Pegylated amyloid beta fab
Abstract
FAB GENTLEED FOR ABET. The present invention relates to a method for treating conditions associated with prophylactic and prophylactic Aβ2 activity. The method employs humanized antibody fragments which specifically bind to the human Aβ peptide between the amino acid positions 13-28, wherein the antibody fragments are covalently attached to a polyethylene glycol (PEG) molecule.

Term
Projected expiry 9 January 2028.
- Priority
- Filed
- Granted
- Today
- Projected expiry
19 claims: 7 independent, 12 dependent
- 1Molécula compreendendo um fragmento de anticorpo que se liga especificamente ao peptídeo Αβ humano entre as posições de aminoácido 13-28, em que o dito fragmento de anticorpo é cova lente mente ligado a 5 uma molécula de polietileno glicol.
- 2Molécula de acordo com a reivindicação 1, em que o fragmento de anticorpo é um fragmento Fab.
- 3Molécula de acordo com a reivindicação 1 ou 2, em que o fragmento de anticorpo compreende uma região variável de cadeia leve da 10 SEQ ID NO:1 e uma região variável de cadeia pesada da SEQ ID NO: 2.
- 4Molécula de acordo com qualquer uma das reivindicações 13, em que a molécula de polietileno glicol é covalentemente ligada à região variável de cadeia pesada do fragmento de anticorpo.
- 5Molécula de acordo com a reivindicação 4, em que a molécu15 Ia de polietileno glicol é covalentemente ligada a uma CDR da região variável de cadeia pesada do fragmento de anticorpo.
- 6Molécula de acordo com qualquer uma das reivindicações 35, em que a molécula de polietileno glicol é covalentemente ligada à cisteína na posição de aminoácido 56 da região variável de cadeia pesada da dita ‘ 20 SEQ ID NO:2.
- 7Molécula de acordo com a reivindicação 1 ou 2 compreendendo uma região variável de cadeia leve da SEQ ID NO:1 e uma região variável de cadeia pesada da SEQ ID NO: 3.
- 8Molécula de acordo com a reivindicação 7, em que a molécu25 Ia de polietileno glicol é covalentemente ligada à região de dobradiça do fragmento de anticorpo.
- 9Molécula de acordo com qualquer uma das reivindicações 18, em que a molécula de polietileno glicol tem um peso molecular de cerca de 0,5 kD a cerca de 30 kD. 30
- 10Molécula de acordo com a reivindicação 9, em que a molécula de polietileno glicol tem um peso molecular de cerca de 20 kD.
- 11Molécula que consiste em um fragmento de anticorpo que especificamente liga-se ao peptídeo Αβ humano entre as posições de aminoácido 13-28, em que o dito fragmento de anticorpo é covalentemente ligado a uma molécula de polietileno glicol.
- 12Molécula compreendendo um fragmento de anticorpo com uma região variável de cadeia leve da SEQ ID NO:1 e uma região variável de cadeia pesada da SEQ ID NO: 2, em que o dito fragmento de anticorpo é covalentemente ligado a uma molécula de polietileno glicol de 20 kD na posição 56 da região variável de cadeia pesada da SEQ ID NO: 2.
- 13Composição compreendendo a molécula como definida em qualquer uma das reivindicações 1-12.
- 14Composição de acordo com a reivindicação 13 adicionalmente compreendendo um veículo farmaceuticamente aceitável.
- 15Método de tratar ou prevenir uma condição associada com atividade do peptídeo Αβ, compreendendo administrar a um sujeito humano em necessidade do mesmo uma quantidade eficaz de uma molécula como definida em qualquer uma das reivindicações 1-12.
- 16Método de tratar uma condição selecionada de um grupo que consiste na doença de Alzheimer clínica ou pré-clínica, síndrome de Down, ou angiopatia amilóide clínica ou pré-clínica (CAA), déficit cognitivo, acidente vascular cerebral, hemorragia cerebral, e debilitação mental geral, compreendendo administrar ao sujeito humano uma quantidade eficaz de uma molécula como definida em qualquer uma das reivindicações 1-12.
- 17Molécula de acordo com qualquer uma das reivindicações 112, para o uso como um medicamento.
- 18Uso de uma molécula como definida em qualquer uma das reivindicações 1-12 na preparação de um medicamento para o tratamento ou prevenção de uma condição associado à atividade do peptídeo Αβ.
- 19Uso de uma molécula como definida em qualquer uma das reivindicações 1-12 na preparação de um medicamento para o tratamento ou prevenção de uma condição selecionada de um grupo que consiste na doença de Alzheimer clínica ou pré-clínica, síndrome de Down, angiopatia amilóide clínica ou pré-clínica (CAA), déficit cognitivo, acidente vascular cerebral, hemorragia cerebral, e debilitação mental geral. RESUMO Patente de Invenção:FAB PEGUILADO PARA ABETA. A presente invenção refere-se a um método para tratar condições associadas à atividade do peptídeo Αβ profilática e terapeuticamente é 5 descrito. O método emprega fragmentos de anticorpo humanizados que especificamente se ligam ao peptídeo Αβ humano entre as posições de aminoácido 13-28, em que os fragmentos de anticorpo são covalentemente ligados a uma molécula de polietileno glicol (PEG). Novo quadro reivindicatório (total de 13 reivindicações) para processamento na Fase Nacional Brasileira.
Independent claims19
309 paragraphs in 13 sections, as filed
Descriptive Report of the Invention Patent for FAB PEGUILADO PARA ABETA.
The present invention relates to an antibody fragment that binds to the beta-amyloid peptide (Αβ) and is covalently linked to one or more polyethylene glycol (PEG) molecules.
The circulating Αβ peptide is composed of 39-43 amino acids (mostly 40 or 42 amino acids) resulting from the cleavage of a precursor protein, amyloid precursor protein (APP). Conversion of Αβ from soluble to insoluble forms with high β-sheet content and its deposition as neuritic and cerebrovascular plaques in the brain appears to be associated with various conditions and diseases, including Alzheimer's disease, Down syndrome, and cerebral amyloid angiopathy (CAA). Prevention and/or reversal of Aβ deposition may treat conditions associated with the Aβ peptide.
Therapeutic agents that affect Aβ deposition include antibodies to Aβ peptide, such as humanized antibodies and fragments discussed in WO 2001/62801, WO 2004/071408 and Tamura, Y., et al, Neurobiol. of Dis. (2005) 20:541-545.
Although many antibodies and their derivatives can be useful in diagnosis and therapy, optimal antibody pharmacokinetics are often not achieved for a particular application. Therapeutic antibodies targeted to combat various conditions and diseases associated with the Aβ peptide are generally immunoglobulins with intact Fc regions. Fc regions are responsible for prolonging the antibody's half-life in plasma. However, this prolongation can be a disadvantage since it prevents the antibody bound to the target peptide from being effectively cleared, resulting in the antigen-antibody complex being present in the plasma circulation for extended periods of time. Subsequent administration of the antibody also leads to the accumulation of the unwanted complex in the plasma. The Fc portion of an antibody may have certain undesirable effector functions and may need to be modified to eliminate such functions. Also, the Fc portion adds substantial size to the overall therapeutics, which often creates problems associated with the delivery route, delivery devices, and large-scale manufacturing processes.
Antibody fragments lacking an Fc portion, including Fabs, have been studied in vivo to determine if such fragments could be potential therapeutics. However, studies suggest that the usefulness of therapies involving fragments such as Fabs is limited due to a rapid release rate and a short half-life. Therefore, there is a need for a therapeutically active anti-Αβ peptide antibody molecule with pharmacokinetics and pharmacodynamics that allow for an improved dosing regimen while avoiding potential side effects that can be created through complex formation in plasma and potential effector functions.
The present invention overcomes several problems associated with therapeutic antibodies or antibody fragments that can be targeted to Aβ peptide. The compounds of the present invention comprise an antibody fragment that binds to Aβ and is covalently linked to one or more polyethylene glycol (PEG) molecules. These compounds can be produced in bacterial or yeast cell systems, which eliminates several problems associated with antibody production in mammalian cell lines, such as cost issues, purification problems, and contamination problems from endogenously produced antigens. Furthermore, the compounds of the present invention can be administered subcutaneously and have an ideal pharmacokinetic (PK) and pharmacodynamic (PD) profile while preserving the affinity and selectivity of the antibody fragment for Aβ.
Completely unpredictably and unexpectedly, the applicants also discovered that covalently linking PEG molecules to the complementarity-determining region (CDR) of the antibody fragment did not alter the activity, affinity, or selectivity of the antibody fragment for Aβ.
This invention provides a molecule comprising an antibody fragment that specifically binds to the human Aβ peptide between amino acid positions 13-28, wherein the antibody fragment is covalently linked to a PEG molecule. Preferably, the antibody fragment is a Fab fragment.
In one embodiment, the invention provides a molecule comprising an antibody fragment having a variable heavy chain region and a variable light chain region, wherein the variable light chain region comprises CDR regions with the following amino acid sequences: CDRL1: SSSQSLIYSDGNAYLH (SEQ ID NO: 6), CDRL2: KVSNRFS (SEQ ID NO: 7) and CDRL3: TQSTHSPWT (SEQ ID NO: 8) and wherein the variable heavy chain region comprises CDR regions with the following amino acid sequences: CDRH1: GYTFSRYSMS (SEQ ID NO: 9), CDRH2: QINIRGÇNTYYPDTVKG (SEQ ID NO: 10) or QINIRGNNTYYPDT VKG (SEQ ID NO: 11), and CDRH3: GDF (SEQ ID NO: 12). Preferably, such a molecule has a PEG molecule that is covalently linked either to the variable heavy chain region or to the variable light chain region of the antibody fragment. More preferably, such a molecule has a PEG molecule that is covalently linked to a CDR. Even more preferably, such a molecule has a PEG molecule that is covalently linked to a cysteine residue within the CDR. Most preferably, such a molecule has a PEG molecule that is covalently linked to a CDRH2: QINIRGÇNTYYPDTVKG (SEQ ID NO: 10) of the variable heavy chain region of the antibody fragment.
In another embodiment, the invention provides a molecule comprising an antibody fragment having a variable light chain region of SEQ ID NO: 1, and a variable heavy chain region of SEQ ID NO: 2. Preferably, such a molecule has a PEG molecule that is covalently linked either to the variable heavy chain region or to the variable light chain region of the antibody fragment. More preferably, such a molecule has a PEG molecule that is covalently linked to a CDR of a variable heavy chain region of the antibody fragment. Even more preferably, such a molecule has a PEG molecule that is covalently linked to a cysteine residue within the
CDR of a variable heavy chain region of the antibody fragment. Most preferably, such a molecule has a PEG molecule that is covalently linked to cysteine at amino acid position 56 of the variable heavy chain region of SEQ ID NO: 2.
In another embodiment, the invention provides a molecule comprising a Fab fragment or an ScFv fragment, wherein the Fab fragment or ScFv fragment is covalently linked to a PEG molecule and has a variable light chain region of SEQ ID NO: 1, and a variable heavy chain region of SEQ ID NO: 2. Preferably, such a molecule has a PEG molecule that is covalently linked to cysteine at amino acid position 56 of the variable heavy chain region of SEQ ID NO: 2. Also preferably, in such a molecule the molecular weight of PEG is about 0.5 kD to about 30 kD, more preferably 20 kD.
