CYTOTOXIC IMMUNOGLOBULIN
The invention relates to a cytotoxic modular antibody with a molecular weight of up to 6OkD, specifically binding to a cell surface target with a binding affinity of Kd<10 −8 M, a method of producing such antibody and its use as a therapeutic.
1 . A cytotoxic immunoglobulin having a binding affinity for an erbB receptor of Kd less than 10 −8 M comprising a CH3 constant domain, wherein said CH3 constant domain specifically binds said erbB receptor and comprises at least one modified structural loop region, said modified structural loop region comprising at least three amino acid modifications with respect to a corresponding structural loop region of a wild type CH3 constant domain which does not bind said erbB receptor
wherein said cytotoxic immunoglobulin is produced by a process comprising the steps of:
(a) providing a library of modified CH3 constant domains, wherein three or more amino acid positions in one or more structural loops can be any amino acid,
(b) contacting said library with said erbB receptor,
(c) selecting a library member having:
(i) erbB receptor binding affinity of Kd less than 10 −8 M or IC 50 less than 10 −8 M and
(ii) cytotoxic activity selected from at least one of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDC), or apoptotic activity.
2 . The cytotoxic immunoglobulin produced according to claim 1 , wherein said modified structural loop region comprises at least four amino acid modifications in a single structural loop.
3 . The cytotoxic immunoglobulin produced according to claim 1 , wherein said modified structural loop region comprises at least three amino acid modifications at least two structural loops.
4 . The cytotoxic immunoglobulin produced according to claim 1 , wherein said cytotoxic immunoglobulin is capable of triggering at least one activity selected from the group consisting of: antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), apoptotic activity, and complement dependent cytotoxicity (CDC), upon binding to a cell displaying said cell surface target.
5 . The cytotoxic immunoglobulin produced according to claim 1 , wherein said library of step (a) is a CH2/CH3 oligomer library.
6 . The cytotoxic immunoglobulin produced according to claim 1 , wherein said library of step (a) is a CH3/CH3 oligomer library.
7 . A cytotoxic immunoglobulin produced according to claim 1 , further comprising the step of: (d) affinity maturation of said selected library member.
8 . A cytotoxic immunoglobulin produced according to claim 1 ,
wherein said three or more amino acid positions of step (a) comprise amino acids which are naturally occurring but foreign to the site of modification or are substitutes of naturally occurring amino acids.
9 . A cytotoxic immunoglobulin produced according to claim 1 , wherein said three or more amino acid positions of step (a) include positions selected from:
7 to 21, 25 to 39, 41 to 81, 83 to 85, 89 to 103 and 106 to 117,
wherein the numbering of the amino acid positions is according to the IMGT numbering scheme.
10 . A cytotoxic immunoglobulin produced according to claim 1 , wherein said cytotoxic immunoglobulin comprises a CH2/CH3 dimer.
11 . A cytotoxic immunoglobulin produced according to claim 9 , wherein said cytotoxic immunoglobulin comprises a CH2/CH3 dimer.
12 . A cytotoxic immunoglobulin produced according to claim 11 , wherein said CH2/CH3 dimer is an IgG1 isotype.
13 . A cytotoxic immunoglobulin produced according to claim 1 , wherein said cell surface target is selected from EGFR, Her2, Her2neu, HER3 and HER4.
14 . A cytotoxic immunoglobulin produced according to claim 9 , wherein said cell surface target is selected from EGFR, Her2, Her2neu, HER3 and HER4.
15 . A cytotoxic immunoglobulin produced according to claim 1 , wherein said cell surface target is Her2.
16 . A cytotoxic immunoglobulin produced according to claim 1 , wherein said CH3 constant domain comprising at least one modified structural loop region is of an isotype selected from the group consisting of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 and IgM.
17 . A cytotoxic immunoglobulin produced according to claim 1 , wherein said CH3 constant domain is a human IgG1 CH3 constant domain.
18 . A cytotoxic immunoglobulin produced according to claim 1 , wherein said antigen binding site specifically binds to Her2 and contains an amino acid sequence selected from the group consisting of SEQ ID numbers as listed in Table 4 and Table 5, contained in an EF and/or AB and/or CD structural loop.
19 . A cytotoxic immunoglobulin produced according to claim 1 , wherein said library is selected from the group consisting of a cellular and a non-cellular library.
20 . A cytotoxic immunoglobulin produced according to claim 19 , wherein said cellular library is selected from the group consisting of a yeast display library, a phage display library, a virus display library, and an insect display library.
21 . A cytotoxic immunoglobulin produced according to claim 19 , wherein said non-cellular library is selected from the group consisting of a ribosome display library, an mRNA display library and a nucleic acid display library.