Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • 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
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/77—Internalization into the cell

Definitions

• the present invention relates to an isolated and/or purified antibody, antibody fragment or derivative thereof able to block the gamma-secretase-dependent processing of the amyloid precursor protein (APP) and to pharmaceutical compositions containing said antibody.
• APP amyloid precursor protein
• AD Alzheimer's disease
• ⁇ amyloid- ⁇
• tau amyloid- ⁇
• the amyloid cascade hypothesis of AD pathogenesis proposes that polymerization of ⁇ into soluble oligomeric and/or insoluble amyloid deposits is a critical and early event that triggers hyperphosphorylation of tau and formation of neurofibrillary lesions, neuroinflammation and neuronal death ultimately leading to dementia (2, 3).
• ⁇ peptides are derived from the amyloid precursor protein (APP) that undergoes a first shedding step by either alpha- or beta-secretase, leading to the formation of the membrane bound 83- or 99-amino-acid-long APP-carboxy-terminal fragments (APP-CTFs) designated APP-C83 and APP-C99, respectively.
• APP-C83 and APP-C99 are further processed by the intramembrane gamma-secretase to generate APP intracellular domains (AICDs), and extracellular nontoxic p3 fragments from APP-C83 or amyloidogenic ⁇ peptides from APP-C99 (3-5).
• AICDs intramembrane gamma-secretase
• a rational strategy to lower ⁇ production consists in selectively inhibiting gamma-secretase-dependent APP-C99 processing without affecting the cleavage of other gamma-secretase substrates such as Notch, involved in cell proliferation and differentiation (6, V) .
• This object has been achieved by providing an isolated and/or purified antibody, antibody fragment or derivative thereof able to block the gamma-secretase-dependent cleavage of the amyloid precursor protein (APP) without impairing the Notch processing.
• APP amyloid precursor protein
• the present invention provides an isolated and/or purified antibody, antibody fragment or derivative thereof able to block the gamma-secretase-dependent processing of the amyloid precursor protein (APP) but not the Notch processing, wherein said isolated and/or purified antibody, antibody fragment or derivative thereof i) specifically recognizes at least one sequence of said APP consisting essentially in the amino acid sequence set forth in SEQ ID No 1 , a fragment thereof and/or a conservative variant thereof.
• APP amyloid precursor protein
• a further object of the present invention is to provide an isolated and/or purified antibody, antibody fragment or derivative thereof for the treatment and/or prevention of APP- associated diseases.
• the present invention also relates to an isolated and/or purified nucleic acid sequence comprising
• nucleotide sequence encoding an isolated and/or purified antibody, an antibody fragment or derivative of said antibody
• nucleic acid sequence having substantial sequence identity or homology to a nucleic acid sequence encoding an isolated and/or purified antibody, an antibody fragment of or derivative of said antibody,
• Another object concerns a pharmaceutical composition
• a pharmaceutical composition comprising a pharmaceutically effective amount of an isolated and/or purified antibody, antibody fragment or derivative thereof of the invention.
• An expression vector comprising an isolated and/or purified nucleic acid sequence of the invention, a host cell comprising said expression vector and a hybridoma and/or clone secreting the monoclonal antibody of any one of the invention are also contemplated.
• a method for treating and/or preventing an APP- associated disease, in a patient in need thereof comprising administering a pharmaceutically effective amount of an isolated and/or purified antibody, antibody fragment or derivative thereof of any one of the invention.
• the invention relates to an immune composition for the treatment and/or prevention of an APP-associated disease comprising at least one or several isolated and/or purified antibody, antibody fragment or derivative thereof of any one of the invention.
• the invention in another aspect, relates to a kit for treating and/or preventing an APP- associated disease, comprising a pharmaceutically effective amount of an isolated and/or purified antibody, antibody fragment or derivative thereof of any one of the invention, optionally with reagents and/or instructions for use.
• the invention in another aspect, relates to a method for reducing ⁇ production or delaying ⁇ plaque formation in a patient in need thereof, comprising administering a pharmaceutically effective amount of an isolated and/or purified antibody, antibody fragment or derivative thereof of any one of the invention.
• Fig. 1 Epitopes determination for the 4 groups of antibodies generated after mice immunization with recombinant and active human APP-C99 (APP-C99).
• mC99(l-7) (comprising 5 clones) targeting the epitope 1-7, 5 clones designated mC99(70-80) for the epitope 70-80 , 1 clone mC99(80-90) for the epitope 80-90, and 1 clone mC99(90-99) for the epitope 90-99.
• the different classes of epitopes obtained, as well as the number of clones per group of epitope relates the accessible versus non-accessible regions of APP-C99 in a membrane bilayer.
• Fig. 2 Monoclonal antibodies targeting APP-C99 decrease its processing by gamma- secretase and reduce the production of secreted ⁇ after their internalization in human cell lines.
• D Incubations of HEK NotchAE cells with 500 nM of anti-APP-C99 antibodies during 24 hours showed no effect on Notch ⁇ processing, whereas APP-CTFs were accumulated, thus emphasizing the specific effect of the antibodies on APP-C99/APP- 1 length (APP-FL) processing.
• Monoclonal antibodies targeting APP-C99 decrease its intracellular processing by gamma-secretase in a dose-dependent and non-toxic manner.
• Lactate Deshydrogenase (LDH) and crystal violet assays were performed to assess cell death in HEK, HeLa, HEK APP-C99-Gal4-VP16 (T-20), HEK APPSwe, and HEK NotchAE cells, after treatments with 250 nM or 500 nM of the indicated antibodies (250 nM for HEK and HeLa cells, and 500 nM for NotchAE and HEK APPSwe cells).
• the term "patient” is well-recognized in the art, and, is used herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human.
• the term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.
• Alzheimer's disease refers to a devastating neurodegenerative disorder for which the prevalence has been predicted to quadruple by 2050 (1). As the average lifespan increases, it has become a major socio-economical priority to identify new disease-modifying treatments to prevent or cure this disease. Although several underlying mechanisms that could explain the etiology of the disease are still unclear. The amyloid cascade remains so far the major and most documented hypothesis to explain the pathogenesis of AD.
• ⁇ peptides are the cleavage products of the 99 membrane-tethered APP-CTF (APP-C99) by gamma-secretase, an
• ⁇ -lowering compounds might be therapeutically beneficial if they do not interfere with other gamma-secretase functions. Indeed, impairment of Notch processing and signaling leads to severe toxicity because of interference with cell renewal and differentiation (8, 14).