In another embodiment, the invention provides a molecule comprising an antibody fragment with a variable light chain region of SEQ ID NO: 1 and a variable heavy chain region of SEQ ID NO: 2, wherein said antibody fragment is covalently linked to a 20 kD PEG molecule at position 56 of the variable heavy chain region of SEQ ID NO: 2. Preferably, in such a molecule, the PEG molecule is covalently linked via a maleimide linkage.
In another embodiment, the invention provides a molecule comprising an antibody fragment that specifically binds to the human Aβ peptide between amino acid positions 13-28, wherein the antibody fragment is covalently linked to a PEG molecule and has a variable light chain region of SEQ ID NO: 1, and a variable heavy chain region of SEQ ID NO: 3. Preferably, such a molecule has a PEG molecule that is covalently linked to the hinge region of the antibody fragment. More preferably, the PEG is covalently linked to the hinge region via a maleimide bond.
The invention also includes a molecule comprising antibody fragments, preferably humanized antibody fragments, in which a PEG molecule is covalently linked to the antibody fragment, resulting in an active therapeutic molecule with pharmacokinetics and pharmacodynamics that allow for a weekly dosing regimen. At the same time, minimizing potential side effects that can be created through complex formation in plasma and preserving or improving the activity, affinity, and selectivity of the antibody fragment for Aβ.
The invention also includes methods of treating, preventing, or reversing conditions and diseases associated with the Aβ peptide, including preclinical and clinical Alzheimer's disease, Down syndrome, and preclinical and clinical cerebral amyloid angiopathy (CAA), cognitive impairment, stroke, cerebral hemorrhage, and general mental debilitation. These methods comprise administering to a subject an effective amount of a molecule described and claimed herein.
This invention provides a molecule comprising an antibody fragment that specifically binds to the Aβ peptide between amino acid positions 13-28, wherein the antibody fragment is covalently linked to a PEG molecule. It has been found that covalently linking a PEG molecule to an Aβ-binding antibody fragment does not negatively alter the activity, affinity, or selectivity of the fragment.
- 20 antibody for Aβ. More surprisingly, it was found that covalently linking a PEG molecule having a molecular weight up to 20 kD to a CDR of an antibody fragment that binds Aβ also does not negatively alter the activity, affinity, or selectivity of the antibody fragment for Aβ peptide. These antibody fragments can be administered subcutaneously and have an improved PK/PD profile for therapeutic use that supports a flexible dosing regimen. Furthermore, these antibody fragments can be produced in bacterial or yeast cell systems, eliminating several problems associated with the production of full-compliance antibodies in mammalian cells. The pegylated antibody fragments of this invention offer the opportunity to prevent and treat, prophylactically and therapeutically, conditions in humans associated with the Aβ peptide.
A naturally occurring full-compliance antibody is an immunoglobulin molecule comprised of four peptide chains: two heavy (H) chains (approximately 50–70 kDa in total length) and two light (L) chains (approximately 25 kDa in total length) interconnected by disulfide bonds. The amino-terminal portion of each chain includes a variable region of approximately 100–110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for the effector function.
Light chains are classified as either kappa or lambda and are characterized by a particular constant region. Each light chain comprises an N-terminal variable light chain region (here LCVR) and a constant light chain region comprising a domain, CL. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody isotype as IgG, IgM, IgA, IgD, and IgE, respectively, and several of these can also be divided into subclasses (isotypes), for example, IgGi, IgG<sub>2</sub>, lgG<sub>3</sub>, lgG<sub>4</sub>IgA-ι and lgA<sub>2</sub>Each type of heavy chain is characterized by a particular constant region. Each heavy chain is comprised of an N-terminal variable heavy chain region (here HCVR) and a constant heavy chain region. The constant heavy chain region is comprised of three domains (CH1, CH2, and CH3) for IgG, IgD, and IgA; and four domains (CH1, CH2, CH3, and CH4) for IgM and IgE.
The HCVR and LCVR regions can also be subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with regions that are more conserved, called structure regions (FRs). Each HCVR and LCVR is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Here, the three heavy chain CDRs are referred to as CDRH1, CDRH2, and CDRH3, and the three light chain CDRs are referred to as CDRL1, CDRL2, and CDRL3. The CDRs contain most of the residues that form specific interactions with the antigen. The numbering and positioning of the CDR amino acid residues within the HCVR and LCVR regions are in accordance with the well-known Kabat numbering convention.
The variable region of each light-heavy chain pair forms an antigen-binding site of the antibody. As used herein, the antigen-binding portion or antigen-binding region or antigen-binding domain or antigen-binding site refers alternately to that portion of an antibody molecule that contains the amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen. This antibody portion includes the structural amino acid residues necessary to maintain the appropriate conformation of the antigen-binding residues. Preferably, the structural regions of the antibodies of the invention are of human origin or substantially of human origin (at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% human origin) and follow Kabat numbering. Alternatively, the antigen-binding region may be derived from a human sequence.
As used herein, the term antibody fragment refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., Aβ). Examples of molecules encompassed within the term antibody fragment of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, and (v) a dAb fragment (Ward, et al., (1989) Nature 341:544-546) consisting of a VH domain. Furthermore, although the two Fv fragment domains, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that allows them to be made as a single protein chain in which the VL and VH regions pair up to form monovalent molecules (known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be included within the term antibody fragment. Other forms of single-chain antibodies, such as diabodies, are also included within the term antibody fragment. Diabodies are bivalent, bispecific binding proteins in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, thus forcing the domains to pair with complementary domains of another chain and creating two antigen-binding sites (see for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).
Furthermore, an antibody or antibody fragment can also be part of a larger immunoadhesion molecule, formed by covalent or non-covalent association of the antibody or antibody fragment with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide, and a C-terminal polyhistidine marker to make the scFv molecules bivalent and biotinylated (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31:1047-1058). Antibody fragments, such as Fab and F(ab') fragments<sub>2</sub>Antibodies, antibody fragments, and immunoadhesion molecules can be prepared from whole antibodies using conventional techniques, such as digestion with papain or pepsin, respectively. Furthermore, antibodies, antibody fragments, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as are well known in the art. Antibodies, antibody fragments, and immunoadhesion molecules may or may not be glycosylated and still fall within the limits of the invention. Preferably, the antibody fragment is a Fab fragment.
The term humanized antibody refers to an antibody that is partially or completely composed of amino acid sequences derived from a human antibody germline or a rearranged sequence made by altering the sequence of an antibody that has non-human CRDs. The structural regions of the variable regions may be replaced by corresponding human structural regions. Human structure regions include genomic structure regions, as well as those containing one or more amino acid substitutions. In particular, such substitutions include mutations in which an amino acid at a particular position in the human structure is replaced with the amino acid at the corresponding position in the natural structure for the non-human CDR. For example, a humanized antibody having mouse CDRs may contain one or more substitutions that replace a particular human-structured amino acid with the corresponding mouse-structured amino acid. References also describing methods involved in humanizing a mouse antibody that can be used are, for example, Queen et al., Proc. Natl. Acad. Sci. USA 88:2869, 1991; Pat. US 5,693,761; Pat. US 4,816,397; Pat. US 5,225,539; ABMOD and ENCAD computer programs as described in Levitt, M., J. Mol. Biol. 168:595-20620, 1983; humanization can essentially be performed following the method of Winter and colleagues (Jones et al., Nature, 321:522-525, 1986; Riechmann et al., Nature, 332:323-327, 1988; Verhoeyen et al., Science, 239:1534-1536, 1988). Preferably, an antibody of the invention is a humanized antibody fragment. More preferably, an antibody of the invention is a humanized antibody Fab fragment.
The present invention also includes antibody fragments that are covalently linked to one or more PEG molecules. It is intended that the terms polyethylene glycol and PEG be used interchangeably and refer to polyethylene glycol or a derivative thereof as known in the art (see, for example, US Patents: 5,445,090; 5,900,461; 5,932,462; 6,436,386; 6,448,369; 6,437,025; 6,448,369; 6,495,659;
6,515,100 and 6,514,491). Preferably, PEG is covalently linked to one or more lysine or cysteine residues of the antibody fragment. More preferably, PEG is covalently linked to one or more lysine or cysteine residues in the variable region of the heavy chain of the antibody fragment. Even more preferably, PEG is covalently linked to one or more lysine or cysteine residues within the CDR of the antibody fragment. Most preferably, PEG is linked to a cysteine residue at amino acid position 56 of the variable heavy chain region of said SEQ ID NO: 2. Alternatively, PEG molecules can be linked to the antibody fragment by means of a linker or spacer molecule to the hinge region of the antibody fragment. Addition of linkers and spacer molecules to hinge regions is well known in the art. Furthermore, a PEG can be covalently linked to modified non-natural amino acids of the antibody fragment by techniques well known in the art.
In its typical form, PEG is a linear polymer with terminal hydroxyl groups and has the formula HO-CH<sub>2</sub>CH<sub>2</sub>-(CH<sub>2</sub>CH<sub>2</sub>THE)<sub>n</sub>-CH<sub>2</sub>CH<sub>2</sub>-OH where n is from about 8 to about 4000. The terminal hydrogen can be replaced with a protecting group such as an alkyl or alkanol group (MPEG). Preferably, PEG has at least one hydroxyl group, more preferably a terminal hydroxyl group. It is this hydroxyl group that is preferably activated to react with the peptide. A variety of chemical modifications are used to prepare an active PEG derivative with a functional group, such as an active carbonate, active ester, aldehyde, tresylate, or using suitable PEG-propionaldehyde to couple to a given target molecule. The activated PEG derivative is then covalently linked to a reactive group in the polypeptide drug. There are many forms of PEG useful for the present invention. Numerous PEG derivatives exist in the art and are suitable for use in the invention. The PEG molecule covalently linked to an antibody fragment of the present invention is not intended to be limited to a particular type or size. The molecular weight of the PEG is preferably from about 0.5 kilodaltons (kD) to about 100 kD, more preferably from about 5 kD to about 30 kD, and most preferably from about 1 kD to about 20 kD. PEG can be linear or branched, and the anti-Αβ peptide antibody fragment of the invention can have 1, 2, 3, 4, 5, or 6 PEG molecules attached to the peptide. It is much preferable that there be one PEG molecule antibody fragment; however, when more than one PEG molecule per peptide molecule is present, it is preferred that there be no more than six. It is also contemplated that both ends of the PEG molecule can be adapted to crosslink two or more anti-αβ peptide antibody fragment molecules together. Methods of linking PEG molecules to proteins, antibodies, and fragments thereof are well known in the art.
The term Kd, as used here, is intended to refer to the dissociation constant of a particular antibody-antigen interaction. It is calculated using the formula:
Kd = koff/kon (measurement in M)
The term k<sub>on</sub> As used herein, it is intended to refer to the association rate constant, or specific reaction rate, of the forward, or complex-forming, reaction, measured in units: M'<sup>1</sup>sec'<sup>1</sup>The term k<sub>off</sub>, as used herein, is intended to refer to the dissociation rate constant, or specific reaction rate, for the dissociation of an antibody from the antibody/antigen complex, measured in units: sec'<sup>1</sup>.
