Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
  • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/32—Immunoglobulins specific features characterized by aspects of specificity or valency specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/775—Apolipopeptides
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/28—Neurological disorders
  • G01N2800/2814—Dementia; Cognitive disorders
  • G01N2800/2821—Alzheimer

Definitions

• apolipoprotein E to methods of producing such an antibody or antigen binding portion thereof, and to therapeutic and diagnostic uses thereof.
• a known genetic risk factor for late-onset AD is the APOE e4 allele, although its precise role in the disease remains unclear.
• the APOE gene encodes apolipoprotein E (ApoE), which is a glycoprotein of 35 kDa
• ApoE exists in three different isoforms, ApoE2, ApoE3 and ApoE4, of which ApoE3 is the most common, ApoE2 has been shown to decrease the risk of AD, and ApoE4 increases it.
• the present invention provides an antibody or antigen binding portion thereof that binds to a fragment of apolipoprotein E (ApoE), wherein the fragment has
• the ApoE fragment consists of the amino acid sequence of any one of SEQ ID NOs: 1 , 2 or 3.
• the present invention provides methods of producing antibodies or antigen binding portions thereof, the methods comprising a step of immunizing a host mammal with a peptide immunogen comprising an N-terminal amino acid sequence selected from the group consisting of LAGQPL (SED ID NO:4), AGQPLQ (SEQ ID NO:5), GQPLQE (SEQ ID NO:6), LAGQPLQ (SEQ ID NO:7), AGQPLQE (SEQ ID NO:8) and LAGQPLQE (SEQ ID NO:9).
• a peptide immunogen comprising an N-terminal amino acid sequence selected from the group consisting of LAGQPL (SED ID NO:4), AGQPLQ (SEQ ID NO:5), GQPLQE (SEQ ID NO:6), LAGQPLQ (SEQ ID NO:7), AGQPLQE (SEQ ID NO:8) and LAGQPLQE (SEQ ID NO:9).
• the present invention provides antibodies, antigen binding portions thereof and/or pharmaceutical compositions comprising the same for use in methods of treatment or for use in methods of detection or diagnosis as described herein.
• the invention can be further understood with reference to the following illustrative embodiments.
• An antibody or antigen binding portion thereof that binds to a fragment of apolipoprotein E (ApoE), wherein the fragment has
• GQPLQE apolipoprotein E
• a method of producing an antibody or an antigen binding portion thereof comprising a step of immunizing a suitable host mammal with a peptide immunogen comprising an N-terminal amino acid sequence selected from the group consisting of LAGQPL (SED ID NO:4), AGQPLQ (SEQ ID NO:5), GQPLQE (SEQ ID NO:6), LAGQPLQ (SEQ ID NO:7), AGQPLQE (SEQ ID NO:8) and LAGQPLQE (SEQ ID NO:9).
• a peptide immunogen comprising an N-terminal amino acid sequence selected from the group consisting of LAGQPL (SED ID NO:4), AGQPLQ (SEQ ID NO:5), GQPLQE (SEQ ID NO:6), LAGQPLQ (SEQ ID NO:7), AGQPLQE (SEQ ID NO:8) and LAGQPLQE (SEQ ID NO:9).
• N-terminal amino acid sequence is GQPLQE (SEQ ID NO:6).
• N-terminal amino acid sequence is selected from LAGQPL (SED ID NO:4), LAGQPLQ (SEQ ID NO:7) and LAGQPLQE (SEQ ID NO:9).
• N-terminal amino acid sequence is selected from AGQPLQ (SEQ ID NO:5) and AGQPLQE (SEQ ID NO:8).
• - CDR-H1 selected from the group consisting of SEQ ID NO: 10, 15, 18 and 21 ;
• VL variable light chain domain
• - CDR-L3 selected from the group consisting of SEQ ID NO: 26, 28, 30 and 33.
• - CDR-H2 selected from the group consisting of SEQ ID NO: 1 1 , 13, 16, 19 and 22; and - CDR-H3 selected from the group consisting of SEQ ID NO: 12, 14, 17, 20 and 23.