• antibodies selected after immunization with an APP sequence preferably a natively folded sequence of said APP consisting essentially in the amino acid sequence set forth in SEQ ID No 1, a fragment of said sequence and/or a
• the present invention relates to an isolated and/or purified antibody, antibody fragment or derivative thereof able to block the gamma-secretase-dependent processing of the amyloid precursor protein (APP) but not the Notch processing, wherein said isolated and/or purified antibody, antibody fragment or derivative thereof specifically recognizes at least one sequence of said APP consisting essentially in the amino acid sequence set forth in SEQ ID No 1, a fragment thereof and/or a conservative variant thereof.
• said at least one APP sequence consisting essentially in the amino acid sequence set forth in SEQ ID No 1 , a fragment thereof and/or a conservative variant thereof is a natively folded sequence.
• Amyloid precursor protein is an integral membrane protein expressed in many tissues and concentrated in the synapses of neurons. Its primary function is not known, though it has been implicated as a regulator of synapse formation, neural plasticity and iron export. APP is best known and most commonly studied as the precursor molecule whose proteolysis generates beta amyloid ( ⁇ ), a 39- to 42-amino acid peptide whose amyloid fibrillar form is the primary component of amyloid plaques found in the brains of Alzheimer's disease patients.
• this APP sequence of the invention consists essentially in the amino acid sequence set forth in SEQ ID No 1 and containing 99 amino acids.
• the phrase "consisting essentially in” when referring to a particular amino acid means a sequence having the properties of a given SEQ ID NO.
• the phrase when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the essential characteristics of the sequence.
• the amino acid sequence will have substantial sequence identity for example at least 50%, 55%, 60%>, 65%, 70%), 75%), 80%), or 85% amino acid sequence identity; more preferably 90%> amino acid sequence identity; and most preferably at least 95%, 96%, 97%, 98%, or 99% % amino acid sequence identity.
• the present invention also refers to a fragment of the amyloid precursor protein (APP) SEQ ID No 1, preferably human APP.
• APP amyloid precursor protein
• SEQ ID No 1 preferably human APP.
• This refers to a biologically active sequence containing less amino acids in length than the corresponding sequence.
• This biologically active fragment can be used as long as it exhibits the same properties as the sequence from which it derives.
• this sequence contains less than 90%, preferably less than 60%, in particular less than 30% amino acids in length than the respective sequence.
• these sequences contain at least 5 to 20, most preferably 7 to 11 contiguous amino acids in length in common with the APP sequence of the invention.
• the fragment of said amino acid sequence set forth in SEQ ID No 1 is selected from the group of sequences comprising the N-terminal sequence 1-7 (SEQ ID No 2), the C- terminal sequence 70-80 (SEQ ID No 3), the C-terminal sequence 80-90 (SEQ ID No 4) and the C-terminal sequence 90-99 (SEQ ID No 5), and/or a combination of said sequences.
• the present invention further refers to a conservative variant of the amyloid precursor protein (APP), or of a fragment thereof as disclosed above.
• This conservative variant refers to polypeptides having amino acid sequences that differ to some extent from the native sequence polypeptide, that is amino acid sequences that vary from the native 3D sequence whereby one or more amino acids are substituted by another one.
• the variants can occur naturally (e.g.
• amino acid substitutions are conservative, i.e. occur within one of the above- identified group and do not affect the biological activity/ies of said APP sequence, i.e. its immunogenicity property.
• an isolated and/or purified antibody, antibody fragment or derivative thereof of the present invention is/are able to block the gamma-secretase- dependent processing of the amyloid precursor protein (APP) but not the Notch processing.
• APP amyloid precursor protein
• an "antibody” is a protein molecule that reacts with a specific antigenic determinant or epitope and belongs to one or five distinct classes based on structural properties: IgA, IgD, IgE, IgG and IgM.
• the antibody may be a polyclonal (e.g. a polyclonal serum) or a monoclonal antibody, including but not limited to fully assembled antibody, single chain antibody, antibody fragment, and chimeric antibody, humanized antibody as long as these molecules are still biologically active and still bind to at least one peptide of the invention.
• the antibody is a monoclonal antibody.
• the monoclonal antibody will be selected from the group comprising the IgGl, IgG2, IgG2a, IgG2b, IgG3 and IgG4 or a combination thereof.
• the monoclonal antibody is selected from the group comprising the IgGl, IgG2, IgG2a, and IgG2b, or a combination thereof.
• Non- limiting examples of antibodies are those listed in table 4.
• a typical antibody is composed of two immunoglobulin (Ig) heavy chains and two Ig light chains.
• Ig immunoglobulin
• Each light chain is composed of two tandem immunoglobulin domains: one constant (CL) domain and one variable domain (VL) that is important for antigen binding.
• isolated when used as a modifier of an antibody of the invention means that the antibody is made by the hand of man or is separated, completely or at least in part, from their naturally occurring in vivo environment. Generally, isolated antibodies are substantially free of one or more materials with which they normally associate with in nature, for example, one or more protein.
• isolated does not exclude alternative physical forms of the antibodies, such as multimers/oligomers, modifications (e g , phosphorylation, glycosylation, lipidation) or derivatized forms, or forms expressed in host cells produced by the hand of man
• an “isolated” antibody can also be “substantially pure” or “purified” when free of most or all of the materials with which it typically associates with in nature.
• an isolated antibody that also is substantially pure or purified does not include polypeptides or polynucleotides present among millions of other sequences, such as antibodies of an antibody library or nucleic acids in a genomic or cDNA library.
• the inventors have generated and selected 12 monoclonal antibodies after BALB/c mice immunization with active human APP-C99 (processed in vitro by gamma-secretase) purified in a lipidic environment to stabilize its native structure. Based on immunogenicity predictions (15- 18) as well as on APP-C99 structural studies (19, 20), the different groups of epitopes (1-7, 70- 80, 80-90, and 90-99) were interpreted as being the most accessible, hydrophilic, and flexible regions of APP-C99 embedded in a lipid-bilayer. Further immunohistochemical characterization confirmed these binding regions on cerebral amyloid deposits in Alzheimer brain slices (data not shown).
• Antibodies used in the present invention are not limited to whole antibody molecules and may be antibody fragments or derivatives as long as they are able to block the gamma-secretase dependent processing of the amyloid precursor protein (APP) and that they specifically recognizes at least one sequence of said APP, a fragment thereof and/or a conservative variant thereof without affecting the Notch processing.