<sup>1</sup> · 20 The term specifically binds as used here refers to the situation where one member of a specific bonding pair does not bind significantly to molecules other than its specific bonding pair(s). The term is also applicable where, for example, an antigen-binding domain of an antibody of the invention is specific for a particular epitope that is carried by several antigens, in which case the specific antibody carrying the antigen-binding domain may bind to the various antigens carrying the epitope. Consequently, a molecule of the invention specifically binds to the Aβ peptide while specifically not binding to APP. Furthermore, a molecule of the invention specifically binds between a linear, non-linear or conformational Αβ epitope comprising the amino acids HHQKLVFFAEDVGSNK (13-28) (SEQ ID NO: 4).
The term activity in reference to a molecule of the present invention includes, but is not limited to, epitope/antigen affinity and specificity, ability to neutralize or antagonize an Aβ peptide activity in vivo or in vitro, IC<sub>5</sub>The in vivo stability of the antibody and the immunogenic properties of the antibody. Other identifiable biological properties or characteristics of an antibody recognized in the technique include, for example, cross-reactivity (i.e., with non-human homologs of the targeted peptide, or with other proteins or tissues in general), and the ability to maintain high levels of protein expression in mammalian cells. The aforementioned properties or characteristics can be observed, measured, or evaluated using techniques recognized in the art, including but not limited to ELISA, competitive ELISA, Biacore or KinExA surface plasmon resonance analysis, in vitro or in vivo neutralization assays without limitation, receptor binding, cytokine or growth factor production and/or secretion. Signal transduction and immunohistochemistry with tissue sections from different sources including human, primate, or any other source.
The terms individual, subject, and patient, used interchangeably herein, refer to a mammal, preferably human. In a certain embodiment, the subject is also characterized by a disease or disorder or condition that would benefit from decreased activity of the Aβ peptide.
As used herein, the terms host cell, host cell line, and host cell culture are used interchangeably and include an individual cell or cell culture that is a recipient of any isolated polynucleotide of the invention or any recombinant vector(s) comprising a sequence encoding an HCVR, LCVR, or monoclonal antibody of the invention. Host cells include progeny of a single host cell. The progeny may not be completely identical, in morphology or in total DNA complement, to the original parent cell due to mutation and/or natural, accidental, or deliberate alteration. A host cell includes cells transformed, transduced, or infected with a recombinant vector or a polynucleotide expressing an antibody fragment of the invention or a light chain or heavy chain thereof. A host cell comprising a recombinant vector of the invention, whether stably incorporated into the host chromosome or not, may also be referred to as a recombinant host cell. Preferred cells for generating host cells of the invention are CHO cells (e.g., ATCC CRL-9096), NSO cells, SP2/0 cells, COS cells (ATCC, e.g., CRL-1650, CRL-1651) and HeLa cells (ATCC CCL-2). Additional host cells for use in the invention include plant cells, yeast cells, other mammalian cells, and prokaryotic cells. More preferably, the cells for use in the invention are yeast or prokaryotic cells.
The term Αβ-related condition or disease, or conditions associated with Αβ-activity disease, is meant to include all conditions, disorders, and diseases that are associated with: 1) the development of β-amyloid plaques in the brain, 2) the synthesis of abnormal forms of Αβ, 3) the formation of particularly toxic forms of Αβ, or 4) abnormal rates of synthesis, degradation, or release of Αβ. Conditions and diseases such as Alzheimer's disease, Down syndrome, and angiopathy are examples.
-20 cerebral amyloid, certain vascular dementias, and moderate cognitive impairment are known or suspected to have such a relationship with Aβ.
This invention provides a molecule comprising an antibody fragment that specifically binds to the Aβ peptide between amino acid positions 13-28, wherein the antibody fragment is covalently linked to a PEG molecule. The antibody fragment is preferably a humanized antibody fragment, such as a Fab fragment and/or scFv fragment. Most preferably, the antibody fragment is a Fab fragment. Specific binding of the molecules of the invention to the Aβ peptide allows the use of said molecules as a therapeutic for diseases and disorders associated with the Aβ peptide, that is, conditions, diseases or disorders that benefit from the inhibition of a biological activity of the Aβ peptide.
In one embodiment of the invention, the antibody fragment has a variable heavy chain region and a variable light chain region, wherein the variable light chain region comprises CDR regions with the following amino acid sequences: CDRL1: SSSQSLIYSDGNAYLH (SEQ ID NO: 6), CDRL2: KVSNRFS (SEQ ID NO: 7) and CDRL3: TQSTHSPWT (SEQ ID NO: 8) and/or wherein the variable heavy chain region comprises CDR regions with the following amino acid sequences: CDRH1: GYTFSRYSMS (SEQ ID NO: 9), CDRH2: QINIRGÇNTYYPDTVKG (SEQ ID NO: 10) or QINIRGNNTYYPDTVKG (SEQ ID NO: 11), and CDRH3: GDF (SEQ ID NO: 12). Preferably, the six CDRs of an antibody fragment of the invention exist together. The composition comprising a CDR of the invention will generally be an antibody heavy or light chain sequence or a substantial portion thereof in which the CDR is located at a location consistent with Kabat numbering. The three CDR regions for each chain, heavy and light, are provided in a structure region as a contiguous sequence represented by the following formula: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The heavy chain or light chain FR1, FR2, FR3, and FR4 combine to form the complete structure region of an antibody fragment when arranged as a contiguous sequence with the CDRs in the stated order. Preferably, the structural regions of an antibody of the invention are of human origin or substantially of human origin (i.e., more than about 80, 82, 85, 87, 90, 92, 95, 97%).
Preferably, the antibody fragment of the invention comprises an LCVR comprising a peptide of the following sequence:
DIVMTQT PLS LS VTPG QP AS ISCSSSQSLIYSDGNAYLHWYLQ KPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCT QSTHSPWTFGGGTKVEIK (SEQ ID NO: 1) and an HCVR comprising a peptide with a sequence selected from the group consisting of the following sequences:
EVQLVESGGGLVKPGGSLRLSCAASGYTFSRYSMSWVRQAP
GKGLEWVGQINIRGÇNTYYPDTVKGRFTISRDDSKNTLYLQMNSLKTEDTA < VYYCTTGDFWGQGTLVTVSS (SEQ ID NO: 2)
EVQLVESGGGLVKPGGSLRLSCAASGYTFSRYSMSWVRQAP
GKGLEWVGQINIRGNNTYYPDTVKGRFTISRDDSKNTLYLQMNSLKTEDTA VYYCTTGDFWGQGTLVTVSS (SEQ ID NO: 3)
Alternatively, the antibody fragment comprises a
LCVR comprising a peptide with a sequence consisting of SEQ ID NO: 1 and an HCVR comprising a peptide with a sequence selected from the group consisting of SEQ ID NO: 2 or SEQ ID NO: 3, wherein the HCVR and LCVR exist together in an antibody fragment. The skilled craftsman will appreciate that the antibody fragments of the invention are not limited to specific HCVR and LCVR sequences, but also include variants of these sequences which, when present in a molecule of the invention, retain or enhance antigen-binding ability and at least one other functional property of the parent antibody, for example, epitope specificity, ability to compete with the parent antibody to bind to the Aβ peptide, IC50 values.<sub>50</sub> and/or K<sub>D</sub> or k<sub>of</sub>f to bind to the Aβ peptide.
In another embodiment of the invention, all or a portion of the variable region is bounded by a particular LCVR sequence as shown.
-- 20 by a SEQ ID NO: 1 and HCVR as shown in SEQ ID NO: 2 or SEQ ID NO: 3 and is also characterized by antagonizing or neutralizing at least one activity of the Aβ peptide in vivo or in vitro. An antibody of the invention, wherein all or a portion of the variable region is bounded by a particular sequence as shown by an LCVR SEQ ID NO: 1 and HCVR
SEQ ID NO: 2 or SEQ ID NO: 3 is also characterized here by specifically binding to human Aβ peptide but not binding to human APP.
In one aspect of the present invention, PEG (or a derivative thereof) is covalently linked to one or more lysine, cysteine, or non-natural modified amino acid residues of an antibody fragment.
Preferably, the PEG molecule is covalently linked to either the variable heavy chain region or the variable light chain region of the antibody fragment. More preferably, such a molecule has a molecule of
PEG that is covalently linked to a CDR of a variable heavy chain region of the antibody fragment. Most preferably, such a molecule has a PEG molecule that is covalently linked to cysteine at amino acid position 56 of the variable heavy chain region of SEQ ID NO: 2. Alternatively, PEG molecules can be linked to the anti-Αβ peptide Fab antibody fragment via a linker or spacer molecule to the hinge region of the antibody fragment.
In another aspect of the present invention, a PEG molecule is covalently linked to one or more lysine, cysteine, or engineered non-natural modified amino acid residues of the antibody of the present invention to replace a glycosylation present in the antibody molecules without significantly affecting the affinity and selectivity of the antibody fragment for Aβ. Preferably, the PEG molecule replaces the glycosylation signal in the variable region of the heavy chain or in the variable region of the light chain of the antibody fragment. More preferably, the PEG molecule replaces the glycosylation signal in the CDR of a variable region of the heavy chain of the antibody fragment. Most preferably, the PEG molecule replaces the glycosylation signal at position 56 of the variable region of the heavy chain of SEQ ID NO: 2.
The PEG molecule covalently linked to an antibody in the present invention is not intended to be limited to a particular type or size. The molecular weight of PEG is preferably from about 0.5 kD to about 100 kD, and more preferably from about 0.5 kD to about 30 kD, and most preferably from about 1 kD to about 20 kD. Alternatively, the molecular weight of the PEG can be selected from a group consisting of about 0.5 kD, about 1 kD, about 5 kD, about 10 kD, and about 20 kD. PEG can be linear or branched, and the PEGylated anti-α peptide antibody of the invention can have more than one PEG molecule attached to the peptide. Preferably, there is one PEG molecule per PEGylated anti-α peptide antibody.
Most preferably, the antibody molecule of this invention comprises an antibody fragment with a variable light chain region of SEQ ID NO: 1 and a variable heavy chain region of SEQ ID NO: 2, wherein said antibody fragment is covalently linked to a 20 kD PEG molecule at position 56 of the variable heavy chain region of SEQ ID NO: 2.
The antigenic Αβ peptide epitope, between which the antibodies of the invention bind, is a linear, non-linear, or conformational epitope comprising HHQKLVFFAEDVGSNK amino acids (SEQ ID NO: 4). Antibodies that bind to said epitope specifically and preferentially bind to the Αβ peptide when compared to its APP ligand. The monoclonal antibodies of the invention bind to the Aβ peptide at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times more (e.g., higher affinity or higher specificity) than they would bind to human APP; more preferably at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 times more than they would bind to APP, or even more preferably do not bind to it.
APP at levels higher than baseline levels as determined, for example, by ELISA assay, competition ELISA assay or K values.<sub>D</sub>in a Biacore or KinExA assay.
The antibody fragments of the invention bind to an epitope between the amino acids HQKLVFFAEDVGSNK (SEQ ID NO: 5) at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times more (e.g., higher affinity or higher specificity) than an epitope not comprising the amino acids HQKLVFFAEDVGSNK (SEQ ID NO: 5). More preferably, at least 150, 200, 250, 300, 350, 400, 450, 500, 550 or 600 times more than with an epitope not comprising the amino acids HQKLVFFA25 EDVGSNK (SEQ ID NO: 5), even more preferably does not bind to an epitope not comprising the amino acids HQKLVFFAEDVGSNK (SEQ ID NO: 5) at levels greater than baseline levels as determined, for example, by ELISA assay, competitive ELISA assay or Kd values in a Biacore or KinExA assay.