• CDR-H1 comprising or consisting of SEQ ID NO: 10 (SYAMS);
• CDR-H2 comprising or consisting of SEQ ID NO: 1 1
• CDR-H3 comprising or consisting of SEQ ID NO: 12
• CDR-L1 comprising or consisting of SEQ ID NO: 24
• CDR-L3 comprising or consisting of SEQ ID NO: 26 (FQGSHLPYT).
• CDR-L2 comprising or consisting of SEQ ID NO: 25 (KVSNRFS);
• CDR-FI2 comprising or consisting of SEQ ID NO: 16
• CDR-FI1 comprising or consisting of SEQ ID NO: 18 (RYAMS);
• CDR-FI3 comprising or consisting of SEQ ID NO: 12
• CDR-L1 comprising or consisting of SEQ ID NO: 31
• CDR-L3 comprising or consisting of SEQ ID NO: 28 (FQGSHVPYT). 28.
• CDR-H3 comprising or consisting of SEQ ID NO: 20
• CDR-L1 comprising or consisting of SEQ ID NO: 31
• CDR-L3 comprising or consisting of SEQ ID NO: 28 (FQGSHVPYT).
• CDR-L3 comprising or consisting of SEQ ID NO: 33 (LQGSFIIPFT).
• VFI heavy chain variable domain
• VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 36 or an amino acid sequence having at least 80%, 90%,
• VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 40, and a VL domain comprising or consisting of the amino acid sequence of SEQ ID NO: 41 ;
• CDR-H1 comprising or consisting of SEQ ID NO: 59;
• CDR-H2 comprising or consisting of SEQ ID NO: 60;
• CDR-H3 comprising or consisting of SEQ ID NO: 61 ;
• CDR-H2 comprising or consisting of SEQ ID NO: 63;
• CDR-H1 comprising or consisting of SEQ ID NO: 65;
• CDR-H3 comprising or consisting of SEQ ID NO: 67;
• CDR-L2 comprising or consisting of SEQ ID NO: 77;
• variable heavy chain domain VH
• VL variable light chain domain
• VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 86 or an amino acid sequence having at least 80%, 90%,
• VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 90 or an amino acid sequence having at least 80%, 90%,
• VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 84, and a VL domain comprising or consisting of the amino acid sequence of SEQ ID NO: 85;
• - CDR-H1 selected from the group consisting of SEQ ID NO: 62, 94 and 97;
• CDR-H1 comprising or consisting of SEQ ID NO: 94;
• CDR-L1 comprising or consisting of SEQ ID NO: 105;
• CDR-L2 comprising or consisting of SEQ ID NO: 106;
• VL light chain variable domain
• VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 1 11 and a VL domain comprising or consisting of the amino acid sequence of SEQ ID NO: 1 12;
• VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 1 13 and a VL domain comprising or consisting of the amino acid sequence of SEQ ID NO: 1 14;
• AD Alzheimer’s disease
• MCI mild cognitive impairment
• D dementia with Lewy body
• Down’s syndrome hereditary cerebral hemorrhage with amyloidosis (Dutch type), cerebral amyloid angiopathy, Parkinson’s disease, and cataract due to amyloid beta deposition.
• AD Alzheimer’s disease
• MCI mild cognitive impairment
• D dementia with Lewy body
• Down’s syndrome hereditary cerebral hemorrhage with amyloidosis (Dutch type), cerebral amyloid angiopathy, Parkinson’s disease, and cataract due to amyloid beta deposition.
• Figure 9 shows the result of LysC cleavage site analysis of the ApoE sequence as described in Example 4.
• Figure 18 shows a sample of binding interactions for recombinant antibodies against the G200 neo-epitope, characterized via bio-layer interferometry as described in Example 12.
• Figure 27 shows the binding of purified monoclonal antibodies against the L198 neo-epitope to a brain extract from an Alzheimer’s disease patient either by (A) direct Western blot or by (C) I P/Western blot, as described in Example 15; (B) shows re-staining of the Western blot membrane with a polyclonal anti-ApoE antibody, demonstrating staining of full-length ApoE.