• APP amyloid precursor protein
• Examples of isolated and/or purified antibody fragment or derivative thereof are selected amongst the group comprising a Fab-fragment, a F(ab2)'- fragment, a single-chain antibody, a chimeric antibody, a CDR-grafted antibody, a bivalent antibody-construct, a humanized antibody, a synthetic antibody, a chemically modified derivative thereof, a multispecific antibody, a diabody, a scFv- fragment; a dsFv- fragment, a labeled antibody, or another type of recombinant antibody.
• an antibody fragment is synthesized by treating the antibody with an enzyme such as papain or pepsin, or genes encoding these antibody fragments are constructed, and expressed by appropriate host cells as known to the skilled artisan.
• the isolated and/or purified antibody of the present invention consists essentially in an amino acid sequence selected from the group comprising
• combination may comprise a gamma VH chain of a first clone, a biologically active fragment thereof and/or a variant thereof, associated with a kappa VL chain of another clone, a biologically active fragment thereof and/or a variant thereof.
• a "Biologically active fragment" of the amino acid sequence of an antibody of the invention refers to a sequence containing less amino acids in length than the corresponding sequence.
• This biologically active fragment can be used as long as it exhibits the same properties as the sequence from which it derives, i.e. able to block the gamma-secretase-dependent processing of the amyloid precursor protein (APP) but not the Notch processing.
• the biologically active fragment of the invention is selective and can neutralize the APP/gamma-secretase (in particular the human APP C99/gamma-secretase) binding site, and/or block the entrance of the substrate into the internal chamber of the complex, and/or inhibit its cleavage to subsequently inhibit the release of ⁇ .
• this biologically active fragment contains less than 90%, preferably less than 60%, in particular less than 30% amino acids in length than the respective sequence.
• this biologically active fragment represents the complementary determining region.
• complementary determining region or “CDR” is well-defined in the art (see, for example ref 27) and refers to the stretches of amino acids within the variable region of an antibody that primarily makes contact with the antigen.
• the isolated and/or purified antibody, antibody fragment or derivative thereof of the invention comprises:
• CDR1 complementary determining region 1
• CDR2 complementary determining region 2
• CDR3 complementary determining region 3
• the isolated and/or purified antibody, antibody fragment or derivative thereof of the invention may also comprise:
• CDR1 complementary determining region 1
• the amino acid sequence determining said CDR1 consists essentially in an amino acid sequence selected from the group comprising SEQ ID No 16, SEQ ID No 18, SEQ ID No 20, SEQ ID No 22, SEQ ID No 24, a biologically active fragment thereof and/or a variant thereof, and/or a combination of said sequences
• CDR2 complementary determining region 2
• the amino acid sequence determining said CDR2 consists essentially in an amino acid sequence selected from the group comprising SEQ ID No 61, SEQ ID No 62, SEQ ID No 63, SEQ ID No 64, SEQ ID No 65, a biologically active fragment thereof and/or a variant thereof, and/or a combination of said sequences, and/or
• CDR3 complementary determining region 3
• the biologically active fragment contains less than 90%, preferably less than 60%, in particular less than 30% amino acids in length than the respective sequence(s) of CDR of the VH and/or CDR of the VL regions.
• Fragments or derivatives of the above antibodies which are able to block the gamma- secretase-dependent processing of the amyloid precursor protein (APP) can be obtained by using methods which are described, e.g., in (27).
• derivatives of said antibodies are obtained by the phage display technique, surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to an epitope of EAG1 (28)".
• CDR-grafted antibody refers to an antibody in which the CDR from one antibody, or a biologically active fragment of said CDR, is inserted into the framework of another antibody.
• the antibody from which the CDR is derived and the antibody from which the framework is derived are of different species.
• the antibody from which the CDR is derived and the antibody from which the framework is derived are of different isotypes.
• humanized antibody refers to an antibody in which all or part of an antibody framework region is derived from a human, but all or part of one or more CDR regions is derived from another species, for example, including, but not limited to, a mouse.
• humanized antibody refers to antibodies in which the framework or "complementarity determining regions” (CDR) have been modified to comprise the CDR of an immunoglobulin of different specificity as compared to that of the parent immunoglobulin.
• CDR complementarity determining regions
• Humanized antibodies can be produced using synthetic or recombinant DNA technology using standard methods or other suitable techniques. Nucleic acid (e.g.
• cDNA sequences coding for humanized variable regions can also be constructed using PCR mutagenesis methods to alter DNA sequences encoding a human or humanized chain, such as a DNA template from a previously humanized variable region (see e.g. (29), (30), (31) and (32)). Using these or other suitable methods, variants can also be readily produced.
• cloned variable regions e.g., dAbs
• sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see e.g., Krebber et al. , U.S. 5,514,548; Hoogenboom et al. , WO 93/06213, published April 1, 1993). Methods for the production of humanized antibodies are also described in, e.g., EP-A1 0 239 400 and W090/07861.
• bivalent or bispecific antibody refers to an antibody in which each of the two pairs of heavy chain and light chain (HC/LC) is specifically binding to a different antigen, i.e. the first heavy and the first light chain (originating from an antibody against a first antigen) are specifically binding together to a first antigen, and , the second heavy and the second light chain (originating from an antibody against a second antigen ) are specifically binding together to a second antigen;
• bivalent, bispecific antibodies are capable of specifically binding to two different antigens at the same time, and not to more than two antigens, in contrary to, on the one hand a monospecific antibody capable of binding only to one antigen, and on the other hand e.g. a tetravalent, tetraspecific antibody which can bind to four antigen molecules at the same time.
• Fab, F(ab')2 and the like can be obtained by treating an antibody of the invention with a proteolytic enzyme such as papain or pepsin, or alternatively, can be prepared by constructing a gene encoding the resulting antibody fragment and introducing this construct into an expression vector, followed by expression in an appropriate host cell.
• a proteolytic enzyme such as papain or pepsin
• recombinant antibody is intended to include all antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell (such as a NSO or CHO cell) or from an animal (e.g. a mouse) that is transgenic for, for example, human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell.
• a host cell such as a NSO or CHO cell
• animal e.g. a mouse
• Such recombinant antibodies have variable and constant regions in a rearranged form.
• the “diabody” refers to an antibody produced in accordance with the technology described in (33). These authors have provided an alternative mechanism for making bispecific antibody fragments.
• the fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.
• VH heavy-chain variable domain
• VL light-chain variable domain
• Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See ref. 34.
• Antibodies with more than two valencies are also contemplated.
• multispecific antibodies such as trispecific antibodies can be prepared as known to the skilled artisan.
