, HLA-A1, HLA-A3, HLA-A25, HLA-B7, HLA-B8, HLA-B35, HLA-B44, HLA-Cw3, HLA-Cw4, and HLA-Cw7;   and/or said antigen A2 is an antigen that is specific for a certain cell type or cell lineage selected from the group consisting of:   CD45; CD34; CD33; CD138; CD15; CD1a; CD2; CD3; CD4; CD5; CD8; CD20; CD23; CD31; CD43; CD56; CD57; CD68; CD79a; CD146; surfactant proteins; synaptophysin; CD56; CD57; nicotinic acetylcholine receptor; muscle-specific kinase MUSK; voltage-gated calcium channel (P/Q-type); voltage-gated potassium channel (VGKC); N-methyl-D-aspartate receptor (NMDA); TSH; amphiphysin; HepPar-1; ganglioside GQ1B, GD3 or GM 1; and glycophorin-A.   
     
     
         10 .- 13 . (canceled) 
     
     
         14 . The set of polypeptides according to  claim 1 , wherein any of said antigens A1 and A2 is selected from the group consisting of: HLA-A2; HLA-Cw6; EpCAM; CD20; CD33; CD38; CD45; Her2; EGFR; CD138; CEA; CD19; PSMA; E-cadherin; Ca-125; Her-2/neu; gross cystic disease fluid protein; BCA-225; CA 19-9; CD117; CD30; Epithelial antigen BER-EP4, Epithelial membrane antigen and Epithelial Related Antigen MOC-31; Epidermal growth factor receptor HER1; Platelet derived growth factor receptor PDGFR alpha; Melanoma associated marker/Mart 1/Melan-A; CD133; TAG 72; aquaporin-2 and a clonotypic antibody on the surface of a B cell. 
     
     
         15 . The set of polypeptides according to  claim 1 , wherein
 (i) one of said antigens A1 and A2 is EpCAM and the other one is EGFR, HER2/neu, CD 10, VEGF-R or MDR;   (ii) one of said antigens A1 and A2 is MCSP and the other one is melanoferrin or EpCAM;   (iii) one of said antigens A1 and A2 is CA125 and the other one CD227;   (iv) one of said antigens A1 and A2 is CD56 and the other one is CD140b or GD3 ganglioside;   (v) one of said antigens A1 and A2 is EGFR and the other one is HER2;   (vi) one of said antigens A1 and A2 is PSMA and the other one is HER2;   (vii) one of said antigens A1 and A2 is Sialyl Lewis and the other one is EGFR;   (viii) one of said antigens A1 and A2 is CD44 and the other one is ESA, CD24, CD133, MDR or CD117;   (ix) one of said antigens A1 and A2 is CD34 and the other one is CD19, CD79a, CD2, CD7, HLA-DR, CD 13, CD 117, CD33 or CD 15;   (x) one of said antigens A1 and A2 is CD33 and the other one is CD19, CD79a, CD2, CD7, HLA-DR, CD13, CD117 or CD15;   (xi) one of said antigens A1 and A2 is MUC1 and the other one is CD10, CEA or CD57;   (xii) one of said antigens A1 and A2 is CD38 and the other one is CD138;   (xiii) one of said antigens A1 and A2 is CD 24 and the other one is CD29 or CD49f;   (xiv) one of said antigens A1 and A2 is carbonic anhydrase IX and the other one is aquaporin-2;   (xv) one of said antigens A1 and A2 is HLA-A2 and the other one is EpCAM;   (xvi) one of said antigens A1 and A2 is HLA-A2 and the other one is CD45;   (xvii) one of said antigens A1 and A2 is HLA-A2 and the other one is EGFR;   (xviii) one of said antigens A1 and A2 is HLA-A2 and the other one is Her2;   (xix) one of said antigens A1 and A2 is HLA-A2 and the other one is CEA;   (xx) one of said antigens A1 and A2 is EpCAM and the other one is CEA;   (xxi) one of said antigens A1 and A2 is CD45 and the other one is CD138;   (xxii) one of said antigens A1 and A2 is EGFR and the other one is CEA;   (xxiii) one of said antigens A1 and A2 is Her2 and the other one is CEA; or   (xxiv) one of said antigens A1 and A2 is CD19 and the other one is a clonotypic antibody on the surface of a B cell.   
     
     
         16 . The set of polypeptides according to  claim 1 , wherein said targeting moiety T1 and/or T2 comprises an immunoglobulin module; or wherein said targeting moiety T1 and/or T2 comprises an aptamer or a natural ligand of said antigen A1 or antigen A2, respectively. 
     