In a preferred embodiment, the invention provides an antibody fragment that has a strong binding affinity for the Aβ peptide, that is, it binds to the Aβ peptide, or a portion thereof, comprising the sequence HQKLVFFAEDVGSNK (SEQ ID NO: 5) [i.e., antibody contacts the HQKLVFFAEDVGSNK polypeptide], with a binding affinity (Kd) for human Aβ peptide of less than about 200 pM, 100 pM, 50 pM, 40 pM, or 30 pM, preferably less than about 20 pM when measured by the KinExA method. Alternatively, the binding affinity (Kd)<sub>D</sub>The antibody affinity for human Aβ peptide ranges from 0.1 pM to 200 pM. Antibody affinities can be determined as described in the examples below and by other methods available in the art.
The route of administration of an antibody of the present invention may be oral, parenteral, via inhalation, or topical. Preferably, the antibodies of the invention may be incorporated into a pharmaceutical composition suitable for parenteral administration. The term parenteral as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, or intraperitoneal administration. Peripheral systemic release by intravenous, intraperitoneal, or subcutaneous injection is preferred. More preferably, the route of administration of an antibody of the present invention is by subcutaneous injection. Suitable vehicles for such injections are obvious in the art.
The antibody fragment of the present invention has a shorter half-life than the corresponding full-compliance anti-Aβ peptide antibody in plasma and is cleared from plasma more rapidly than the corresponding full-compliance anti-Aβ peptide antibody. Alternatively, the antibody of the present invention has a longer plasma half-life than the corresponding anti-Αβ peptide Fab fragment that is not covalently linked to a PEG molecule, and is cleared less rapidly from the plasma than the corresponding anti-Αβ peptide Fab fragment that is not covalently linked to a PEG molecule (Examples 1, 2 and 3). The term "corresponding" in reference to an antibody as used herein refers to an antibody with the same LCVR and HCVR. For example, the full-compliance antibody corresponding to a Fab fragment of antibody having an LCVR of SEQ ID NO: 1 and an HCVR consisting of SEQ ID NO: 2 would have the same LCVR of SEQ ID NO: 1 and an HCVR consisting of SEQ ID NO: 2 along with an intact Fc domain.
In another aspect, the present invention is directed to recombinant polynucleotides encoding antibodies which, when expressed, comprise the LCVR of SEQ ID NO: 1 and an HCVR consisting of SEQ ID NO: 2. Due to codon degeneracy, other polynucleotide sequences can be easily substituted for those sequences. Particularly preferred polynucleotides of the present invention encode antibodies that, when expressed, comprise the light chain CDRs of SEQ ID NO: 6-8, and heavy chain CDRs of SEQ ID NO: 9, 10 or 11, and 12, or any of the variable regions of SEQ ID NO: 1-SEQ ID NO: 3. Examples of polynucleotide sequences encoding for LCVR of SEQ ID NO: 1 and HCVR of SEQ ID NO: 2 are represented in SEQ ID NO: 13 (LCVR) and SEQ ID NO: 14 (HCVR), respectively.
The polynucleotides will typically also include an expression control polynucleotide sequence operably linked to humanized immunoglobulin-coding sequences, including naturally associated or heterologous promoter regions. Preferably, the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic cells, but control sequences for prokaryotic cells can also be used. Once the vector has been incorporated into the appropriate host cell line, the host cell is propagated under conditions suitable for expressing the nucleotide sequences, and, as desired, collection and purification of light chains, heavy chains, light/heavy chain dimers or intact antibodies, binding fragments or other forms of immunoglobulin can follow.
The nucleic acid sequences of the present invention capable of ultimately expressing the desired antibodies or antibody fragments can be formed from a variety of different polynucleotides (genomic oligonucleotides or cDNA, RNA, synthetic, etc.) and components (e.g., V, J, D, and C regions), using any of a variety of well-known techniques. Combining the appropriate genomic and synthetic sequences is a common production method, but cDNA sequences can also be used.
Human constant region DNA sequences can be isolated, according to well-known procedures, from a variety of human cells, but preferably from immortalized B cells. Suitable source cells for the polynucleotide sequences and host cells for immunoglobulin expression and secretion can be obtained from several well-known sources in the art.
In addition to the humanized antibodies or antibody fragments specifically described herein, other substantially modified homologous antibodies can be readily designed and produced using various recombinant DNA techniques well known to those skilled in the art. For example, the structural regions can vary from the native sequences at the primary structural level by various amino acid substitutions, terminal and intermediate additions and deletions, and the like. Furthermore, a variety of different human structural regions can be used individually or in combination as a basis for the humanized antibodies of the present invention. In general, gene modifications can be easily performed by a variety of well-known techniques, such as directed mutagenesis.
As previously stated, polynucleotides will be expressed in hosts after the sequences have been operably ligated (i.e., positioned to ensure the functioning of) an expression control sequence. These expression vectors are typically replicable in host cells either as episomes or as an integral part of the host chromosomal DNA. Typically, expression vectors will contain selection markers, for example, tetracycline or neomycin, to allow detection of those host cells transformed with the desired DNA sequences. Expression vectors for these cells may include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, Adeno5 virus, Bovine Papillomavirus, cytomegalovirus, and similar genes.
Vectors containing the polynucleotide sequences of interest (e.g., heavy and light chain coding sequences and expression control sequences) can be transferred to the host cell by well-known methods, varying depending on the cell type. A variety of hosts can be employed to express the antibodies of the present invention using techniques well known in the art. Preferred cell lines include COS, CHO, SP2/0, NSO (available from public repositories such as ATCC, American Type Culture Collection, Manassas, VA) and yeast cell lines. Preferably, a host cell of the invention comprises one or more vectors or constructs comprising a nucleic acid molecule of the present invention. The host cell of the invention is a cell into which a vector of the invention has been introduced, said vector comprising a polynucleotide encoding an LCVR of the invention and/or a polynucleotide encoding...<sup>k</sup> · 20 is an HCVR of the invention. The invention also provides a host cell to which two vectors of the invention have been introduced: one comprising a polynucleotide encoding an LCVR of an antibody of the invention and one comprising a polynucleotide encoding an HCVR present in an antibody of the invention, each operably linked to a promoter sequence. Cell types include mammalian, bacterial, plant, and yeast cells. Preferably, the cell is a CHO cell, a COS cell, an SP2/0 cell, an NSO cell, a yeast cell, or a derivative or progeny of any preferred cell type.
Once expressed, the intact antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present invention can be purified according to standard art procedures, including ammonium sulfate precipitation, ion exchange, affinity, reverse phase, hydrophobic interaction column chromatography, gel electrophoresis, and the like. Substantially pure immunoglobulins of at least about 90%, 92%, 94%, or 96% homogeneity are preferred, and 98 to 99% or more homogeneity being more preferred, for pharmaceutical uses. Once purified, partially or to homogeneity as desired, the peptides can then be used therapeutically or prophylactically, as directed herein.
Several symptoms resulting in cognitive deficits, stroke, cerebral hemorrhage, and general mental impairment appear to be associated with neuritic and cerebrovascular plaques in the brain containing the Aβ peptide. These conditions include preclinical and clinical Alzheimer's disease, Down syndrome, and preclinical and clinical cerebral amyloid angiopathy (CAA). Amyloid plaques are formed from Aβ peptide. These peptides circulate in the blood and cerebrospinal fluid (CSF), typically in complexed form with lipoproteins. The circulating Αβ peptide is composed of 39-43 amino acids (mostly 40 or 42 amino acids) and is the result of the cleavage of a common precursor protein, amyloid precursor protein, often designated APP. Some soluble forms of APP are themselves neurotoxic and can determine the severity of neurodegeneration and/or cognitive decline (McLean, C. A., et al., Ann. Neturol. (1999) 46:860-866; Lambert, Μ. P., et al. (1998) 95:6448-6453; Naslund, J., J. Am. Med. Assoe. (2000) 283:1571).
Therefore, a pharmaceutical composition comprising a molecule of the invention may be useful for the treatment or prevention of conditions in which the presence of peptide Aβ causes or contributes to undesirable pathological effects or decreased activity of peptide Aβ has a therapeutic benefit in mammals, preferably humans, including, but not limited to, clinical or preclinical Alzheimer's disease, Down syndrome, clinical or preclinical amyloid angiopathy (CAA), Prodromal Alzheimer's disease, moderate cognitive impairment (MCI) and cognitive deficits, stroke, cerebral hemorrhage, and general mental impairment appear to be associated with neuritic and cerebrovascular plaques in the brain containing the Aβ peptide. The use of a molecule of the present invention to treat or prevent at least one of the aforementioned disorders in which the activity of the Aβ peptide is detrimental or which benefits from decreased levels of the bioactive Aβ peptide is contemplated herein. Additionally, the use of a molecule of the present invention for use in the manufacture of a medicament for the treatment of at least one of the aforementioned disorders is contemplated.
As used herein, the terms treatment, treating, and similar terms refer to achieving a desired pharmacological and/or physiological effect. The effect may be a partial or complete cure for a disease and/or an adverse effect attributable to disease progression. Treatment, as used herein, involves administering a compound, particularly to a human, and includes: (a) inhibiting the disease, that is, halting its development; or (b) alleviating the disease, that is, causing regression of the disease or disorder or alleviating its symptoms or complications. Dosage regimens may be adjusted to provide the desired optimal response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be given over time, or the dose may be proportionally reduced or increased as indicated.<sup>k</sup> · 20 due to the demands of the therapeutic situation.
A molecule of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. The molecules of the invention can be administered alone or in combination with a pharmaceutically acceptable vehicle, diluent, and/or excipient, in single or multiple doses. Pharmaceutical compositions for administration are designed to be appropriate for the selected route of administration, and pharmaceutically acceptable diluent, vehicle, and/or excipients such as dispersing agents, buffers, surfactants, preservatives, solubilizing agents, isotonic agents, stabilizing agents, and others are used when appropriate (See, for example, Example 14 here). These compositions are designed according to conventional techniques, such as those described in Remington, The Science and Practice of Pharmacy.
19<sup>the</sup> Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA 1995, which provides a compendium of formulation techniques as they are generally known to practitioners.
A pharmaceutical composition comprising a molecule of the present invention may be administered to a subject at risk of or exhibiting pathologies as described herein using standard administration techniques including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. Preferably, a molecule of the present invention can be administered to a subject at risk of or exhibiting the pathologies as described herein by subcutaneous administration.
A pharmaceutical composition of the invention is preferably a therapeutically effective amount or a prophylactically effective amount of a molecule of the invention. A therapeutically effective amount refers to an effective quantity, in dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of a molecule can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the molecule's ability to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or harmful effects of the molecule are exceeded in value by the therapeutically beneficial effects. A prophylactically effective amount refers to an amount that is effective, in dosages and for the time periods required, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects before or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.
