Bispecific anti-cxcr7 immunoglobulin single variable domains

Abstract

The present invention relates to particular polypeptides, nucleic acids encoding such polypeptides; to methods for preparing such polypeptides; to host cells expressing or capable of expressing such polypeptides; to compositions and in particular to pharmaceutical compositions that comprise such polypeptides, for prophylactic, therapeutic or diagnostic purposes. In particular, the present invention provides immunoglobulin single variable domains inhibiting CXCR7 mediated tumour growth.

1 . A construct comprising at least one immunoglobulin single variable domain (ISVD) that binds to and/or recognizes amino acid residue M33, and optionally amino acid residue V32 and/or amino acid residue M37 in CXCR7 (SEQ ID NO: 1) and at least one ISVD that binds to and/or recognizes amino acid residue W19, and optionally S23 and/or D25 of CXCR7 (SEQ ID NO: 1). 
     
     
         2 . The construct according to  claim 1  for use as a medicament to reduce tumour growth and/or to treat cancer, preferably head and neck cancer or GBM. 
     
     
         3 . An immunoglobulin single variable domain that can specifically displace SDF-1 and/or I-TAC on human CXCR7 (SEQ ID NO: 1) with an average Ki of less than 100 nM and an average SDF-1 and I-TAC displacement of 50% or more. 
     
     
         4 - 8 . (canceled) 
     
     
         9 . An immunoglobulin single variable domain that can bind human CXCR7 (SEQ ID NO: 1) with a Kd of less than 50 nM. 
     
     
         10 . An immunoglobulin single variable domain selected from the group consisting of
 immunoglobulin single variable domains that bind to and/or recognize amino acid residue M33, and optionally amino acid residue V32 and/or amino acid residue M37 in CXCR7 (SEQ ID NO: 1); and   immunoglobulin single variable domains that bind to and/or recognize amino acid residue W19, and optionally amino acid residue S23 and/or amino acid residue D25 of CXCR7 (SEQ ID NO: 1).   
     
     
         11 . (canceled) 
     
     
         12 . The immunoglobulin single variable domain according to claim  5  for use as a medicament to reduce tumour growth and/or to treat cancer, preferably head and neck cancer or GBM. 
     
     
         13 . The immunoglobulin single variable domain of  claim 3 , wherein the immunoglobulin single variable domain is selected from the group consisting of:
 immunoglobulin variable domains that comprise an amino acid sequence with the formula 1
   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1);
 
   wherein FR1 to FR4 refer to framework regions 1 to 4 and are framework regions of an immunoglobulin single variable domain; and   wherein CDR1 is chosen from the group consisting of:
 a) the immunoglobulin single variable domain of SEQ ID NO: 9, 
 b) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 9, 
 c) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 9, 
   and wherein CDR2 is chosen from the group consisting of:
 d) the immunoglobulin single variable domain of SEQ ID NO: 19; 
 e) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 19; 
   f) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 19;   and wherein CDR3 is chosen from the group consisting of:
 g) the immunoglobulin single variable domain of SEQ ID NO: 29; 
 h) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 29; 
 i) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 29; 
   immunoglobulin single variable domains that comprise an amino acid sequence with the formula 1
   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1);
 
   wherein FR1 to FR4 refer to framework regions 1 to 4 and are framework regions of an immunoglobulin single variable domain; and   wherein CDR1 is chosen from the group consisting of:
 a) the immunoglobulin single variable domain of SEQ ID NO: 10, 
 b) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 10, 
 c) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 10, 
   and wherein CDR2 is chosen from the group consisting of:
 d) the immunoglobulin single variable domain of SEQ ID NO: 20; 
 e) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 20; 
 f) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 20; 
   and wherein CDR3 is chosen from the group consisting of:
 g) the immunoglobulin single variable domain of SEQ ID NO: 30; 
 h) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 30; 
 i) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 30; 
   immunoglobulin single variable domains that comprise an amino acid sequence with the formula 1
   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1);
 