• single chain Fv can be prepared by linking together an H chain V region and an L chain V region from an antibody of the present invention by using an appropriate peptide linker or the like.
• scFv can be prepared by constructing a DNA segment encoding the entire sequences or desired amino acid sequences of a gene encoding an H chain or H chain V region from the above antibody and a gene encoding an L chain or L chain V region from the antibody, and introducing this construct into an expression vector, followed by expression in an appropriate host cell.
• disulfide-stabilized antibody is an antibody fragment in which polypeptides modified to replace one amino acid residue by a cysteine residue in both H and L chain V regions from an antibody of the present invention are linked together between these cysteine residues via a disulfide linkage.
• An amino acid residue to be replaced by a cysteine residue can be selected by stereostructural estimation of the antibody.
• dsFv can be prepared by constructing a DNA segment encoding the entire sequence or a desired amino acid sequence of a gene encoding the antibody fragment, and introducing this construct into an expression vector, followed by expression in an appropriate host cell.
• a CDR-containing peptide comprises at least one or more CDR regions selected from CDR regions in H or L chains of an antibody of the invention. Also, multiple CDR regions may be linked together by techniques using an appropriate peptide linker or the like.
• the CDR-containing peptide may also be prepared by constructing a DNA segment encoding the entire sequence or a desired amino acid sequence of a gene encoding the peptide, and introducing this construct into an expression vector, followed by expression in an appropriate host cell.
• the CDR-containing peptide can also be prepared by chemical synthesis such as Fmoc or tBoc method.
• chimeric antibodies are described, for example, in W089/09622.
• a further source of antibodies to be utilized in accordance with the present invention are so-called xenogenic antibodies.
• the general principle for the production of xenogenic antibodies such as human antibodies in mice is described in, e.g., WO 91/10741, WO 94/02602, WO 96/34096 and WO 96/33735.
• the antibody of the invention may exist in a variety of forms besides complete antibodies; including, for example, Fv, Fab and F(ab)2, as well as in single chains; see e.g. W088/09344.
• the Notch receptor in particular the Notch-I receptor, is a known substrate of gamma -secretase and crucial signaling molecule involved in the differentiation and proliferation of a wide variety of cell types (6, 21). Indeed, significant adverse effects of gamma-secretase inhibitors have been described in preclinical and clinical studies, including gastrointestinal, haematopoetic, and skin toxicity, all of which have been attributed to impaired Notch processing (8, 9, 11, 12). Thus, it was important for the Applicants to develop drugs that lower ⁇ production without adversely affecting other gamma-secretase substrates and associated functions. As discussed above, the 12 monoclonal antibodies have been obtained after mice immunization with a natively folded sequence of human APP-C99.
• the present invention further contemplates an isolated and/or purified nucleic acid sequence comprising
• nucleotide sequence encoding an isolated and/or purified antibody, an antibody fragment or derivative of said antibody of the invention
• nucleic acid sequence having substantial sequence identity or homology to a nucleic acid sequence encoding an isolated and/or purified antibody, an antibody fragment of or derivative of said antibody of the invention
• v a nucleic acid sequence capable of hybridizing under stringent conditions to i), ii), iii) or iv).
• nucleic acid sequence refers to nucleic acid free or substantially free of material with which it is naturally associated such as other polypeptides or nucleic acids with which it is found in its natural environment, or the environment in which it is prepared (e. g. cell culture) when such preparation is by recombinant nucleic acid technology practiced in vitro or in vivo.
• nucleic acid is intended to refer either to DNA or to RNA.
• DNA which can be used herein is any DNA
• polydeoxynuclotide sequence including, e.g. double-stranded DNA, single-stranded DNA, double-stranded DNA wherein one or both strands are composed of two or more fragments, double-stranded DNA wherein one or both strands have an uninterrupted phosphodiester backbone, DNA containing one or more single-stranded portion(s) and one or more double- stranded portion(s), double-stranded DNA wherein the DNA strands are fully complementary, double-stranded DNA wherein the DNA strands are only partially complementary, circular DNA, covalently-closed DNA, linear DNA, covalently cross-linked DNA, cDNA, chemically- synthesized DNA, semi-synthetic DNA, biosynthetic DNA, naturally-isolated DNA, enzyme- digested DNA, sheared DNA, labeled DNA, such as radiolabeled DNA and fluorochrome- labeled DNA, DNA containing one or more non-naturally occurring species of nucleic acid.
• DNA sequences that encode the isolated and/or purified antibody, an antibody fragment of or derivative of said antibody of the invention, or a biologically active fragment thereof can be synthesized by standard chemical techniques, for example, the phosphotriester method or via automated synthesis methods and PCR methods.
• the purified and/or isolated DNA sequence encoding an isolated and/or purified antibody, an antibody fragment of or derivative of said antibody of the invention according to the invention may also be produced by enzymatic techniques.
• restriction enzymes which cleave nucleic acid molecules at predefined recognition sequences can be used to isolate nucleic acid sequences from larger nucleic acid molecules containing the nucleic acid sequence, such as DNA (or RNA) that codes for the isolated and/or purified antibody, an antibody fragment of or derivative of said antibody of the invention or for a fragment thereof.
• nucleic acid in the form of a
• RNA polyribonucleotide
• RNA including, e.g., single-stranded RNA, double-stranded RNA, double- stranded RNA wherein one or both strands are composed of two or more fragments, double- stranded RNA wherein one or both strands have an uninterrupted phosphodiester backbone, RNA containing one or more single-stranded portion(s) and one or more double-stranded portion(s), double-stranded RNA wherein the RNA strands are fully complementary, double- stranded RNA wherein the RNA strands are only partially complementary, covalently crosslinked RNA, enzyme-digested RNA, sheared RNA, mRNA, chemically-synthesized RNA, semi- synthetic RNA, biosynthetic RNA, naturally-isolated RNA, labeled RNA, such as radiolabeled RNA and fluorochrome-labeled RNA, RNA containing one or more non-naturally
• the isolated and purified nucleic acid sequence also comprises an isolated and/or purified nucleic acid sequence having substantial sequence identity or homology to a nucleic acid sequence encoding an isolated and/or purified antibody, an antibody fragment of or derivative of said antibody of the invention.
• the nucleic acid will have substantial sequence identity for example at least 50%, 55%, 60%>, 65%, 70%, 75%, 80%, or 85% nucleic acid identity; more preferably 90% nucleic acid identity; and most preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity.