• Another object of the invention is to enable the diagnosis of AD and other neurodegenerative disorders via detection of ApoE fragments
• Another object of the invention is to provide antibodies, or antigen binding portions thereof, having a novel and useful binding specificity.
• the antibodies and antigen binding portions thereof of the first aspect bind selectively to the ApoE fragments described herein.
• the term“bind selectively” refers to the preferential binding of the antibody or antigen binding portion thereof to the ApoE fragment target.
• the antibodies and antigen binding portions thereof of the first aspect do not bind to full-length apolipoprotein E, particularly full-length human apolipoprotein E.
• the antibodies and antigen binding portions of the first aspect bind to neo-epitopes at the N-terminus of the ApoE fragments described herein.
• the antibody or antigen binding portion thereof binds to an epitope comprising amino acid residues 200-205 in full-length apolipoprotein E (GQPLQE).
• the antibody or antigen binding portion thereof binds to an epitope comprising amino acid residues 199-204 in full- length apolipoprotein E (AGQPLQ).
• the antibody or antigen binding portion thereof binds to an epitope comprising amino acid residues 199-205 in full-length apolipoprotein E (AGQPLQE).
• embodiments of the antibody or antigen binding portion thereof of the first and third aspects of the disclosure may be characterized by specific amino acid sequences in the regions determining its binding capability, such as the CDRs of the variable light and variable heavy chains, or indeed the entire variable light and/or heavy chain domains or regions.
• specific amino acid sequences are provided herein for the specific antibodies generated as described in Examples 9-18. It is contemplated that the specific sequence information provided for the generated antibodies enables the skilled person to define combinations and variations of these sequences within the scope of the invention.
• the antibody or antigen binding portion thereof comprises the six CDRs CDR-H1 / CDR-H2 / CDR-H3 / CDR-L1 / CDR- L2 / CDR-L3 selected from the sequences listed above, in any combination thereof.
• the antibody or antigen binding portion thereof comprises the following three CDRs in any combination of CDR-H1 / CDR- H2 / CDR-H3, e.g. in a heavy chain variable region when present:
• the antibody or antigen binding portion thereof comprises a heavy chain variable region (VH) sequence selected from the group consisting of SEQ ID NOs: 82, 84, 86, 88 and 90, and sequences having at least 70 % identity thereto.
• VH heavy chain variable region
• the combinations of VH/VL are those present in the antibodies exemplified in Examples 9-18 (see Tables 3, 7 and 12 in particular).
• antigen binding portions include, but are not limited to: (1 ) a Fab fragment, which is a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) a Fab’ fragment, which is a Fab fragment with the heavy chain hinge region, (3) a F(ab’)2 fragment, which is a dimer of Fab’ fragments joined by the heavy chain hinge region, for example linked by a disulfide bridge at the hinge region; (4) an Fc fragment; (5) an Fv fragment, which is the minimum antibody fragment having the VL and VH domains of a single arm of an antibody; (6) a single chain Fv (scFv) fragment, which is a single polypeptide chain in which the VH and VL domains of an scFv are linked by a peptide linker; (7) an (scF
• an antibody or antigen binding portion thereof according to the first or third aspect, or a pharmaceutical composition according to the fourth aspect, for use as a diagnostic agent for use as a diagnostic agent.
• Such diseases or conditions include but are not limited to Alzheimer’s disease (AD), mild cognitive impairment (MCI), dementia with Lewy body, Down’s syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); as well as other diseases which are based on or associated with amylogenic proteins, such as cerebral amyloid angiopathy, Parkinson’s disease, and cataract due to amyloid beta deposition.
• AD Alzheimer’s disease
• MCI mild cognitive impairment
• dementia with Lewy body Down’s syndrome
• Dutch type hereditary cerebral hemorrhage with amyloidosis
• other diseases which are based on or associated with amylogenic proteins such as cerebral amyloid angiopathy, Parkinson’s disease, and cataract due to amyloid beta deposition.
• This example describes the hcmcgenizaticn cf human brain tissues and the fcllcwing Western blct analysis cf ApcE fragments frcm brain extracts in Radic-lmmuncprecipitaticn Assay (RIPA) buffer with 2% scdium dcdecyl sulfate (SDS).