   wherein FR1 to FR4 refer to framework regions 1 to 4 and are framework regions of an immunoglobulin single variable domain; and   wherein CDR1 is chosen from the group consisting of:
 a) the immunoglobulin single variable domain of SEQ ID NO: 11, 
 b) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 11, 
 c) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 11, 
   and wherein CDR2 is chosen from the group consisting of:
 d) the immunoglobulin single variable domain of SEQ ID NO: 21; 
 e) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 21; 
 f) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 21; 
   and wherein CDR3 is chosen from the group consisting of:
 g) the immunoglobulin single variable domain of SEQ ID NO: 31; 
 h) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 31; 
 i) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 31; 
   immunoglobulin single variable domains that comprise an amino acid sequence with the formula 1
   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1);
 
   wherein FR1 to FR4 refer to framework regions 1 to 4 and are framework regions of an immunoglobulin single variable domain; and   wherein CDR1 is chosen from the group consisting of:
 a) the immunoglobulin single variable domain of SEQ ID NO: 12, 
 b) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 12, 
 c) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 12, 
   and wherein CDR2 is chosen from the group consisting of:
 d) the immunoglobulin single variable domain of SEQ ID NO: 22; 
 e) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 22; 
 f) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 22; 
   and wherein CDR3 is chosen from the group consisting of:
 g) the immunoglobulin single variable domain of SEQ ID NO: 32; 
 h) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 32; 
 i) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 32; 
   immunoglobulin single variable domains that comprise an amino acid sequence with the formula 1
   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1);
 
   wherein FR1 to FR4 refer to framework regions 1 to 4 and are framework regions of an immunoglobulin single variable domain; and   wherein CDR1 is chosen from the group consisting of:
 a) the immunoglobulin single variable domain of SEQ ID NO: 13, 
 b) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 13, 
 c) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 13, 
   and wherein CDR2 is chosen from the group consisting of:
 d) the immunoglobulin single variable domain of SEQ ID NO: 23; 
 e) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 23; 
 f) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 23; 
   and wherein CDR3 is chosen from the group consisting of:
 g) the immunoglobulin single variable domain of SEQ ID NO: 33; 
 h) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 33; 
 i) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 33; 
   immunoglobulin single variable domains that comprise an amino acid sequence with the formula 1
   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1);
 
   wherein FR1 to FR4 refer to framework regions 1 to 4 and are framework regions of an immunoglobulin single variable domain; and   wherein CDR1 is chosen from the group consisting of:
 a) the immunoglobulin single variable domain of SEQ ID NO: 93, 
 b) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 93, 
 c) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 93; 
   and wherein CDR2 is chosen from the group consisting of:
 d) the immunoglobulin single variable domain of SEQ ID NO: 95; 
 e) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 95; 
 f) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 95; 
   and wherein CDR3 is chosen from the group consisting of:
 g) the immunoglobulin single variable domain of SEQ ID NO: 97; 
 h) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 97; 
 i) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 97; 
   immunoglobulin single variable domains that comprise an amino acid sequence with the formula 1
   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1);
 
   wherein FR1 to FR4 refer to framework regions 1 to 4 and are framework regions of an immunoglobulin single variable domain; and   wherein CDR1 is chosen from the group consisting of:
 a) the immunoglobulin single variable domain of SEQ ID NO: 107, 
 b) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 107, 
 c) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 107, 
   and wherein CDR2 is chosen from the group consisting of:
 d) the immunoglobulin single variable domain of SEQ ID NO: 115; 
 e) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 115; 
 f) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 115; 
   and wherein CDR3 is chosen from the group consisting of:
 g) the immunoglobulin single variable domain of SEQ ID NO: 123; 
 h) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 123; 
 i) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 123; 
   immunoglobulin single variable domains that comprise an amino acid sequence with the formula 1
   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1);
 
   wherein FR1 to FR4 refer to framework regions 1 to 4 and are framework regions of an immunoglobulin single variable domain; and   wherein CDR1 is chosen from the group consisting of:
 a) the immunoglobulin single variable domain of SEQ ID NO: 108, 
 b) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 108, 
 c) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 108, 
   and wherein CDR2 is chosen from the group consisting of:
 d) the immunoglobulin single variable domain of SEQ ID NO: 116; 
 e) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 116; 
 f) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 116; 
   and wherein CDR3 is chosen from the group consisting of:
 g) the immunoglobulin single variable domain of SEQ ID NO: 124; 
 h) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 124; 
 i) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 124; and 
   immunoglobulin single variable domains that comprise an amino acid sequence with the formula 1
   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1);
 