• Identity as known in the art and used herein, is a relationship between two or more amino acid sequences or two or more nucleic acid sequences, as determined by comparing the sequences. It also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. Identity and similarity are well known terms to skilled artisans and they can be calculated by conventional methods (for example see 35-41).
• Methods which are designed to give the largest match between the sequences are generally preferred. Methods to determine identity and similarity are codified in publicly available computer programs including the GCG program package (42); BLASTP, BLASTN, and FASTA (43). The BLAST X program is publicly available from NCBI and other sources (43).
• nucleic acid sequence complementary to the isolated and purified nucleic acid sequence encoding an isolated and/or purified antibody, an antibody fragment of or derivative of said antibody/ies of the invention.
• nucleic acid sequence having a sequence which differs from a nucleic acid sequence encoding the isolated and/or purified antibody, an antibody fragment or derivative of said antibody of the invention, or a derivative or complementary sequence thereof, due to degeneracy in the genetic code.
• nucleic acid encodes functionally equivalent isolated and/or purified antibody, antibody fragment or derivative of said antibody of the invention but differs in sequence from the sequence due to degeneracy in the genetic code. This may result in silent mutations which do not affect the amino acid sequence. Any and all such nucleic acid variations are within the scope of the invention.
• nucleic acid sequence capable of hybridizing under stringent conditions, preferably high stringency conditions, to a nucleic acid sequence encoding an isolated and/or purified antibody, an antibody fragment of or derivative of said antibody of the invention, a nucleic acid sequence complementary thereof or a degenerated nucleic acid sequence thereof.
• Appropriate stringency conditions which promote DNA hybridization are known to those skilled in the art, or can be found in ref. 44. For example, 6. OX sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0XSSC at 50°C may be employed.
• the stringency may be selected based on the conditions used in the wash step.
• the salt concentration in the wash step can be selected from a high stringency of about 0.2XSSC at 50°C.
• the temperature in the wash step can be at high stringency conditions, at about 65° C.
• the present invention also includes an isolated and/or purified nucleic acid encoding an antibody, an antibody fragment or derivative of said antibody of the invention comprising a nucleic acid sequence encoding a truncation or an analog of the antibody, antibody fragment or derivative of said antibody of the invention.
• truncation refers to a sequence encoding a peptide containing less amino acid than the native but exhibiting the same properties.
• the invention also encompasses allelic variants of the disclosed isolated and/or purified nucleic sequence; that is, naturally-occurring alternative forms of the isolated and/or purified nucleic acid that also encode peptides that are identical, homologous or related to that encoded by the isolated and/or purified nucleic sequences.
• allelic variants may be produced by mutagenesis techniques or by direct synthesis.
• a biologically active fragment of the disclosed isolated and/or purified nucleic sequence is also considered and refers to a sequence containing less nucleotides in length than the nucleic acid sequence encoding an isolated and/or purified antibody, an antibody fragment of or derivative of said antibody of the invention, a nucleic acid sequence complementary thereof or a degenerated nucleic acid sequence thereof.
• This sequence can be used as long as it exhibits the same properties as the native sequence from which it derives.
• this sequence contains less than 90%, preferably less than 60%, in particular less than 30% amino acids in length than the respective isolated and/or purified nucleic sequence of the antibody, antibody fragment or derivative of said antibody of the invention.
• Yet another concern of the present invention is to provide an expression vector comprising the isolated and/or purified nucleic acid sequence encoding an isolated and/or purified antibody, an antibody fragment or derivative of said antibody of the invention.
• the choice of an expression vector depends directly, as it is well known in the art, on the functional properties desired, e.g., an isolated and/or purified antibody, an antibody fragment of or derivative of said antibody of the invention expression and the host cell to be transformed or transfected.
• a further concern of the present invention is to provide a host cell comprising the expression vector of the invention.
• the host cell is a bacterium, a fungal, a plant, or an animal cell.
• the animal cell is a mammalian cell and most preferably a human cell or a human cell line.
• the present invention also provides one or more hybridoma and/or clones secreting the monoclonal antibody of the invention.
• Non limiting examples of clones include the folio wings: 2C5, 1D8, 2F9, 1H9, 1H10 which produce antibodies that bind to human and murine APP and/or a fragment of said APP and block the gamma-secretase-dependent processing of the amyloid precursor protein (APP).
• APP amyloid precursor protein
• the term "monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art.
• Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
• monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in (27), (45) (said references incorporated by reference in their entireties).
• the term "monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
• One additional aspect of the present invention is to provide a pharmaceutical composition
• a pharmaceutical composition comprising a pharmaceutically effective amount of an isolated and/or purified antibody, antibody fragment or derivative thereof as disclosed herein.
• the pharmaceutical agent can be in a variety of well known formulations and administered using any of a variety of well known methods of administration such as intra-nasal, oral, subcutaneous, intravenous, intraarterial, intraperitoneal and/or intramuscular are also contemplated or the like.
• compositions wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.
• compositions adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurized aerosols, nebulizers or insufflators.
• the formulation may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, among others.
• suspending agents as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, among others.
• Useful intranasal formulations may contain at least one stabilizer and surfactant.
• the pharmaceutically acceptable surfactants are polyoxyethylene castor oil derivatives, such as polyoxyethylene-glycerol-triricinoleate, also known as polyoxyl 35 caster oil (CREMOPHOR EL), or poloxyl 40 hydrogenated castor oil (CREMOPHOR RH40) both available from BASF Corp.; mono-fatty acid esters of polyoxyethylene (20) sorbitan, such as polyoxyethylene (20) sorbitan monolaurate (TWEEN 80), polyoxyethylene monostearate (TWEEN 60),
• the surfactant will be between about 0.01% and 10%> by weight of the pharmaceutical composition.
• antioxidants such as sodium sulfite, sodium metabisulfite, sodium thiosulfate, sodium formaldehyde sulfoxylate, sulfur dioxide, ascorbic acid, isoascorbic acid, thioglycerol, thioglycolic acid, cysteine hydrochloride, acetyl cysteine, ascorbyl palmitate, hydroquinone, propyl gallate, nordihydroguaiaretic acid, butylated hydroxytoluene, butylated hydroxyanisole, alpha-tocopherol and lecithin.
• the stabilizer will be between about 0.01% and 5% by weight of the pharmaceutical composition.
• Suspensions may also include chelating agents such as ethylene diamine tetraacetic acid, its derivatives and salts thereof, dihydroxyethyl glycine, citric acid and tartaric acid among others. Additionally, proper fluidity of a suspension can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants, such as those previously mentioned.