A therapeutically or prophylactically effective amount is at least the minimum dose, but less than a toxic dose, of an active agent that is necessary to give a therapeutic benefit to a subject. Stated otherwise, a therapeutically effective amount of a molecule of the invention is an amount that, in mammals, preferably humans, <· decreases the activity of the Aβ peptide, for example, by binding to the Aβ peptide, wherein the presence of the Aβ peptide causes or contributes to undesirable pathological effects or a decrease in the Aβ peptide results in a beneficial therapeutic effect in a mammal, preferably a human.
The route of administration of a molecule of the present invention may be oral, parenteral, via inhalation, or topical. Preferably, the antibodies of the invention may be incorporated into a pharmaceutical composition suitable for parenteral administration. The term parenteral as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, or intraperitoneal administration. Peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection is preferred. Subcutaneous injection is the most preferred method. Suitable vehicles for such injections are obvious in the technique.
The pharmaceutical composition must typically be sterile and stable under the manufacturing and storage conditions in the supplied container, including, for example, a sealed vial or syringe. Therefore, pharmaceutical compositions may be sterile filtered after formulation, or otherwise made microbiologically acceptable. A typical intravenous infusion solution could contain a volume of up to 250-1000 ml of fluid, such as sterile Ringer's solution, physiological saline solution, etc.<sup>k</sup> 20. Mix dextrose and Hank's solution with a therapeutically effective dose (e.g., 1 to 100 mg/ml, or more) of the therapeutic agent to release the typical dosages listed below. The dose may vary depending on the type and severity of the disease. As is well known in medical techniques, dosages for any subject depend on many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs that are administered concurrently. A typical dose might be, for example, in the range of 0.001 to 1000 pg; however, doses below or above this exemplary range are targeted, especially considering the factors mentioned above. The daily parenteral dosage regimen may be approximately 0.1 pg/kg to approximately 100 mg/kg of total body weight, preferably approximately 0.3 pg/kg to approximately 10 mg/kg, and more preferably approximately 1 pg/kg to 1 mg/kg, even more preferably approximately 0.5 to 10 mg/kg of body weight per day. Progress may be monitored by periodic assessment. For repeated administrations over several days or much longer, depending on the condition, treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and are not excluded. The desired dosage may be delivered by a single bolus administration, by multiple bolus administrations, or by continuous infusion of the molecule, depending on the pharmacokinetic decay pattern the physician wishes to achieve.
These suggested quantities of the molecules of the invention are subject to considerable therapeutic discretion. The key factor in selecting an appropriate dose and schedule is the outcome obtained. Factors to consider in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the antibody release site, the particular type of antibody, the method of administration, the administration schedule, and other factors known to physicians.
The therapeutic agents of the invention can be frozen or lyophilized for storage and can be reconstituted in a suitable sterile vehicle before use. Lyophilization and reconstitution may lead to varying degrees of loss of antibody activity. Dosages may need to be adjusted to compensate.
The following examples are intended to illustrate, but not limit, the invention. The examples below employ, among others, a murine monoclonal antibody designated 266 (m266) that was originally prepared by immunization with a peptide composed of residues 13-28 of human Aβ peptide and a Fab fragment of the murine monoclonal antibody designated 266 (m266-Fab). The antibody is confirmed to immunoreact with this peptide. The preparation of m266 has been previously described. To covalently link a PEG molecule to m266-Fab, Fab can be mutated to introduce a cysteine residue at CDR2 (N56C) of the variable heavy chain and PEGylated in a manner shown below (Example 4). As the examples here describe experiments conducted in murine systems, the use of murine monoclonal antibodies is satisfactory. However, in the treatment methods of the invention intended for human use, humanized forms of the antibodies of the present invention, or fragments thereof, are preferred. The 1A1-Fab referred to in the examples below is a humanized antibody Fab fragment comprising LCVR from SEQ ID NO: 1 and HCVR from SEQ ID NO: 2.
EXAMPLE 1
Studies of subcutaneous PK/PD of m266-FAB PEG in PDAPP mice
Young (3 months old) transgenic PDAPP mice are used to investigate the plasma pharmacokinetic/pharmacodynamic response of antibody and antibody-Αβ complex. Several antibodies are investigated including mouse 266 Fab (m266-Fab), m266Fab+5KD PEG, m266-Fab+10KD PEG, m266-Fab+20KD PEG, and intact full-length m266 IgG antibody. PDAPP+/- mice are injected subcutaneously with 1 mg/kg of antibody, and plasma is subsequently isolated at the following time points: 1, 4, 8, 24, 48, 96, 168, and 240 hours post-dose. Animals receiving the m266-Fab antibody are analyzed with additional early time points due to the rapid turnover of this half. The time points for m266-Fab are as follows: 1, 4, 8, 12, 16, 24, and 48 hours post-dose. A total of five animals are analyzed per antibody per time point. Whole blood is obtained via cardiac puncture using 23-gauge needles connected to 1CC syringes that have been previously rinsed with 0.5 M EDTA. Blood samples are incubated on ice during the isolation procedure and subsequently centrifuged at 14,000 RPM in a microcentrifuge refrigerated at 4 degrees for 15 minutes. The resulting plasma samples are aliquoted and stored at -80 degrees.
A. Methodology for PK analyses of Fab
Plasma Fab concentrations are determined using an antigen capture ELISA. Briefly, plates are coated with Αβ-BSA conjugate overnight at 4°C or 1 hour at 37°C then blocked with Pierce casein buffer. Standards, control samples, and study samples are added to the plates followed by a one-hour incubation at room temperature. A goat anti-mouse HRP is used for detection and a colorimetric response is developed with OPD substrate. Plates are read at an absorbance of A493 with a reference of A700. Immunoreactivity concentrations of plasma samples are determined from standard curves prepared from known quantities of m266 Fab in mouse plasma using a 4/5 parameter algorithm. The assay range for m266 Fab is 0.05 to 0.5 pg/ml. The range for PEGylated Fabs is 0.075 to 0.8 pg/ml.
The immunoreactivity concentrations of plasma samples are determined from standard curves prepared from known quantities of m266 Fab in mouse plasma using a 4/5 parameter algorithm. The assay range for m266 Fab is 0.05 to 0.5 pg/mL. The range for PEGylated Fabs is 0.075 to 0.8 pg/mL. Results clearly demonstrate that adding a PEG molecule and increasing the size of the PEG molecule increases the retention of PEGylated Fabs in plasma (2545 ng/ml after 8 hours for 20K PEGylated m266-Fab) when compared to non-PEGylated m266-Fabs (350 ng/ml after 8 hours).
B. m266 Αβ ELISA assay
To measure the amount of Aβ in plasma in the absence or presence of therapeutic antibody (full length or Fab fragment), an ELISA assay is developed and used. The Aβ peptides being measured in these assays are full-length Aβ1-40 or Aβ1-42. 96-well Immulon 4HBX ELISA plates (ThermoLabsystems) are coated overnight at 4 degrees with C-terminal capture antibody (m2G3 for Aβ40 plates or m21F12 for Aβ42 plates) at 10 pg/ml in PBS (100 µl per well). The test plates are sealed to prevent evaporation during overnight incubation. The following day, the well solution is removed and the wells are washed three times with PBS (400 µl per well) using a Labsystems 96-well plate washer. Blocking buffer (360 pl of 1% milk-PBS) is added and the plates incubated at 37 degrees for one hour. Samples are prepared by diluting plasma in sample diluents to yield the following: 20% plasma, 0.5 M guanidine, 5 mM Tris pH 8.0, 0.5X protease inhibitor cocktail, 25 pg/ml m266, and PBS. The volume of plasma used in the assay may need to be reduced at certain time points due to the high levels of Aβ peptide present, and in these circumstances, the residual plasma volume is adjusted with rat plasma (percentage of final volume is maintained at 20%). The Aβ standards are generated with concentrations ranging from 250 pg/ml to 3.9 pg/ml in standard diluent (20% rat plasma, 0.5 M guanidine, 5 mM Tris pH 8.0, and 0.5X EDTA-free Complete protease inhibitor cocktail (Roche Diagnostics), 25 pg/ml m266, and PBS). The incorporation of 25 pg/ml of intact m266 into the sample and standard diluents is required to neutralize any negative interference that varying levels of core domain antibodies may exert on the assay. After blocking, the plates are washed 4 times with PBS. Samples and standards are loaded in triplicate (100 µl per well) and the plate is sealed and incubated overnight at 4 degrees. The following morning, the plates are washed 4 times with PBS-T (PBS + 0.05% Tween-20) and the wells incubated with the biotinylated secondary antibody m3D6 (100 µl per well diluted in 0.5% BSA/PBS-T) for 2 hours at room temperature. After the plates are washed 4 times with PBS-T, they are incubated with streptavidin-polyHRP (1:5000 in 0.5% BSA/PBS-T) for 1.5 hours at room temperature. The plates are washed 4 times with PBS-T and 100 µl per well of TMB substrate (Sigma) are added. Colorimetric progression is monitored at 650 nm at 15, 30, and 60 minutes.
Table 1. Pharmacodynamic results: Mean Plasma Concentration for Aβ 40 (pg/ml)
<td>Time (h)</td><td>m266 Fab</td><td>m266 Fab + 5KD PEG</td><td>m266 Fab + 10KD PEG</td><td>m266 Fab + 20KD PEG</td><td>m266 Intact</td>
<td> 1</td><td> 246,7</td><td> 253,5</td><td> 178,4</td><td> 196,3</td><td> 223,7</td>
<td> 4</td><td> 498,8</td><td> 693,2</td><td> 898,1</td><td> 816,6</td><td> 1110</td>
<td> 8</td><td> 576,7</td><td> 997,8</td><td> 1011</td><td> 1259</td><td> 1852</td>
<td> 12</td><td> 530,9</td><td></td><td></td><td></td><td></td>
<td> 18</td><td> 344,1</td><td></td><td></td><td></td><td></td>
<td> 24</td><td> 181,2</td><td> 914,7</td><td> 1728</td><td> 2966</td><td> 6919</td>
<td> 48</td><td></td><td> 200,1</td><td> 789,6</td><td> 2642</td><td> 8557</td>
<td> 96</td><td></td><td> 79,1</td><td> 104,8</td><td> 329,3</td><td> 10792</td>
<td> 168</td><td></td><td> 62,64</td><td> 76,49</td><td> 143,3</td><td> 9923</td>
<td> 240</td><td></td><td> 50,14</td><td> 98,24</td><td> 101,2</td><td> 6114</td>
In addition to a more flexible dosing program that can be manipulated based on PEG size, the results demonstrate that the pegylated Fab-antigen complex does not accumulate in the plasma circulation for extended periods of time like the intact antibody (intact m266). The intact antibody prolongs the antibody half-life in plasma and results in the antigen:antibody complex being present in the plasma circulation for extended periods of time (> 240 hours).
Native Fabs (m266 Fab), on the other hand, have a rapid release rate and a short half-life (< 24 hours) that limits them as a therapy. In contrast, as demonstrated in Table 1, PEGylated Fabs provide an antibody molecule with pharmacokinetics and pharmacodynamics that allow for an improved dosing regimen.
EXAMPLE 2
Studies of subcutaneous PK/PD PEG of 1A1-Fab in PDAPP mice
Studies are being conducted in young (3-month-old) transgenic PDAPP mice to investigate the pharmacokinetic/plasma pharmacodynamic response of the antibody and antibody-Αβ complex.