     
         17 . The set of polypeptides according to  claim 16 , wherein said targeting moiety T1 comprises an immunoglobulin module I1 comprising a V L  domain linked to a V H  domain or comprising a variable domain V H H of a llama antibody, camel antibody or shark antibody; and/or
 said targeting moiety T2 comprises an immunoglobulin module I2 comprising a V L  domain linked to a V H  domain or comprising a variable domain V H H of a llama antibody, camel antibody or shark antibody.   
     
     
         18 . The set of polypeptides according to  claim 17 , wherein said immunoglobulin module I1 comprises a scFv (single-chain variant fragment), a Fab or a F(ab′) 2  of an antibody or a complete antibody; and/or
 said immunoglobulin module I2 comprises a scFv (single-chain variant fragment), a Fab or a F(ab′) 2  of an antibody or a complete antibody. 
 
     
     
         19 .- 21 . (canceled) 
     
     
         22 . The set of polypeptides according to  claim 1 , wherein any one of said targeting moiety T1 and T2 comprises an allergen or substrate which binds to a clonotypic antibody on the surface of a B cell. 
     
     
         23 . The set of polypeptides according to  claim 1 , wherein said functional domain F is or comprises an immunoglobulin module, or a fluorescent molecule, or a molecule capable of mediating bioluminescence. 
     
     
         24 . The set of polypeptides according to  claim 23 , wherein said functional domain F is a Fv (variant fragment) or a scFv (single-chain variant fragment) of an antibody. 
     
     
         25 .- 28 . (canceled) 
     
     
         29 . The set of polypeptides according to  claim 1 , wherein said fragment F1 comprises a V L  domain of an antibody and said fragment F2 comprises a V H  domain of the same antibody; or wherein said fragment F1 comprises a V H  domain of an antibody and said fragment F2 comprises a V L  domain of the same antibody. 
     
     
         30 . The set of polypeptides according to  claim 1 , wherein said fragment F1 comprises a V L  domain of an anti-CD3, anti-His or anti-DIG antibody and said fragment F2 comprises a V H  domain of the same antibody, or wherein said fragment F1 comprises a V H  domain of an anti-CD3 anti-His or anti-DIG antibody and said fragment F2 comprises a V L  domain of the same antibody. 
     
     
         31 . (canceled) 
     
     
         32 . The set of polypeptides according to  claim 23 , wherein said immunoglobulin module comprises a V domain selected from the group consisting of:
 (i) a V domain of an anti-CD3 antibody comprising a V L  domain comprising SEQ ID NO: 2 and/or a V H  domain comprising SEQ ID NO: 1;   (ii) a V domain of an anti-CD3 antibody comprising a V L  domain comprising SEQ ID NO: 4 and/or a V H  domain comprising SEQ ID NO: 3;   (iii) a V domain of an anti-CD3 antibody comprising a V L  domain comprising SEQ ID NO: 6 and/or a V H  domain comprising SEQ ID NO: 5;   (iv) a V domain of an anti-CD3 antibody comprising a V L  domain comprising SEQ ID NO: 8 and/or a V H  domain comprising SEQ ID NO: 7;   (v) a V domain of an anti-CD3 antibody comprising a V L  domain comprising SEQ ID NO: 10 and/or a V H  domain comprising SEQ ID NO: 9;   (vi) a V domain of an anti-His antibody comprising a V L  domain comprising SEQ ID NO: 12 and/or a V H  domain comprising SEQ ID NO: 11; and   (vii) a V domain of an anti-DIG antibody comprising a V L  domain comprising SEQ ID NO: 14 and/or a V H  domain comprising SEQ ID NO: 30.   
     
     
         33 . The set of polypeptides according to  claim 1 , wherein any of the polypeptides P1 and P2 is or comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 114-129 and 197. 
     
     
         34 . A method of treating a patient who is suffering from cancer and/or a tumour or method of diagnosing a patient who is suffering from cancer and/or a tumour, said method comprising the step of administering to said patient an effective amount of the set of polypeptides according to  claim 1 . 
     
     
         35 .- 41 . (canceled) 
     
     
         42 . A nucleic acid molecule or a set of nucleic acid molecules encoding the set of polypeptides or one of the polypeptides of the set of polypeptides according to  claim 1 . 
     
     
         43 . The nucleic acid molecule or set of nucleic acid molecules according to  claim 42  comprising a nucleotide sequence as depicted in any one of SEQ ID NOS: 135-150 and 196. 
     
     
         44 .- 45 . (canceled) 
     
     
         46 . A kit comprising the set of polypeptides according to  claim 1 . 
     
     
         47 . A kit comprising the nucleic acid molecule or the set of nucleic acid molecules according to  claim 42 .