• chelating agents such as ethylene diamine tetraacetic acid, its derivatives and salts thereof, dihydroxyethyl glycine, citric acid and tartaric acid among others.
• proper fluidity of a suspension can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants, such as those previously mentioned.
• Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
• the pharmaceutically effective amount of an agent of the invention may be mixed with at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants such as glycerol; (d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (e) solution retarding agents such as paraffin; (f) absorption accelerators such as quaternary ammonium compounds; (g) wetting agents such as cetyl alcohol and glycerol
• compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
• the solid dosage forms of tablets, capsules, pills and granules can be prepared with coatings and shells such as enteric coating and other coatings well-known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a
• composition such that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
• compositions which can be used include polymeric substances and waxes.
• Liquid dosage forms for oral administration or for spray formulation include
• the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof.
• inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol
• This invention also envisages the use of an isolated and/or purified antibody, antibody fragment or derivative thereof of the invention in a pharmaceutically acceptable salt form.
• salts may include sodium, potassium, calcium, aluminum, gold and silver salts.
• salts formed with pharmaceutically acceptable amines such as ammonia, alkyl amines, hydroxyalkylamines, N-methylglucamine and the like.
• Certain basic compounds also form pharmaceutically acceptable salts, e.g., acid addition salts.
• pyrido-nitrogen atoms may form salts with strong acid, while compounds having basic substituents such as amino groups also form salts with weaker acids.
• acids for salt formation are hydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, malonic, salicylic, malic, fumaric, succinic, ascorbic, maleic, pamoic, methanesulfonic and other mineral and carboxylic acids well known to those skilled in the art.
• the salts are prepared by contacting the free base form with a sufficient amount of the desired acid to produce a salt in the conventional manner.
• the free base forms may be regenerated by treating the salt with a suitable dilute aqueous base solution such as dilute aqueous NaOH, potassium carbonate, ammonia and sodium bicarbonate.
• a suitable dilute aqueous base solution such as dilute aqueous NaOH, potassium carbonate, ammonia and sodium bicarbonate.
• the free base forms differ from their respective salt forms somewhat in certain physical properties, such as solubility in polar solvents, but the acid and base salts are otherwise equivalent to their respective free base forms for purposes of the invention.
• composition comprising an isolated and/or purified antibody, antibody fragment or derivative thereof of the invention, as described herein, as an active ingredient may also be incorporated or impregnated into a bioabsorbable matrix, with the matrix being administered in the form of a suspension of matrix, a gel or a solid support.
• the matrix may be comprised of a biopolymer.
• Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi permeable matrices of solid hydrophobic polymers containing a pharmaceutically effective amount of an agent of the invention, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
• a pharmaceutically effective amount of an isolated and/or purified antibody, antibody fragment or derivative thereof of the present invention will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any and the nature of the effect desired.
• the appropriate dosage form will depend on the disease, the isolated and/or purified antibody, antibody fragment or derivative thereof of the invention, and the mode of
• the pharmaceutically effective amount of an isolated and/or purified antibody, antibody fragment or derivative thereof of the invention is present in an amount between about 0.001% and 20% by weight of the pharmaceutical composition.
• an alternative pharmaceutical composition may contain an isolated and/or purified nucleic acid sequence encoding said isolated and/or purified antibody, antibody fragment or derivative thereof, as described herein, as an active ingredient (alone or in combination with an isolated and/or purified antibody, antibody fragment or derivative thereof) as an active ingredient.
• This pharmaceutical composition may include either the sole isolated and/or purified DNA sequence, an expression vector comprising said isolated and/or purified DNA sequence or a host cell previously transfected or transformed with an expression vector described herein.
• host cell will preferably be isolated from the patient to be treated in order to avoid any antigenicity problem.
• gene and cell therapy approaches are especially well suited for patients requiring repeated administration of the pharmaceutical composition, since the said purified and/or isolated DNA sequence, expression vector or host cell previously transfected or transformed with an expression vector can be incorporated into the patient's cell which will then produce the protein endogenously.
• compositions of the invention are preferably for the treatment and/or prevention of diseases of aging based on or associated with amyloid-like proteins and
• AD Alzheimer's Disease
• LBD Lewy body dementia
• Down's syndrome hereditary cerebral hemorrhage with
• amyloidosis Dutch type
• Guam Parkinson- Dementia complex Other diseases which are based on or associated with amyloid- like proteins are progressive supranuclear palsy, multiple sclerosis; Creutzfeld Jacob disease, Parkinson's disease, HIV-related dementia, ALS (amyotropic lateral sclerosis), Adult Onset Diabetes, senile cardiac amyloidosis, endocrine tumors, and others, including ocular diseases associated with pathological abnormalities/changes in the tissues of the visual system, particularly associated with amyloid-beta-related pathological abnormalities/changes in the tissues of the visual system, such as neuronal degradation.
• Said pathological abnormalities may occur, for example, in different tissues of the eye, such as the visual cortex leading to cortical visual deficits; the anterior chamber and the optic nerve leading to glaucoma; the lens leading to cataract due to beta-amyloid deposition; the vitreous leading to ocular amyloidosis; the retina leading to primary retinal degeneration, and macular degeneration, for example age-related macular degeneration; the optic nerve leading to optic nerve drusen, optic neuropathy and optic neuritis; and the cornea leading to lattice dystrophy.
• the diseases is selected among the group comprising Alzheimer's disease, Down syndrom, diseases caused by an extra copy of Chromosome 21 or extra copy(ies) of the gene APP, cerebral amyloid angiopathy (CAA), hereditary cerebral hemorrhage with amyloidosis Dutch type (HCHWAD), amyloid microangiopathy in the vascular variant of Alzheimer's disease, Abeta related angiitis (ABRA), fronto temporal dementias (FTD), Parkinson's disease and amyotrophic lateral sclerosis .
• ABRA cerebral amyloid angiopathy
• HHWAD hereditary cerebral hemorrhage with amyloidosis Dutch type
• ABRA Abeta related angiitis
• FTD fronto temporal dementias
• Parkinson's disease and amyotrophic lateral sclerosis .
• Also encompassed in the present invention is a method for treating and/or preventing a disease disclosed herein, in a patient in need thereof, comprising administering a pharmaceutically effective amount of an isolated and/or purified antibody, antibody fragment or derivative thereof of the invention.
• the present invention is also directed to an immune composition for the treatment and/or prevention of an APP-associated disease comprising at least one or several antibodies or antibody fragments of the invention, e.g. two or three antibodies or antibody fragments of the invention.