Several antibodies are being investigated, including humanized 1A1-Fab, 1A1Fab+5KD PEG, 1A1-Fab+10KD PEG, and 1A1-Fab+20KD PEG. PDAPP+/- mice are injected subcutaneously with 1 mg/kg of antibody, and plasma is subsequently isolated at different time points depending on the antibody injection group. The following time points are used for the various antibodies:
1A1-Fab patients are bled at 1, 4, 8, 12, 18, 24, and 48 hours post-dose.
1A1-Fab+ 5KD PEG are bled at 1, 4, 8, 24, 48, 96, and 168 hours post-dose.
1A1-Fab+ 10KD PEG are bled at 1, 4, 8, 24, 48, 96, and 168 hours post-dose.
1A1-Fab+ 20KD PEG are bled at 1, 8, 24, 48, 96, 168, and 240 hours post-dose.
A total of five animals are analyzed for antibody at each time point. The resulting plasma samples are aliquoted and stored at -80 degrees.
A. Methodology for PK analysis of Fab
Plasma concentrations of 1A1 Fab are determined using a sandwich ELISA. Plates are coated with goat anti-human IgG, standards, control samples, and study samples are added to the plates after incubation for one hour at room temperature. A goat anti-human IgG is used for detection followed by OPD for a colorimetric response. Plates are read at an absorbance of A493 with a reference of A700.
Concentrations of samples in plasma are determined from standard curves prepared with known amounts of 1A1 Fab in mouse plasma using a 4/5 parameter algorithm; the range for the Fab and Fab-5K PEG assays is 0.003 to 0.3 pg/ml; the ranges for the Fab-10K PEG assays are 0.006 to 0.2 and 0.04 to 0.4 pg/ml; the ranges for the Fab 20K PEG assays are 0.02-0.4 and 0.04-0.4 pg/ml. The results clearly demonstrate that the addition of a PEG molecule and increasing the size of the PEG molecule increases the retention of PEGylated Fabs in plasma (77 ng/ml after 96 hours for 20K Pegylated 1A1 Fab) when compared to non-Pegylated 1A1 Fab (undetectable after 24 hours).
B. 1A1 Aβ ELISA assay
The ELISA test is essentially the same as described above for m266.
Samples are prepared by diluting plasma in sample diluents to yield the following: 20% plasma, 0.5 M guanidine, 5 mM Tris pH 8.0, 0.5X protease inhibitor cocktail, 20 pg/ml 1A1, and PBS. The Αβ peptides being measured in these assays are full-length Αβ1-40 or Αβ1-42. Colorimetric progression is monitored at 650 nm at 15, 30, and 60 minutes. The results are presented in Table 2 below. Table 2. Pharmacodynamic results: Mean Plasma Concentration for Aβ 40 (pg/ml)
<td>Time (h)</td><td>1A1 Fab</td><td>1A1 Fab + 5KD PEG</td><td>1A1 Fab + 10KD PEG</td><td>1A1 Fab + 20KD PEG</td>
<td> 1</td><td> 136,8</td><td> 117,1</td><td> 116,3</td><td> 111,2</td>
<td> 4</td><td> 153,6</td><td> 208,2</td><td> 281,6</td><td></td>
<td> 8</td><td> 96,65</td><td> 198,4</td><td> 406,3</td><td> 529,2</td>
<td> 12</td><td> 131,9</td><td></td><td></td><td></td>
<td> 18</td><td> 105,9</td><td></td><td></td><td></td>
<td> 24</td><td> 114,7</td><td> 133,6</td><td> 585,1</td><td> 1243</td>
<td> 48</td><td> 106,8</td><td> 95,12</td><td> 170,7</td><td> 642</td>
<td> 96</td><td></td><td> 88,48</td><td> 113,7</td><td> 177,9</td>
<td> 168</td><td></td><td> 93,96</td><td> 110,8</td><td> 125,4</td>
<td> 240</td><td></td><td></td><td></td><td> 200</td>
In a manner similar to the m266 Fabs in Example 1, the data in Table 2 demonstrate that humanized Fabs that are covalently linked to a PEG molecule also provide an ideal PK/PD profile that allows for a flexible dosing schedule while preventing the antibody-antigen complex from accumulating in the plasma circulation for extended periods of time.
EXAMPLE 3
PURIFICATION OF MURINE FAB 266 AND HUMANIZATION ANALOGUES
Supernatants from cell cultures transfected with mouse 266 Fab or humanized 1A1 Fab and analogs are purified using a two-step chromatography strategy consisting of cation exchange chromatography followed by size exclusion chromatography using Superdex 75 resin (GE Healthcare). Following collection, the culture supernatant is concentrated using TFF and subjected to dialysis along with a 20-fold excess volume of 10 mM pH 5 sodium acetate overnight at 4°C. Precipitate is removed by centrifugation and the supernatant is loaded onto a stacked SP sepharose bed (GE Healthcare) loaded with 10 mM pH 5 sodium acetate. The column is successively washed with 10 mM pH 5 sodium acetate containing larger amounts of NaCl until the Fab fragment elutes in approximately 90 to 110 mM NaCl. The column fractions containing active Fab are identified and pooled. The volume is reduced and buffer exchanged (PBS) using a >20 centrifugal concentrating device (Millipore). The final volume is adjusted to 13 mL and loaded onto a Superdex 75 size column. Fractions containing Fab eluting at approximately 50 kD are identified and grouped for further characterization and PEGylation.
EXAMPLE 4
PEGUILATION AND IN VITRO CHARACTERIZATION
Cysteine N56C in purified 1A1-Fab cell culture is blocked for PEGylation. Pierce's Reduce-IMM® Immobilized Reductant beads are used to selectively reduce cysteine from N56C. These reducing beads are extracted from the manufacturer's supplied column and used in a batch mode. ~4 ml of beads are first activated with 8 ml of 10 mM DTT in Reduce-IMM Equilibration Buffer #1 (sodium phosphate + EDTA, pH 8.0) for 30 m. The beads are then washed 3 times with PBS.
1.7 mg/ml of 1A1 N56C Fab in PBS pH 7.4 is added to the beads, and 10 mM EDTA is added to the mixture. The mixture is spun and incubated at room temperature for 4-5 hours. Fab is separated from the beads using Handee™ resin separators, and the beads are washed with PBS. Fab and washes are combined and reacted with 5 times molar excess of Peg-maleimide (20kPEG from NOF; 10kPEG from Sunbio; 5kPEG from Nektar) for one hour. The reaction mixture is dialyzed with 4 L of 10 mM sodium acetate buffer pH 5.0 so that Fab and Fab-PEG can be captured on a Sepharose SP column that is equilibrated with 10 mM sodium acetate buffer pH 5.0. Unreacted Fab and Fab-PEG are eluted with a salt gradient. They are eluted in 50 mM to 70 mM NaCl. The protein is also purified by size exclusion chromatography (Superdex75 column, GE Healthcare) with PBS as the mobile phase. The reduction reaction can be graded up and down. Similar methods can be used to prepare Pegylated Murine 266 Fab N56C.
Samples are analyzed using size exclusion chromatography to confirm the addition of PEG to the Fab. Size exclusion chromatography is performed using a TSK G3000PW XL column (Tosoh Bioscience). The column is operated at 0.5 ml/min with PBS plus 0.35 M NaCl at pH 7.4 using an Agilent HP1100 series analytical HPLC operating at 214 nm. Additionally, samples are analyzed using SDS-PAGE. 10 Pages of purified material are loaded into a 4-12% NuPage® Bis-Tris gel and stained with SimplyBlue® SafeStain.
EXAMPLE 5
Measuring kinetic constants with Biacore
The Biacore® 2000 instrument is also used to measure binding kinetics. The Biacore® utilizes the optical properties of surface plasmon resonance to detect changes in protein concentration of interacting molecules within a dextran biosensor array. Except as noted, all reagents and materials are purchased from Biacore® AB (Uppsala, Sweden). All measurements are performed at 25°C. Samples are dissolved in HBS-EP buffer (150 mM sodium chloride, 3 mM EDTA, 0.005% (w/v) P-20 surfactant, and 10 mM HEPES, pH 7.4). Goat anti-human capillary antibody is immobilized on flow cells 1 to 4 of a CM5 sensor integrated circuit at a level of 8000 response units (Ru) using an amine coupling kit.
Binding is evaluated using multiple analytical cycles. Each cycle is run at a flow rate of 50 pL/minute and consists of the following steps: injection of ~20 pL of an antibody binding composition at 10 pg/ml targeting a capture of 400-500 Rus, injection of 250 pL of Abeta
Human (1-40) (starting at 200 nM and using serial dilutions twice during each cycle) followed by 20 minutes for dissociation, and regeneration using ~30 μL of 10 mM glycine hydrochloride, pH 1.5. Association and dissociation rates were evaluated during each cycle using a 1:1 binding model (Langmuir) in the BIAevaluation software. Results show that
PEguilation at the N56C site has little impact on the binding affinity of Fab to human Abeta.
EXAMPLE 6
Measuring Equilibrium Constants with KinExa
KinExA analysis is used as an orthogonal method to measure binding affinity through equilibrium binding analysis due to the slow rate-off of the antigen-Fab complex. A KinExA 3000 instrument (Sapidyne Inst. Inc.) is used to measure binding kinetics. Briefly, the antigen is covalently coupled to the sepharose beads and the binding of free Fab/Fab-PEG to the beads is detected on the instrument. To measure Kd, individual tubes containing Fab/Fab-PEG (20 pM or 500 pM for 1A1-Fab-20kPEG, 5 pM or 50 pM for 1A1 Fab) with serially diluted human soluble Abeta antigen (1-40) (0-10 nM) are incubated for 30-50 hours at 37°C in PBS containing 1 mg/ml BSA to ensure equilibration. After incubation, free Fab/Fab-PEG in each equilibrated sample is determined on the KinExA 3000 according to the manufacturer's instructions. Kd values are determined by nCurve Analysis using KinExA 3000 software. The results demonstrate that 1A1
Fab binds tightly to human Abeta (19 pM), with ~10 times higher affinity compared to murine 266 Fab (240 pM). Furthermore, covalent binding of 20K PEG at the N56C site has no impact on the affinity of 1A1-Fab (12 pM).
EXAMPLE 7
Amyloid precursor protein (APP) binding analysis using cell-based ELISA
To evaluate the cross-reactivity of 266 Fabs/mAbs with the Abeta APP precursor, HEK 293 cells stably expressing APP (aa 1-751) are used. These cells are created by cloning the APP gene (1751) into a plasmid containing the neomycin resistance marker. The recombinant plasmid is transfected into HEK 293 and the cells are screened at 200 pg/ml of G418 to generate a stable upexpressing cell line. For ligation assays, 75,000 APP 751 cells are bathed in each well of a 96-well plate coated with PDL. Following incubation for 2 days in growth media (DMEM F12, 5% FBS, 10 mM Hepes pH 7.5, 200 pg/ml G418), the liquid is removed and 20 pg/ml Fab or mAb are added in PBS (with Ca/Mg) containing 10 mg/ml BSA. Ligation proceeds for 2 hours at 4°C and the cells are washed 3X with 10 mg/ml BSA. A secondary antibody (horseradish peroxidase-conjugated anti-kappa light chain (hrp)) specific for human or mouse light chain is added in PBS/BSA (Southern Biotech). A 1:5000 dilution in PBS/BSA is used for anti-human light chain and 1:2000 for anti-mouse light chain. Following a one-hour incubation at 4°C, the cells are washed 5X with BSA/PBS. Hrp activity, as a Fab/mAb binding function to APP, is measured by adding TMB substrate for 10 minutes. The reactions are transferred to a clear 96-well plate and absorbance at 650 nm is measured. Data indicate that pegylated 1A1-Fab and m266-Fab (5 kD, 10 kD, and 20 kD) confer selectivity for Abeta peptide in APP.