• RIPA Radic-lmmuncprecipitaticn Assay
• vector-transfected cells were collected for Western blot analysis or seeded again at 2.0 c 10 4 cells/well in a Seahorse XF96 cell culture microplate (Agilent Technologies) 4 hours before mitochondrial respiration measurement.
• FCCP carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone
• the desired antigens were transiently expressed using the Expi293 system (Expi293 cells and ExpiFectamineTM 293 reagent; Thermo Fisher Scientific) and designed to be secreted into the supernatant.
• hybridoma selection medium D (StemCell Technologies), containing HAT for selection.
• G200 N-terminal neo epitope peptide (ApoE sequence 200-205 incorporated in SEQ ID NO:50) coupled to BSA, purified recombinant C-terminal ApoE fragment G200-HIS (SEQ ID NO:48) and recombinant full-length ApoE4 (SEQ ID NO:45).
• Hybridoma supernatants were diluted 2-fold (dilution buffer: PBS with 0.1 % BSA and 0.05 % TWEEN®-20) and screened against binding to G200-HIS fragment.
• “positive” wells were selected based on OD-values of >2 and the presence of clone/s.
• the identified positive clones were then subjected to positive and negative screenings using the same ELISA protocol, with G200-HIS fragment (SEQ ID NO:48), G200- peptide (SEQ ID NO:50) coupled to BSA, and ApoE4 full-length protein (SEQ ID NO:45) as coat for the plates.
• Antibodies that bind selectively to the N-terminal neo-epitope starting at amino acid G200 of the ApoE protein were generated by immunizations using an ApoE specific sequence consisting of the six first amino acids following the N-terminal in the 200-299 ApoE fragment.
• the shortness of the immunization peptide was considered necessary in order to enable generation of antibodies that bind selectively to the N-terminal neo-epitope starting at amino acid G200 of the ApoE protein, without any binding to the linear epitope found in full- length ApoE protein.
• mice were sacrificed and the spleens were collected and used for hybridoma generation.
• Detection antibody HRP-conjugated anti-mouse IgG, Southern Biotech, cat. no. 1030-05, diluted 1/10000 in dilution buffer
• 50 mI/well 50 mI/well of K-Blue® aqueous substrate (Neogen) were added, and the reaction was stopped after 5-15 min with 50 mI/well of 0.5 M H2SO4.
• the optical density at 450 nm was read using an ELISA reader (Tecan). The optical density was plotted against the antibody concentration to generate concentration-response curves ( Figure 14).
• HEK293 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 6-14 days. An appropriate volume of cells were transfected with the aim of obtaining 2 mg of purified antibody.
• the monoclonal antibodies 4E6, 7B10, 7C7, 17G4, 23D5 and 28F2 0 were selected for production as recombinant lgG2c antibodies, whereas 21 C3 was not produced (because of the sequence redundancy with 4E6). All recombinant antibodies were successfully produced and purified to a final concentration of 1 mg/ml. Antibody purity, as defined by SDS-PAGE, was >98 % for all antibodies.
• a second inhibition ELISA with the ApoE peptides conjugated to BSA was run, in which the starting concentration of the antigens in solution was increased 10-fold. i.e. the starting concentration of the N-terminal neo-epitope of synthetic ApoE peptides conjugated to BSA and starting at amino acid L198 (SEQ ID NO:52), A199 (SEQ ID NO:51 ) or G200 (SEQ ID NO:50), or to BSA-conjugated negative control peptide (SEQ ID NO:53), was 10000 ng/ml.
• Membranes were washed and incubated for 1 h at room temperature with the detection antibody anti-mouse-800CW (LI-COR, cat. no. 925-32210) diluted 1 :25000 in Intercept ® PBS Blocking Buffer (LI-COR) with 0.1 % TWEEN®-20. Membranes were washed and images acquired using Odyssey ® FC (LI- COR).