• the present invention is also directed to an acid nucleic vaccine composition for the treatment and/or prevention of an APP-associated disease comprising at least one expression vector comprising at least one copy of the purified and isolated nucleic acid sequence of the invention, fragments thereof, molecular chimeras thereof, combinations thereof and/or variants thereof.
• kits for treating and/or preventing a disease disclosed supra comprising a pharmaceutically effective amount of an isolated and/or purified antibody, antibody fragment or derivative thereof of the invention, optionally with reagents and/or instructions for use.
• the kit may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, and syringes.
• Plasmids The coding regions of human APP-C99, APP-C83, APP-C59, APP-C50 (C-terminal numbering), ⁇ 1-60, ⁇ 1-70, ⁇ 1-80, ⁇ 1-90 ( ⁇ numbering) were amplified by PCR and cloned in the peT21b vector. One part of these fragments was generated with a methionine followed by a His-tag on their N-terminal extremities, giving the resulting constructs: Met-His- ⁇ 1-60, ⁇ - ⁇ 8- ⁇ 1-70, ⁇ - ⁇ - ⁇ - ⁇ , Met-His ⁇ l-90 ( ⁇ numbering).
• the other part of the fragments was generated with a methionine at their N-termini and a His-tag at their C- termini, giving the following constructs: Met-C89-His, Met-C59-His, Met-C50-His ( ⁇ numbering). All sequences were verified by DNA sequencing.
• the obtained vectors were transformed in BL21(DE3) Escherichia Coli bacteria.
• the transformed bacteria were then cultured in Luria Bertani medium supplied with ampicillin (100 ⁇ g/ml), with shaking at 37°C, until OD 6 oonm reached 0.8.
• Expression of recombinant proteins was then induced during 4 hours, by addition of ImM isopropyl thiogalactoside (IPTG, Applichem). After 4 hours induction, bacteria were harvested during 1 hour by centrifugation at 5000 g. After lysis of bacterial pellets, and 3 centrifugation steps, recombinant proteins were purified using Ni-NTA agarose affinity resin (Invitrogen). All fragments were analyzed by SDS-PAGE followed by Coomassie blue staining (Invitrogen).
• In vitro gamma-secretase activity assays In vitro gamma-secretase activity assays. In vitro gamma-secretase activity assays using the recombinant APP-C99 substrate and purified gamma-secretase were performed as previously reported (22). After 4 hours incubation at 37°C, the reaction was stopped by adding Laemmli buffer and the samples were analyzed by western blot for AICD-His.
• mice injection with purified and active human APP-C99 were injected intraperitoneally in 8-week-old female BALB/c mice. Three injections were performed at 2 week-intervals. Four days prior hybridoma fusion, mice with positively reacting sera were reinjected. Spleen cells were fused with Sp2/0 Agl4 myeloma cells as described previously (23). Hybridoma culture supernatants were tested 10 days later by ELISA for cross-reaction with APP-C99 recombinant protein. Positive supernatants were then tested by Western blot.
• ELISA tests for epitopes determination were coated with 100 ⁇ per well of the diluted antigens (2 ⁇ g/ml in PBS) and incubated at 4°C overnight, following what they were washed twice with PBS-tween (0.05% of tween 20). Non-specific binding was blocked by adding 200 ⁇ of 1% gelatin/PBS. After 30 minutes incubation at room temperature, plates were washed twice with PBS-tween, and 100 ⁇ of hybridomas supematants were supplied to appropriate wells for 1 hour at room temperature. Washing steps were repeated and 100 ⁇ of secondary antibody conjugated with Alkaline Phosphatase were subsequently incubated for 1 hour. After 2 washing steps, 100 ⁇ of substrate (pNPP 1 mg/ml in 1M diethanolamine pH 9.8, 0.5 mM MgC ) was added and absorbances were read at 405-410 nm with an ELISA plate reader.
• substrate pNPP 1 mg/ml in 1M
• Luminescence T20-cell-based assay T20 cells, a luciferase-based reporter system (24) (a gift from Dr. M. S. Wolfe) was used for measuring the processing of APP-C99 by gamma-secretase.
• T20 cells are stably overexpressing APP-C99 fused with Gal4-VP16 on its C-terminus and luciferase placed under the control of a Gal4 promoter. In presence of tetracycline (1 ⁇ g/ml), APP-C99 fused with Gal4-VP16 is expressed and cleaved by gamma-secretase.
• the AICD-Gal4- VP16 product is then translocated to the nucleus to activate the expression of luciferase, which generates the luminescence signal reporting APP-C99 processing by gamma-secretase.
• T20-cells were plated in 96-wells plates (Corning), incubated during 24 hours with 350 nM, 500 nM, and 750 nM of each purified monoclonal antibody. Their luminescence signal was subsequently measured with a Tecan Infinite M200 plate reader, using the Bright-Glo luciferase kit (Promega).
• Cell lines culture. HEK cells stably expressing APPSwe (a gift from Dr. D. J.
• T20, HEK and HeLa cells were cultured in DMEM containing 10% FBS and Penicillin-Streptomycin (Invitrogen).
• DMEM fetal calf serum
• Penicillin-Streptomycin Invitrogen
• medium was supplied with 150 ⁇ g/ml G418, for T20 cells with 50 ⁇ g/ml blasticidin, 200 ⁇ g/ml zeocin, and 500 ⁇ g/ml hygromycin.
• Cells treatments and proteins extraction were treated for 24 hours at 37°C with 10 ⁇ DAPT or different concentrations of each monoclonal antibody. Cells were then lysed with Hepes buffer pH7, containing NP40 1% and protease inhibitors (Roche). Protein extracts were quantified by BCA (Pierce) and analyzed by western-blot.
• Proteins extracts obtained from cells treatments were separated by SDS-PAGE or Native- SDS -PAGE and analyzed by western-blot with the following primary antibodies: rabbit monoclonal A8717 antibody (targeting the last 19 amino acids of human APP, Sigma), anti-P-actin antibody (Sigma), Vail 744 anti-Notch cleavage site antibody (Cell signaling). Secondary goat anti-mouse and goat anti-rabbit 680 antibodies (Alexa fluor) were used to probe the membrane and the fluorescence signal was detected with a Li-Cor Odyssey scanner.
• ⁇ peptides ELISA Cultures media from HEKAPPSwe were collected and secreted ⁇ peptides were quantified using human ⁇ 40 and ⁇ 42 ELISA kits from Invitrogen (KHB3482, KHB3544).