4.
<110>
<120> <130> <150> <151>
<160>
<170> <210> <211>
<212> -20 <213>
<220>
<223>
<400>
SEQUENCE LISTING Eli Lilly and Company Bales, Kelly R Bumol, Thomas F Chow, Chi-Kin Demattos, Ronald B Hansen, Ryan Kuchibholta, Uma Lu, Lirong
McDonnell, Peter
Pegylated FAB for ABeta X17087
US 60/885439
2007-01-18
PatentIn version 3.4
112
PRT
Artificial
Synthetic construction
<td>Asp</td><td>ile</td><td>go</td><td>Met</td><td>Thr</td><td>Gin</td><td>Thr</td><td>Pro</td><td>He read</td><td>to be</td><td>He read</td><td>to be</td><td>go</td><td>Thr</td><td>Pro</td><td>Gly</td>
<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>
<td>Gin</td><td>Pro</td><td>Allah</td><td>To be</td><td>Isle</td><td>to be</td><td>Cys</td><td>to be</td><td>To be</td><td>To be</td><td>Gin</td><td>To be</td><td>He read</td><td>Isle</td><td>Tyr</td><td>To be</td>
<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>
<td>Asp</td><td>Gly</td><td>Asn</td><td>Allah</td><td>Tyr</td><td>He read</td><td>HIS</td><td>Trp</td><td>Tyr</td><td>He read</td><td>Gin</td><td>Lys</td><td>Pro</td><td>Gly</td><td>Gl n</td><td>To be</td>
<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>
<td>Pro</td><td>Gin</td><td>He read</td><td>He read</td><td>Isle</td><td>Tyr</td><td>Lys</td><td>go</td><td>To be</td><td>Asn</td><td>Arg</td><td>Phe</td><td>To be</td><td>Gly</td><td>val</td><td>Pro</td>
<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>
<td>Asp</td><td>Arg</td><td>Phe</td><td>To be</td><td>Gly</td><td>To be</td><td>Gly</td><td>to be</td><td>Gly</td><td>Thr</td><td>Asp</td><td>Phe</td><td>Thr</td><td>He read</td><td>Lys</td><td>Isle</td>
<td></td><td> 65</td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>
<td></td><td>Ser Arg</td><td>go</td><td>Glu</td><td>Allah</td><td>Glu</td><td colspan="2">Asp goes</td><td colspan="2">Gly will</td><td>Tyr</td><td>Tyr</td><td>cys</td><td>Thr</td><td>Gin</td><td>To be</td>
<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>
<td></td><td>Thr His</td><td>To be</td><td>Pro</td><td>Trp</td><td>Thr</td><td colspan="4">Phe Gly Gly Gly</td><td>Thr</td><td>Lys</td><td>val</td><td>Glu</td><td>Isle</td><td>Lys</td>
<td> 5</td><td> <210></td><td> 2</td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>
<td></td><td> <211></td><td colspan="2"> 112</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> <212></td><td colspan="2">PRT</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> <213></td><td colspan="4">Artificial</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>
<td> 10</td><td> <220></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> <223></td><td colspan="6">Synthetic construction</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> <400></td><td> 2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>
<td></td><td>Glu Vai</td><td>Gin</td><td>He read</td><td>go</td><td>Glu</td><td>To be</td><td colspan="3">Gly Gly Gly</td><td>He read</td><td>go</td><td>Lys</td><td>Pro</td><td colspan="2">Gly Gly</td>
<td></td><td> 1</td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>
<td> 15</td><td>Be Read</td><td>Arg</td><td>He read</td><td>to be</td><td>cys</td><td>Allah</td><td>Allah</td><td>To be</td><td>Gly</td><td>Tyr</td><td>Thr</td><td>Phe</td><td>to be</td><td>Arg</td><td>Tyr</td>
<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>
<td></td><td>Ser Met</td><td>To be</td><td>Trp</td><td>Go</td><td>Arg</td><td>Gin</td><td>Allah</td><td>Pro</td><td>Gly</td><td>Lys</td><td>Gly</td><td>He read</td><td>Glu</td><td>Trp</td><td>Val</td>
<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>
<td></td><td>Gly Gin</td><td>Isle</td><td>Asn</td><td>ile</td><td>Arg</td><td>Gly</td><td>Cys</td><td>Asn</td><td>Thr</td><td>Tyr</td><td>Tyr</td><td>Pro</td><td>Asp</td><td>Thr</td><td>val</td>
<td> 20</td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>
<td></td><td>Lys Gly</td><td>Arg</td><td>Phe</td><td>Thr</td><td>Isle</td><td>To be</td><td>Arg</td><td>Asp</td><td>Asp</td><td>to be</td><td>Lys</td><td>Asn</td><td>Thr</td><td>He read</td><td>Tyr</td>
<td></td><td> 65</td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>
<td></td><td>Leu Gin</td><td>Met</td><td>Asn</td><td>To be</td><td>He read</td><td>Lys</td><td>Thr</td><td>Glu</td><td>Asp</td><td>Thr</td><td>Allah</td><td>val</td><td>Tyr</td><td>Tyr</td><td>Cys</td>
<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>
<td> 25</td><td>Thr Thr</td><td>Gly</td><td>Asp</td><td>Phe</td><td>Trp</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>He read</td><td>val</td><td>Thr</td><td>Val</td><td>To be</td><td>To be</td>
<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>
<210> 3 <211> 112 <212> PRT <213> Artificial <220>
<223> Synthetic construction <400> 3
<td>Glu</td><td>Go</td><td>Gin</td><td>He read</td><td>go</td><td>Glu</td><td>To be</td><td>Gly</td><td>Gly</td><td>Gly</td><td>He read</td><td>Go</td><td>Lys</td><td>Pro</td><td>Gly</td><td>Gly</td>
<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>
<td>To be</td><td>He read</td><td>Arg</td><td>He read</td><td>To be</td><td>Cys</td><td>Allah</td><td>Allah</td><td>To be</td><td>Gly</td><td>Tyr</td><td>Thr</td><td>Phe</td><td>To be</td><td>Arg</td><td>Tyr</td>
<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>
<td>To be</td><td>Met</td><td>To be</td><td>Trp</td><td>Go</td><td>Arg</td><td>Gin</td><td>Allah</td><td>Pro</td><td>Gly</td><td>Lys</td><td>Gly</td><td>He read</td><td>Glu</td><td>Trp</td><td>val</td>
<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>
<td>Gly</td><td>Gin</td><td>Isle</td><td>Asn</td><td>Isle</td><td>Arg</td><td>Gly</td><td>Asn</td><td>Asn</td><td>Thr</td><td>Tyr</td><td>Tyr</td><td>Pro</td><td>Asp</td><td>Thr</td><td>go</td>
<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>
<td>Lys</td><td>Gly</td><td>Arg</td><td>Phe</td><td>Thr</td><td>Isle</td><td>To be</td><td>Arg</td><td>Asp</td><td>Asp</td><td>To be</td><td>Lys</td><td>Asn</td><td>Thr</td><td>He read</td><td>Tyr</td>
<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>
<td>He read</td><td>Gin</td><td>Met</td><td>Asn</td><td>To be</td><td>He read</td><td>Lys</td><td>Thr</td><td>Glu</td><td>Asp</td><td>Thr</td><td>Allah</td><td>go</td><td>Tyr</td><td>Tyr</td><td>cys</td>
<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>
<td>Thr</td><td>Thr</td><td>Gly</td><td>Asp</td><td>Phe</td><td>Trp</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>He read</td><td>go</td><td>Thr</td><td>go</td><td>To be</td><td>to be</td>
100 105 110 <210> 4 <211> 16 <212> PRT <213> Homo sapiens <400> 4
His His Gin Lys Leu Vai Phe Phe Ala Glu Asp Vai Gly Ser Asn Lys 15 10 15 <210> 5 <211> 15 <212> PRT <213> Homo sapiens <400> 5
His Gin Lys Leu goes Phe Phe Ala Glu Asp goes Gly Ser Asn Lys 15 10 15 <210> 6 <211> 16 <212> PRT
<td> <213></td><td>Artificial</td>
<td> <220></td><td></td>
<td> <223></td><td>Synthetic construction</td>
<td> <400></td><td> 6</td>
Ser be be Gin Ser Leu lie Tyr ser Asp Gly Asn Ala Tyr Leu His
<td> 1</td><td> 5 10 15</td>
<td> <210></td><td> 7</td>
<td> <211></td><td> 7</td>
<td> <212></td><td>PRT</td>
<td> <213></td><td>Artificial</td>
<td> <220></td><td></td>
<td> <223></td><td>Synthetic construction</td>
<td> <400></td><td> 7</td>
<td>Lys Vai</td><td>Ser Asn Arg phe Ser</td>
<td> 1</td><td> 5</td>
<td> <210></td><td> 8</td>
<td> <211></td><td> 9</td>
<td> <212></td><td>PRT</td>
<td> <213></td><td>Artificial</td>
<td> <220></td><td></td>
<td> <223></td><td>Synthetic construction</td>
<td> <400></td><td> 8</td>
Thr Gin be Thr His be Pro Trp Thr
<td> 1</td><td> 5</td>
<td> <210></td><td> 9</td>
<td> <211></td><td> 10</td>
<td> <212></td><td>PRT</td>
<td> <213></td><td>Artificial</td>
<td> <220></td><td></td>
<td> <223></td><td>Synthetic construction</td>
<td> <400></td><td> 9</td>
Gly Tyr Thr Phe Ser Arg Tyr Ser Met Ser
<td></td><td> 1</td><td colspan="2"> 5</td><td colspan="4"> 10</td>
<td></td><td> <210></td><td> 10</td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> <211></td><td> 16</td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> <212></td><td>PRT</td><td></td><td></td><td></td><td></td><td></td>
<td> 5</td><td> <213></td><td>Artificial</td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> <220></td><td></td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> <223></td><td>Synthetic construction</td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> <400></td><td> 10</td><td></td><td></td><td></td><td></td><td></td>
<td></td><td>Gin Ile</td><td>Asn Ile Arg Gly Cys Asn</td><td>Thr</td><td>Tyr Tyr</td><td>Pro Asp</td><td>Thr</td><td>go</td>
<td> 10</td><td> 1</td><td> 5</td><td></td><td> 10</td><td></td><td></td><td> 15</td>
<td></td><td> <210></td><td> 11</td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> <211></td><td> 17</td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> <212></td><td>PRT</td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> <213></td><td>Artificial</td><td></td><td></td><td></td><td></td><td></td>
<td> 15</td><td> <220></td><td></td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> <223></td><td>Synthetic construction</td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> <400></td><td> 11</td><td></td><td></td><td></td><td></td><td></td>