• LI-COR detection antibody anti-mouse-800CW
• I-COR Intercept ® PBS Blocking Buffer
• the membranes were re-stained over night with a polyclonal anti-ApoE antibody (Calbiochem, cat. no. 178479; immunogen ApoE aa 1 -299), diluted 1 :2000 in Intercept ® PBS Blocking Buffer (LI-COR) with 0.1 % TWEEN®-20.
• a polyclonal anti-ApoE antibody Calbiochem, cat. no. 178479; immunogen ApoE aa 1 -299
• diluted 1 :2000 in Intercept ® PBS Blocking Buffer (LI-COR) with 0.1 % TWEEN®-20 Membranes were washed and incubated for 1 h at room temperature with detection antibody anti-goat-680RD (LI-COR, cat. no. 925-68074) diluted 1 :25000 in Intercept ® PBS Blocking Buffer (LI-COR) with 0.1 % TWEEN®-20.
• MI-COR detection antibody anti-goat-680RD
• the immunogen used in this experiment was designed to incorporate one of the N-terminal neo-epitopes of the neurotoxic ApoE fragment identified in Examples 1 -7.
• the immunogen comprised the amino acid residues corresponding to amino acid residues 198-205 in full-length ApoE. This N-terminal sequence was coupled C-terminally to a 6-aminocaproic acid linker (Acp; also denoted
• peptides were AGQPLQ-Acp-C (SEQ ID NO:51 , prepared by Innovagen AB and delivered at 96.7 % purity), GQPLQE-Acp-C (SEQ ID NO:50, prepared by Innovagen AB and delivered at 95.5 % purity) and the negative control peptide AATVGSLAGQPLQER-Acp-C (SEQ ID NO:53, prepared by Innovagen AB and delivered at 97.8 % purity).
• Antibodies were produced from these clones and purified to generate purified monoclonal antibodies (Innovagen AB), and the clones were sent for sequencing (Absolute Antibodies). Results
• Antibodies that bind selectively to the N-terminal neo-epitope starting at amino acid L198 of the ApoE protein were generated by immunizations using an ApoE specific sequence consisting of the eight first amino acids following the N-terminal in the 198-299 ApoE fragment. The shortness of the
• Hvbridoma sequencing Hybridoma clones with a demonstrated selectivity for the N-terminal neo-epitope of ApoE fragment starting at amino acid L198, in addition to a demonstrated binding to human target in brain 5 extracts from Alzheimer’s disease, were sequenced.
• the antibodies were isotyped based on their sequences, and their respective sub-class and light chain are summarized in Table 9 below.
• This example describes the characterization of the purified monoclonal antibodies described in Examples 13 and 14 by various methods, including direct ELISA, inhibition ELISA, surface plasmon resonance, Western blot on human brain extract and immunoprecipitation on human brain extract.
• BSA- conjugated negative control peptide SEQ ID NO:52, 51 , 50 and 53, respectively.
• the mix was added to a microtiter plate coated with the BSA-coupled L198 synthetic ApoE peptide. If the purified monoclonal antibody binds to any of the antigens in the pre-incubation step (the synthetic ApoE peptides), the antibody is prevented from binding to the synthetic L198 ApoE peptide immobilized on the microtiter plate. This leads to inhibition of the ELISA detection signal.
• IP Immunoprecipitation
• ApoE-derived peptides were prepared which incorporated the other two identified putative N-terminal neo epitopes of the neurotoxic ApoE fragment, as well as a negative control peptide without any of the identified neo-epitopes.
• These peptides were LAGQPL-Acp-C (SEQ ID NO:52, prepared by Innovagen AB and delivered at 95.2 % purity), GQPLQE-Acp-C (SEQ ID NO:50, prepared by Innovagen AB and delivered at 95.5 % purity) and the negative control peptide
• Hybridoma sequencing Hybridoma clones with a demonstrated selectivity for the N-terminal neo-epitope of ApoE fragment starting at amino acid A199, in addition to a demonstrated binding to human target in brain extracts from Alzheimer’s disease, were sequenced. The following hybridoma clones were sequenced: 36A12, 38G9, 63F6 and 67G3. The amino acid sequences of the entire antibodies were obtained. Amino acid sequences obtained for the respective variable heavy (VH) and variable light (VL) chains are given in Table 12 below.
