Humanized and de-immunized antibodies
The invention relates to humanized and de-immunized antibodies that bind to an epitope at the N-terminus of pyroglutamated amyloid beta (Aβ N3pE) peptide and to preventive and therapeutic treatment of diseases and conditions that are related to accumulation and deposition of amyloid peptides, such as amyloidosis, a group of disorders and abnormalities associated with pyroglutamated amyloid peptide, like Alzheimer's disease, Down's syndrome, cerebral amyloid angiopathy and other related aspects. More specifically, it pertains to the use of monoclonal antibodies of the invention to bind pyroglutamated amyloid beta peptide in plasma, brain, and cerebrospinal fluid to prevent accumulation or to reverse deposition of Aβ N3pE within the brain and in various tissues in the periphery, and to alleviate amyloidosis. The present invention further pertains to diagnostic assays for the diagnosis of amyloidosis using the antibodies of the invention.
1. An antibody wherein said antibody comprises CDR regions:
(SEQ ID NO: 3)
VLCDR1: SSQSLLYSDGKTYLN;
(SEQ ID NO: 4)
VLCDR2: LVSKLDS;
and
(SEQ ID NO: 5)
VLCDR3: VQGTHFP
in the light chain and CDR regions:
(SEQ ID NO: 6)
VHCDR1: GYSFTGHTMN;
(SEQ ID NO: 7)
VHCDR2: LINPSDGVTRYNQKFQG;
and
(SEQ ID NO: 8)
VHCDR3: EAKREWDETY
in the heavy chain
wherein the antibody specifically binds a pyroglutamate carrying N-terminus of the Aβ N3pE epitope.
2. The antibody of claim 1 , wherein the variable part of the light chain of said antibody comprises the amino acid sequence:
(SEQ ID NO: 1)
DVVMTQSPLSLPVTLGQPASISCKSSQSLLYSDGKTYLNWFQQRPGQSPR
RLTYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHFP
FTFGGGTKVEIK.
3. The antibody of claim 1 , wherein the variable part of the heavy chain of said antibody comprises the amino acid sequence:
(SEQ ID NO: 2)
QVQLVQSGAEVVKPGASVKVSCKASGYSFTGHTMNWVRQAPGQGLEWMGL
INPSDGVTRYNQKFQGRVTITRDTSTTTVHMELTSLTSEDTATYYCTREA
KREWDETYWGQGTLVTVSS.
4. The antibody of claim 1 wherein the light chain comprises the amino acid sequence:
(SEQ ID NO: 17)
DVVMTQSPLSLPVTLGQPASISCKSSQSLLYSDGKTYLNWFQQRPGQSPR
RLTYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHFP
FTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC.
5. The antibody of claim 1 wherein the heavy chain comprises the amino acid sequence:
(SEQ ID NO: 18)
QVQLVQSGAEVVKPGASVKVSCKASGYSFTGHTMNWVRQAPGQGLEWMGL
INPSDGVTRYNQKFQGRVTITRDTSTTTVHMELTSLTSEDTATYYCTREA
KREWDETYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.
6. The antibody of claim 1 wherein the heavy chain comprises the amino acid sequence:
(SEQ ID NO: 19)
QVQLVQSGAEVVKPGASVKVSCKASGYSFTGHTMNWVRQAPGQGLEWMGL
INPSDGVTRYNQKFQGRVTITRDTSTTTVHMELTSLTSEDTATYYCTREA
KREWDETYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.
7. The antibody according to claim 1 wherein the antibody binds specifically to an epitope selected from the group consisting of
pEFRHDSGYEVHHQKLV,
(SEQ ID NO: 23)
pEFRHDSGYEVHHQKL,
(SEQ ID NO: 24)
pEFRHDSGYEVHHQK,
(SEQ ID NO: 25)
pEFRHDSGYEVHHQ,
(SEQ ID NO: 26)
pEFRHDSGYEVHH,
(SEQ ID NO: 27)
pEFRHDSGYEVH,
(SEQ ID NO: 28)
pEFRHDSGYEV,
(SEQ ID NO: 29)
pEFRHDSGYE,
(SEQ ID NO: 30)
pEFRHDSGY,
(SEQ ID NO: 31)
pEFRHDSG,
(SEQ ID NO: 32)
pEFRHDS,
(SEQ ID NO: 33)
pEFRHD
(SEQ ID NO: 34)
pEFRH,
(SEQ ID NO: 35)
and
pEFR.
(SEQ ID NO: 36)
8. The antibody according to claim 1 wherein the antibody binds to an Aβ N3pE variant, wherein the Aβ N3pE variant is defined as pE-Aβ 3-x , and wherein x is defined as an integer between 19 and 42.
9. The antibody of claim 8 , wherein the Aβ N3pE variant is selected from:
pE-Aβ 3-38 ,
pE-Aβ 3-40 ,
pE-Aβ 3-42 .
10. The antibody according to claim 1 wherein said antibody does not bind to epitopes that do not carry a pyroglutamate at the N-terminus.
11. A pharmaceutical composition comprising the antibody according to claim 1 .
12. The pharmaceutical composition of claim 11 , further comprising a further biologically active substance and/or a pharmaceutically acceptable carrier and/or a diluent and/or an excipient.
13. The pharmaceutical composition of claim 12 , wherein said further biologically active substance is selected from the group consisting of neutron-transmission enhancers, psychotherapeutic drugs, acetylcholine esterase inhibitors, calcium-channel blockers, biogenic amines, benzodiazepine tranquillizers, acetylcholine synthesis enhancers, acetylcholine storage enhancers, acetylcholine release enhancers, acetylcholine postsynaptic receptor agonists, monoamine oxidase-A inhibitors, monoamine oxidase-B inhibitors, N-methyl-D-aspartate glutamate receptor antagonists, non-steroidal anti-inflammatory drugs, antioxidants, and serotonergic receptor antagonists.
14. The pharmaceutical composition of claim 12 , wherein said further biologically active substance is selected from the group consisting of compounds effective against oxidative stress, anti-apoptotic compounds, metal chelators, inhibitors of DNA repair, 3-amino-1-propanesulfonic acid (3 APS), 1,3-propanedisulfonate (1,3PDS), α-secretase activators, β-secretase inhibitors, γ-secretase inhibitors, tau proteins, neurotransmitter, β-sheet breakers, attractants for amyloid beta clearing/depleting cellular components, inhibitors of N-terminal truncated amyloid beta, anti-inflammatory molecules, cholinesterase inhibitors (ChEIs), MI agonists, amyloid modifying drugs, tau modifying drugs, nutritive supplements, cholinesterase inhibitors (ChEIs), memantine, and glutaminyl cyclase inhibitors.