• HEK APPSwe cells were lysed with CHAPSO 1% and proteins were extracted. Proteins extracts were then immunoprecipitated with proteins G beads (Invitrogen) and analyzed by immunoblotting.
• Cell death assays Cell death in HEK, HeLa, HEK APPSwe, N7 and T20 cells was assessed with the LDH CytoTox-one kit (Promega) as well as the crystal violet test. Briefly, cells were incubated 10 min at RT with 0.2% crystal violet solution. After 2 washing-steps, 1% SDS was added to solubilize the dye, and the absorbance was read at 570 nm.
• mice immunization with active APP-C99 generates monoclonal antibodies targeting 2 major and 2 minor groups of epitopes.
• APP-C99 All fragments including APP-C99 were protein normalized by silver stained SDS-PAGE and by western blot using an anti Histidine-tag antibody (Fig. ID, Anti His-Tag).
• Fig. ID Anti His-Tag
• 5 were directed against the N-terminal region of APP-C99.
• the 7 others antibodies were directed against the C-terminal region of APP-C99, with 5 of them targeting the region 70- 80 (mC99(70-80)), 1 binding the region 80-90 (mC99(80-90)) and 1 recognizing the region 90- 99 (mC99(90-99), see Fig. 1C and ID).
• the model comprises a long transmembrane helix (residues 30-52), and 2 small helical domains interacting with the membrane bilayer (the first one being either 13-22 or 19-22 depending on the hydrophobic environment of the protein (19, 20), and the second one being 90-99).
• our data strongly support a model in which the most exposed regions of active APP-C99 embedded into the lipid bilayer contain the N- and C-terminal epitopes 1-7 and 70-80, respectively.
• the epitopes 80-90 and 90-99 are less exposed and accessible regions as only one antibody has been identified per group. No antibodies have been found to target the putative natively folded, non- exposed 30-69 region that includes the transmembrane domain (TMD) of APP-C99.
• Anti-APP-C99 monoclonal antibodies reduce gamma-secretase-dependent APP-C99 processing and alter ⁇ production following internalization in human cell lines.
• APP-CTFs including APP-C99 and APP-C83
• gamma-secretase processing common characteristics among known ⁇ -secretase substrates
• Impairment of APP-C99 processing was dose-dependent as observed after 48 hours incubation with 350, 500, or 750 nM of each antibody in a previously described gamma-secretase-based luciferase reporter assay using HEK cells stably expressing APP-C99 fused to Gal4-VP16 (T20 cells, (24) see Fig. 2B).
• the luminescence signal depends on AICD-Gal4-VP16 activating the expression of luciferase, one cannot exclude that reduced luminescence can be partly attributed to the binding of the C- terminal antibodies to AICD thus preventing it from reaching the luciferase promoter.
• mC99(l- 7) antibody showed a significant luminescence reduction (40 ⁇ 2% at 750nM when compared to IgG A control), and recognizes only the N-terminal part of APP-C99.
• the effect of anti- APP-C99 antibodies on APP-C99 processing by gamma-secretase was further validated by measuring ⁇ secretion in HEK293T cells stably overexpressing human APP carrying the Swedish mutation (HEK APPSwe). As shown in Fig. 2C, all antibodies triggered a dose- dependent decrease of both ⁇ 40 and ⁇ 42 production in these cells.
• mC99(70- 80)#10 treatment the C-terminal antibodies induced a significant decrease at 500 nM treatment (27.6 ⁇ 4.2% and 25 ⁇ 6.6% for ⁇ 40 and ⁇ 42, respectively) when compared to IgG A control (Fig. 2C bottom panel), without affecting the ⁇ 40/42 ratio.
• mC99(l-7) targets the ⁇ sequence of APP-C99, it also probably depletes the ⁇ peptides contained in the medium and generates a 92 ⁇ 1% drop of ⁇ 40 and 42 levels when compared to control antibodies, at 500 nM treatment.
• APP-CTFs Consistent with impaired gamma-secretase-dependent APP-C99 processing, dose- dependent accumulations of APP-CTFs were detected in these cells treated for 24 hours with anti-APP-C99 antibodies concentrations as low as 25-75 nM (Fig. 2C top panel and Fig. 3A), but not in cells incubated with IgGA, IgGmix, or a monoclonal antibody targeting the N- terminus of APP full-length (APP-FL) (22C11 against aa 66-81 of APP) (Fig. 3A and 3B). Interestingly, treatments with antibodies also triggered a dose-dependent accumulation of APP full-length (APP-FL, Fig. 2C, top panel).
• the monoclonal antibodies were probed by ELISA and by
• mC99(l-7) and mC99(70-80) antibodies bind to their corresponding epitopes in Alzheimer brain tissue sections.
• mC99(l-7) and mC99(70-80) antibodies were tested for detection of their corresponding epitopes in brain sections from ⁇ -containing Alzheimer brain slices. Strong immunolabeling of ⁇ plaques was achieved with mC99(l-7) ). In contrast, no staining of the congophilic ⁇ plaques could be detected with mC99(70-80), which instead decorated the periphery of Congo Red-positive deposits as dense granular puncta or more elongated filaments likely
• Anti-APP-C99 monoclonal antibodies affect specifically the gamma-secretase cleavage.
• the different chains and CDRs of the antibodies were determined with NCBI Ig blast (IMGT®, the international ImMunoGeneTics information system).
• ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGTTTCCA 1 D8 DNA GAAGTGATGTTTTGATGACCCAAAGTCCACTCTCCCTGCCTGTCAGTCTTGG kappa FULL AGATCAAGCCTCCATCTCTTGCAGATCAAGTCAGAGCATTGTACATAGTAAT GGAAACACCTATTTAGAATGGTACCTGCAGAAATCAGGCCAGTCTCCAAAGG GCCTGATCTACAAAGTTTCCAACCGATTTTCCGGGGTCCCAGACAGGTTCAG TGGCAGTGGA CAGGGACAGAT CACAC CAAGA CAGCAGAG GGAGGC GAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACGTGTTCCGCTCACGT TCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGT ATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTTAACATCTGGAGGTGCCTCAGTCAG
• Alzheimer's disease the amyloid cascade hypothesis. Science 256: 184-185.
• Tumor necrosis factor-alpha, interleukin-lbeta, and interferon-gamma stimulate gamma-secretase- mediated cleavage of amyloid precursor protein through a JNK-dependent MAPK pathway. J Biol Chem 279:49523-49532.

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