DIRECTED SEQUENCE POLYMER COMPOSITIONS AND ANTIBODIES THEREOF FOR THE TREATMENT OF PROTEIN CONFORMATIONAL DISORDERS
The instant invention comprises a process for the solid phase synthesis of directed epitope peptide mixtures useful in the treatment and diagnosis of protein conformational disorders, such process defined by a set of rules regarding the identity and the frequency of occurrence of amino acids that substitute a base or native amino acid of a known epitope. The resulting composition is a mixture of related peptides for therapeutic use. The invention also pertains to the process of generating antibodies using the directed epitope peptide mixtures as the antigens, and antibodies generated by such process, useful in the treatment and diagnostics of the said protein conformational disorder.
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This application claims the benefit of U.S. Provisional Application No. 61/168,555, filed Apr. 10, 2009, and U.S. Provisional Application No. 61/124,689, filed Apr. 17, 2008, the specifications of which are hereby incorporated herein by reference in their entirety.
FIELD OF INVENTIONThis application provides methods of making improved compositions comprising certain mixtures of peptides, which mixtures may be synthesized as a single manufactured entity and designed based on disease-related polypeptides, and compositions comprising antibodies against such mixtures for the prophylactic and/or therapeutic treatment of protein conformational disorders.
BACKGROUND OF THE INVENTION Protein Conformational DisorderIt has been recognized in the recent years that there is a class of diseases and disorders that correlates with the presence of aggregates, whether intra- or extra-cellular, of misfolded or conformationally altered proteins. These proteins exist in a non-diseased environment. In a disease state, however, through certain alterations in the conformation, they adopt a secondary/tertiary structure different from those in the non-diseased state. The amino acid sequence is often unaltered. The misfolded proteins tend to self-associate, aggregating in an ordered fashion, for toxic precipitates, and deposit into tissues. The aggregated protein often takes a fibrillar appearance.
Examples of these disorders, now known as “protein conformational disorders” (PCDs), include but are not limited to Alzheimer's disease (AD), Parkinson disease (PD), Type-2 diabetes, amyotrophic lateral sclerosis (ALS), dialysis-related amyloidosis (DRA), reactive amylosis, cystic fibrosis (CF), sickle cell anemia, Huntington's disease (HD), Creutzfeldt-Jakob disease (CJD) ad related disorders, and systemic and cerebral hereditary amyloidosis. Examples of globular proteins that undergo fibrillogenesis include transthyretin, beta 2 microglobulin, serum amyloid A protein, Ig light chains, insulin, human lysozyme, alpha lactalbumin, and monellin. Examples of natively unfolded proteins that undergo fibrillogenesis include amyloid beta protein, tau protein, alpha-synuclein, amylin and prothymosin alpha.
Pathogenesis and Biochemical Progression of PCDInvestigators have correlated protein aggregate deposition with the degeneration of tissue. Although there remains controversy with regard to the “cause or effect” of the presence of aggregate and the manifestation of the disease pathology, evidence is accumulating that the pathology is caused by aggregates, perhaps by direct toxicity due to the aggregation or by a loss of biological function of the misfolded protein.
The formation of aggregates is referred to as “fibrillogenesis.” Before the start of fibrillogenesis, the protein relevant to PCD pathology is in a naturally folded conformation and in monomeric or defined oligomeric forms, each peptide comprising a mixture of alpha-helices, some beta-sheets, and random coils. By the end of fibrillogenesis, the protein is aggregated, and the peptide has adopted an altered conformation, i.e. mostly a beta-pleated sheet conformation. The conformational changes of the peptides and aggregation appear to coincide, but the cause and effect of conformational change and aggregation, and the sequence of events, remain to be elucidated.
When considering the pathogenesis of a PCD, it has been proposed that the fibrillogenesis is a crystallization-like process: after a “seed” of oligomers forms, an aggregate grows over time through self-association. The protein may take an altered conformation because the aggregate exists and serves as a template, or it may take the altered conformation because of other factors, but once in that conformation, easily participates in fibrillogenesis. In contrast, another proposal hypothesizes that the conformational alterations alone may not cause or promote aggregation, and there is a factor that induces the aggregation. Such underlying factors that promote or induce structural changes in the protein include inflammatory or oxidative environments, nitration, phosphorylation, pH, or metal ion exposure (high concentrations of copper ions can induce the oligomerization of β2 microglobulin monomers, which in turn leads to fibril formation (Eakin et al., Biochemistry 2004, 43, 7808-7815)).
Various treatment modes and possible therapeutic agents for PCDs are currently being investigated. Whether con formational change precedes the start of the fibrillogenesis or vice versa will influence the effectiveness of a treatment strategy. For example, a treatment mode with, an assumption that fibrillogenesis is caused by the beta-sheet conformation will attempt to inhibit the beta-sheet formation. In contrast, if the assumption was that aggregation promotes further formation of proteins with a degenerative conformation, a treatment mode may aim to inhibit aggregation by various means. An illustration of the former approach includes an attempt to inhibit the formation of, or to break, beta-sheets, using peptides. Such peptides are designed from the sequences of areas of proteins most likely involved in the process of nucleation and aggregation, such as the hydrophic core of amyloid-beta, a peptide intimately involved in the pathology of Alzheimer's disease. An illustration of the latter approach is an attempt to manipulate protein conformation and prohibit nucleation and subsequent formation of amyloids, or, “amyloidogenesis,” by creating mini-chaperone peptides from outside of the beta-sheet regions.