   wherein FR1 to FR4 refer to framework regions 1 to 4 and are framework regions of an immunoglobulin single variable domain; and   wherein CDR1 is chosen from the group consisting of:
 a) the immunoglobulin single variable domain of SEQ ID NO: 110, 
 b) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 110, 
 c) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 110, 
   and wherein CDR2 is chosen from the group consisting of:
 d) the immunoglobulin single variable domain of SEQ ID NO: 118; 
 e) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 118; 
 f) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 118; 
   and wherein CDR3 is chosen from the group consisting of:
 g) the immunoglobulin single variable domain of SEQ ID NO: 126; 
 h) immunoglobulin single variable domains that have at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 126; 
 i) immunoglobulin single variable domains that have 3, 2, or 1 amino acid difference with the immunoglobulin single variable domain of SEQ ID NO: 126. 
   
     
     
         14 - 21 . (canceled) 
     
     
         22 . The immunoglobulin single variable domain according to  claim 13 , wherein the framework regions (FRs) have a sequence identity of more than 80% with the FRs of SEQ ID NOs: 4 to 8, 92, 103, 104 or 106 (FR1), 14 to 18, 94, 111, 112 or 114 (FR2), 24 to 28, 96, 119, 120 or 122 (FR3), and/or 34 to 38, 98, 127, 128 or 130 (FR4). 
     
     
         23 . A polypeptide comprising an immunoglobulin single variable domain of  claim 3 . 
     
     
         24 . The polypeptide according to  claim 23 , wherein the immunoglobulin single variable domain is selected from the group consisting of immunoglobulin single variable domains that have an amino acid sequence with a sequence identity of more than 80% with the immunoglobulin single variable domains of SEQ ID NOs: 39 to 43, 91 or 99-102. 
     
     
         25 . The polypeptide according to  claim 23  and additionally comprising at least one human serum albumin binding immunoglobulin single variable domain and optionally comprising a linker selected from the group of linkers with SEQ ID NOs: 49 to 58. 
     
     
         26 . The polypeptide according to  claim 23  and additionally comprising ALB8 (SEQ ID NO: 2), and optionally comprising a linker selected from the group of linkers with SEQ ID NOs: 49 to 58. 
     
     
         27 . The polypeptide according to  claim 23 , wherein the polypeptide is selected from the group consisting of polypeptides that have an amino acid sequence with a sequence identity of more than 80% with the polypeptides of SEQ ID NOs: 44 to 48, 78 to 89 and 131 to 140. 
     
     
         28 . A construct chosen from the group consisting of:
 constructs comprising at least two ISVDs that bind to and/or recognize amino acid residue W19, and optionally amino acid residue S23 and/or amino acid residue D25 of CXCR7 (SEQ ID NO: 1), wherein said at least two ISVDs can be the same or different;   constructs comprising at least two ISVDs that bind to and/or recognize amino acid residue M33, and optionally amino acid residue V32 and/or amino acid residue M37 in CXCR7 (SEQ ID NO: 1), wherein said at least two ISVDs can be the same or different;   constructs comprising at least one group 1 ISVD and at least one group 2 ISVD;   constructs comprising at least one group 1 ISVD and at least one group 3 ISVD;   constructs comprising at least one group 2 ISVD and at least one group 3 ISVD; and   constructs comprising at least one 01C10-like sequence and at least one 14G03-like sequence.   
     
     
         29 . The construct according to  claim 28  for use as a medicament to reduce tumour growth and/or to treat cancer, preferably head and neck cancer or GBM. 
     
     
         30 . A nucleic acid sequence encoding for an immunoglobulin single variable domain according to  claim 3 . 
     
     
         31 . A pharmaceutical composition comprising an immunoglobulin single variable domain according to  claim 3  and optionally a pharmaceutically acceptable excipient. 
     
     
         32 . An immunoglobulin single variable domain according to  claim 3  for use in cancer, preferably head or neck cancer, GBM, inflammatory diseases, rheumatoid arthritis and/or multiple sclerosis. 
     
     
         33 - 34 . (canceled) 
     
     
         35 . Method for producing an immunoglobulin single variable domain, said method at least comprising the steps of:
 a) expressing, in a suitable host cell or host organism or in another suitable expression system, a nucleic acid or nucleotide sequence according to  claim 30 ; optionally followed by:   b) isolating and/or purifying the immunoglobulin single variable domain.