<td></td><td>Gin Ile</td><td>Asn Ile Arg Gly Asn Asn</td><td>Thr</td><td>Tyr Tyr</td><td>Pro Asp</td><td>Thr</td><td>Go</td>
<td></td><td> 1</td><td> 5</td><td></td><td> 10</td><td></td><td></td><td> 15</td>
<td> 20</td><td>Gly</td><td></td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> <210></td><td> 12</td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> <211></td><td> 3</td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> <212></td><td>PRT</td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> <213></td><td>Artificial</td><td></td><td></td><td></td><td></td><td></td>
<td> 25</td><td> <220></td><td></td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> <223></td><td>Synthetic construction</td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> <400></td><td> 12</td><td></td><td></td><td></td><td></td><td></td>
<td></td><td>Gly Asp</td><td>Phe</td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td>
<td> 30</td><td> <210></td><td> 13</td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> <211></td><td> 657</td><td></td><td></td><td></td><td></td><td></td>
<td></td><td> <212></td><td>DNA</td><td></td><td></td><td></td><td></td><td></td>
<td> <213></td><td>Artificial</td>
<td> <220></td><td></td>
<td> <223></td><td>Synthetic construction</td>
<td> <400></td><td> 13</td>
<td>gacatcgtta</td><td>tgactcagac tccattgtcc ttgtctgtta ctccaggtca accagcttct</td>
<td>atttcctgtt</td><td>cctcctccca atctttgatc tactccgacg gtaacgctta cttgcactgg</td>
<td>tacttgcaaa</td><td>agcctggtca atccccacaa ttgttgatct acaaggtttc caacagattc</td>
<td>tctggtgttc</td><td>ctgacagatt ttctggttcc ggttccggta ctgacttcac tttgaagatc</td>
<td>tccagagttg</td><td>aagctgagga tgttggtgtt tactactgta ctcagtccac tcattcccca</td>
<td>tggacttttg</td><td>gtggtggtac taaggttgag atcaagagaa ctgttgctgc tccatccgtt</td>
<td>ttcattttcc</td><td>caccatccga cgaacaattg aagtctggta ctgcttccgt tgtttgtttg</td>
<td>ttgaacaact</td><td>tctacccaag agaggctaag gttcagtgga aggttgacaa cgctttgcaa</td>
<td>tccggtaact</td><td>cccaagaatc cgttactgag caagactcta aggactccac ttactccttg</td>
<td>tcctccactt</td><td>tgactttgtc caaggctgat tacgagaagc acaaggttta cgcttgtgag</td>
<td>gttacacatc</td><td>agggtttgtc ctccccagtt actaagtcct tcaacagagg agagtcc</td>
<td> <210></td><td> 14</td>
<td> <211></td><td> 657</td>
<td> <212></td><td>DNA</td>
<td> <213></td><td>Artificial</td>
<td> <220></td><td></td>
<td> <223></td><td>Synthetic construction</td>
<td> <400></td><td> 14</td>
<td>gaggttcagt</td><td>tggttgaatc tggtggtgga ttggttaagc ctggtggttc tttgagattg</td>
<td>tcctgtgctg</td><td>cttccggtta cactttctcc agatactcca tgtcctgggt tagacaagct</td>
<td>ccaggaaagg</td><td>gattggagtg ggttggtcaa atcaacatca gaggttgtaa cacttactac</td>
<td>ccagacactg</td><td>ttaagggaag attcactatc tccagagatg actccaagaa cactttgtac</td>
<td>ttgcagatga</td><td>actccttgaa aactgaggac actgctgttt actactgtac tactggtgac</td>
<td>ttttggggac</td><td>agggaacttt ggttactgtt tcctccgctt ctactaaggg accatccgtt</td>
<td>tttccattgg</td><td>ctccatcctc taagtctact tccggtggta ctgctgcttt gggatgtttg</td>
<td>gttaaggact</td><td>acttcccaga gccagttact gtttcttgga actccggtgc tttgacttct</td>
<td>ggtgttcaca</td><td>ctttcccagc tgttttgcaa tcttccggtt tgtactcctt gtcctccgtt</td>
<td>gttactgttc</td><td>catcctcttc cttgggtact cagacttaca tctgtaacgt taaccacaag</td>
<td>ccatccaaca</td><td>ctaaggttga caagaaggtt gaaccaaagt cctctgacaa gactcac</td>
Contents13
1 sheet
Sheet 1
108 members in 30 offices
Priority claims3
| Document | Office | Kind | Date |
|---|---|---|---|
| 60885439 | United States of America | – | |
| 88543907 | United States of America | P | |
| 2008050554 | United States of America | W |
Members108
| Document | Office | Kind | |
|---|---|---|---|
| EP1935652A1 | European Patent Office (EPO) | A1 | |
| EP1935659A1 | European Patent Office (EPO) | A1 | |
| AU2007334792A1 | Australia | A1 | |
| WO2008074548A1 | World Intellectual Property Organization (WIPO) | A1 | |
| WO2008074588A1 | World Intellectual Property Organization (WIPO) | A1 | |
| WO2008074589A1 | World Intellectual Property Organization (WIPO) | A1 | |
| WO2008077850A2 | World Intellectual Property Organization (WIPO) | A2 | |
| AU2008206555A1 | Australia | A1 | |
| CA2675847A1 | Canada | A1 | |
| WO2008088983A1 | World Intellectual Property Organization (WIPO) | A1 | |
| CL2008000121A1 | Chile | A1 | |
| TW200836763A | Taiwan Province of China | A | |
| WO2008077850A3 | World Intellectual Property Organization (WIPO) | A3 | |
| PE20081634A1 | Peru | A1 | |
| AR064944A1 | Argentina | A1 | |
| MX2009007691A | Mexico | A | |
| EP2097247A2 | European Patent Office (EPO) | A2 | |
| EP2097265A1 | European Patent Office (EPO) | A1 | |
| KR20090101941A | Republic of Korea | A | |
| CN101563208A | China | A | |
| CN101563233A | China | A | |
| CN101563234A | China | A | |
| CN101573240A | China | A | |
| EP1935659B1 | European Patent Office (EPO) | B1 | |
| AT448086T | Austria | T | |
| ATE448086T1 | Austria | T1 | |
| EP2121754A1 | European Patent Office (EPO) | A1 | |
| DE602006010421D1 | Germany | D1 | |
| US2010007692A1 | United States of America | A1 | |
| US2010015155A1 | United States of America | A1 | |
| US2010026743A1 | United States of America | A1 | |
| US2010039463A1 | United States of America | A1 | |
| US2010047454A1 | United States of America | A1 | |
| EA200970694A1 | Eurasian Patent Organization (EAPO) | A1 | |
| ES2334256T3 | Spain | T3 | |
| IL199838D0 | Israel | D0 | |
| EP1935652B1 | European Patent Office (EPO) | B1 | |
| PL1935659T3 | Poland | T3 | |
| AT465014T | Austria | T | |
| ATE465014T1 | Austria | T1 | |
| DE602006013855D1 | Germany | D1 | |
| JP2010521330A | Japan | A | |
| JP2010522141A | Japan | A | |
| ES2342189T3 | Spain | T3 | |
| PL1935652T3 | Poland | T3 | |
| CN101981053A | China | A | |
| CN101573240B | China | B | |
| BRPI0806715A2This record | Brazil | A2 | |
| UA95996C2 | Ukraine | C2 | |
| US8066999B2 | United States of America | B2 | |
| CN101563233B | China | B | |
| US8142860B2 | United States of America | B2 | |
| US2012140005A1 | United States of America | A1 | |
| KR101160385B1 | Republic of Korea | B1 | |
| US8282197B2 | United States of America | B2 | |
| US8287112B2 | United States of America | B2 | |
| CN101563208B | China | B | |
| US2012277340A1 | United States of America | A1 | |
| AU2007334792B2 | Australia | B2 | |
| AU2012247077A1 | Australia | A1 | |
| CN101563234B | China | B | |
| AU2008206555B2 | Australia | B2 | |
| US8480203B2 | United States of America | B2 | |
| US8529049B2 | United States of America | B2 | |
| US2013235114A1 | United States of America | A1 | |
| JP5307030B2 | Japan | B2 | |
| JP5313163B2 | Japan | B2 | |
| CN101981053B | China | B | |
| JP2013241420A | Japan | A | |
| CN103479998A | China | A | |
| AU2012247077B2 | Australia | B2 | |
| KR20140015123A | Republic of Korea | A | |
| IL199838A | Israel | A | |
| IL230876D0 | Israel | D0 | |
| BRPI0720683A2 | Brazil | A2 | |
| US8757789B2 | United States of America | B2 | |
| KR101414847B1 | Republic of Korea | B1 | |
| HK1190623A1 | Hong Kong, China | A1 | |
| CA2675847C | Canada | C | |
| EP2121754B1 | European Patent Office (EPO) | B1 | |
| DK2121754T3 | Denmark | T3 | |
| EP2842967A1 | European Patent Office (EPO) | A1 | |
| EA020979B1 | Eurasian Patent Organization (EAPO) | B1 | |
| SI2121754T1 | Slovenia | T1 | |
| HRP20150237T1 | Croatia | T1 | |
| US9004663B2 | United States of America | B2 | |
| PT2121754E | Portugal | E | |
| ES2535641T3 | Spain | T3 | |
| PL2121754T3 | Poland | T3 | |
| RS53948B1 | Serbia | B1 | |
| IN3549CHN2009A | India | A | |
| IN3550CHN2009A | India | A | |
| IN3556CHN2009A | India | A | |
| IN3597CHN2009A | India | A | |
| JP5782479B2 | Japan | B2 | |
| CN103479998B | China | B | |
| EP2097247B1 | European Patent Office (EPO) | B1 | |
| IL230876A | Israel | A | |
| EP2842967B1 | European Patent Office (EPO) | B1 | |
| PT2842967T | Portugal | T |
7 legal events, as the office reported them to INPADOC
Over the term
Point at a mark for the eventEvents
| Event | Code | |
|---|---|---|
| Patent lapsed as no evidence of payment of the annual fee has been furnished to inpi [chapter 8.11 patent gazette]LapsedREFERENTE AO ARQUIVAMENTO PUBLICADO NA RPI 2496 DE 06/11/2018.B08K | B08K | |
| Application dismissed because of non-payment of annual fees [chapter 8.6 patent gazette]REFERENTE A 11A ANUIDADE.B08F | B08F | |
| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]B06F | B06F | |
| Others concerning applications: alteration of classificationB15K | B15K | |
| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]B07E | B07E | |
| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPIB07E | B07E | |
| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]B07D | B07D |
Numbers
- Publication
- PI0806715
- Application
- 8067155
Titles2
- Portuguese
- FAB PEGUILADO PARA ABETA
- English
- FAB pegylated FOR ABETA
Classification
- CPC, 15
- C07K16/18
- A61K2039/505
- A61K2039/6093
- B82Y5/00
- C07K2317/24
- C07K2317/56
- C07K2317/565
- C07K2319/31
- Y10S530/866
- A61K47/60
- A61K47/6893
- A61P25/00
- A61P25/28
- A61P9/00
- A61K39/395
- IPC, 5
- C07K16 18
- A61K39 395
- A61K47 48
- C12N15 13
- A61P25 28