• CDRs complementarity determining regions
• This example describes the characterization of the purified monoclonal antibodies described in Examples 16 and 17 by various methods, including direct ELISA, inhibition ELISA, surface plasmon resonance and Western blot on human brain extract. Materials and methods
• the screening was performed according to standard ELISA protocols. Briefly, 1 pg/ml solutions of BSA-conjugated neo-epitope peptides and negative control peptide, and 0.1 mM recombinant C-terminal ApoE fragment and full-length ApoE (Abeam, cat. no. ab50243) were prepared by dilution in PBS. 50 mI/well were added to an ELISA half-area 96 well microtiter plate, and the plate was sealed with adhesive sealer and incubated over night at 4 °C. After discarding the solution, the plates were blocked with 150 mI/well of protein-free blocking solution (Pierce) for 1 h at room temperature with shake (600-900 rpm). The plates were washed four times with washing buffer containing 0.28 mM NaH2P04, 2.5 mM Na2HP04, 150 mM NaCI, 0.1 %
• Detection antibody HRP-conjugated anti-mouse IgG, Southern Biotech, cat. no. 1030-05, diluted 1 :10000 in dilution buffer
• 50 mI/well 50 mI/well of K- Blue® aqueous substrate (Neogen) were added, and the reaction was stopped after 5-15 min with 50 mI/well of 0.5 M H2SO4.
• the optical density at 450 nm was read using an ELISA reader (Tecan). The optical density was plotted against the antibody concentration to generate concentration- response curves ( Figure 28) and the EC50 values were determined from the log agonist concentration response curve.
• the purified monoclonal antibody to be tested was allowed to interact with the N-terminal neo-epitope of synthetic ApoE peptides
• BSA- conjugated negative control peptide SEQ ID NO:51 , 52, 50 and 53, respectively.
• the mix was added to a microtiter plate coated with the BSA-coupled A199 synthetic ApoE peptide. If the purified monoclonal antibody binds to any of the antigens in the pre-incubation step (the synthetic ApoE peptides), the antibody is prevented from binding to the synthetic A199 ApoE peptide immobilized on the microtiter plate. This leads to inhibition of the ELISA detection signal.
• phosphatase conjugated anti-mouse IgG detection antibody (Mabtech, cat. no. 3310-4) was diluted 1 :1000 and added to each plate (50 mI/well). The plate was sealed and incubated with shaking (900 rpm) for 45 min at room temperature and subsequently washed as described above. Alkaline phosphatase substrate (50 mI/well) was added to the plate and the optical density was read every 10 min at a wavelength of 405 nm for up to 120 min. The IC50 values were determined from a log inhibitor concentration response curve (Figure 29).
• Reference (no immobilized antigen) and active surfaces were treated with the same conditions using the amine coupling reagents on flow cell 1 (Fc1 ) and flow cell 2 (Fc2) respectively.
• the immobilization level for the active surfaces was kept at approximately 150- 200 response units (RU).
• the same protocol setup was used to immobilize full-length ApoE on the CM5 chip surface.
• Purified antibodies were prepared in 2-fold serial dilution from 14 to 0.3 nM (in 5-7 steps). Next, the prepared serial dilution of purified antibodies was injected (30 mI/min, contact time 360 s, dissociation time 2500 s) over both flow cells of the sensor chip. The interaction series was done in triplets. Values were blank subtracted, and a bivalent analyte binding kinetics fit model was used for the evaluation.
• Intercept ® PBS Blocking Buffer (LI-COR) with 0.1 % TWEEN®-20).
• Table 16 Summary of results from surface plasmon resonance of purified monoclonal anti-A199 antibodies Selectivity evaluation and human target binding in Alzheimer’s disease brain extracts by Western blot analysis: The monoclonal antibodies selective for the N-terminal neo-epitope of ApoE fragments starting at amino acid A199 (36A12, 38G9, 63F6 and 67G3) were tested for their ability to bind selectively to ApoE fragments ⁇ 12 kDa in human brain extracts from an Alzheimer’s disease patient, and without any binding to full-length ApoE.

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