Another promising approach, regardless of the mechanism of aggregate formation, is to focus on the aggregates themselves. There have been attempts to reduce the level of aggregated protein of interest by antibodies: given sufficient specificity and ability to promote clearance, an antibody has a potential to be an effective therapeutic. To overcome delivery challenges, attempts have been made to express such antibodies intracellularly from a delivered gene. However, despite its potential, currently, the existing antibody therapeutics, if any, do not sufficiently prevent, improve, or even slow progression of the pathology, and there remains largely unmet needs for an effective treatment for a PCD.
Strategy for Creating Synthetic Therapeutic PeptidesThe development and exploitation of combinatorial chemistry (CC) has propelled drug discovery. Drug discovery can be generalized into two major steps, lead generation and lead optimization. Oftentimes, a lead compound is identified that has some of the desired characteristics of a commercially viable therapeutic, but has shortcomings such as a low specific activity, toxicity, instability, etc. Thus, once a lead is identified, practitioners attempt to optimize the lead compound by testing other related compounds with similar structures. CC allows practitioners to create and quickly screen a library made of a vast number of candidates, to identify those with a specific activity against a target of interest.
For peptide based drugs, the goal is to define a single, or a limited set of peptides which demonstrate a particular activity. The art of CC as applied to the synthesis of peptide libraries, too, has advanced, producing highly reliable and pure mixtures of peptides of great diversity. The process of identifying the single or limited set of peptides that were responsible for the observed activity from such diverse libraries, called deconvolution, is schematically represented in
An analogous process applies for development of a therapeutically effective antibody. Traditionally, antibodies were raised by immunizing an animal using a target protein or peptide as an antigen, either directly collecting sera for polyclonal antibodies (i.e. a mixture of antibodies enriched for those that bind to the target) or by creating hybridomas and selecting those hybridomas that produce monoclonal antibodies that bind to the target. In more recent years, phage display libraries have been used to present a large number of antibodies, from which antibodies that bind to the target is selected. In other words, antibody isolation is an initial screening of a lead molecule from a large number of candidates.
It is well known in the art, however, that an antibody that binds to the target is not necessarily one that has a desired therapeutic effect. As such, therapeutically effective antibodies may still have to be created through the process of lead optimization. The optimization may take a form of further screening of an antibody library (e.g. a phage display library), direct manipulation of complementarity determining regions of an immunoglobulin, or renewed immunization of an animal using related but different epitopes in an attempt to create a further variety in the enriched antibodies that the animal produces.
Low Immunogenicity, or Necessity for Highly Specific AntibodiesIn PCD, for therapeutic, prophylactic, and diagnostic purposes, the antibodies that are desirable recognize and specifically bind to proteins of certain altered conformation. The difficulty lies in the fact that these proteins exist as normal parts of the patient's system, were it not for the altered conformation that they are in. Thus, even though these proteins are pathological, they may not elicit strong natural immune responses in the afflicted individuals, and it may be difficult to elicit an immune response (thus to raise antibodies) using the native sequence of the target protein in other subjects of the same species, or in an individual with similar immunological profile, which is often desirable due to the lower probability of adverse immunological reaction.
Another challenge is that the antibody should differentiate between the same protein in a non-pathological conformation and in a pathological conformation. A protein relevant to a PCD may have the same primary structure, whether in a non-pathological condition or in pathological condition. Without the ability to distinguish, the antibody intended for therapeutic purposes may adversely affect the patient by eliminating or interfering with the normal, functioning protein. Thus, a high specificity towards the particular conformation, or series of alterations, is required.
Although immunization with an immunogen having a single epitope may induce multiple antibodies having complementarity determining regions (CDR) different from each other, it may be difficult to strongly elicit (and thus detect and identify) all varieties of antibodies. In addition, even if antibodies are induced, the most easily inducible and detectable antibodies against such epitope may not include those antibodies with a high specificity towards the particular pathological conformation as described in the preceding paragraphs. In an attempt to overcome these challenges, investigators have designed peptides with sequences similar to the target peptides. These variations of the target peptides may induce generation of antibodies that are different from those induced by the target peptides, but may cross-react sufficiently with the target peptides. Thus, these related peptides may be desirable and/or required to identify an antibody that may not be induced by an epitope of the original sequence.
One such approach is the creation of altered peptide ligands (APL). This approach is schematically represented in
Further complicating the application of the technology, an APL that may induce an antagonist-like reaction, may also induce a partial agonist response, or induce a state of anergy in the reactive T cell population. See, for example, Fairchild et al., Curr. Topics Peptide Protein Res. 2004, 6:237-44, who, in discussing APL in the context of allograft rejection therapy, note that an APL acting as an antagonist for one TCR, may become an agonist for another.
The approach using APL, along with other approaches currently known in the art, to identity therapeutic peptides, while recognizing the advantage of variations in the therapeutic peptide compositions, derive from the concept that there is one or more defined peptide sequence evoking a defined immunological response. These strategies have attempted to multiply and diversify modulatory peptides via the introduction of defined, single changes performed one at a time.
An entirely different approach which has evolved alongside the defined sequence peptide immunotherapy approach is the use of limited amino acid diversity, random epitope polymers. Random sequence polymers (RSP) can be described as a random order mixture of amino acid copolymers comprising two or more amino acid residues in various ratios, forming copolymers by random sequence bonding, preferably through peptide bonds, of these amino acid residues, which mixture is useful for invoking or attenuating certain immunological reactions when administered to a mammal. Because of the extensive diversity of the sequence mixture, a large number of therapeutically effective peptide sequences are likely included in the mixture. In addition, because of the additional peptides which may at any given time not be therapeutically effective, but may emerge as effective as the epitope shifting and spreading occurs, the therapeutic composition may remain effective over a time of dosing regimen. This approach is schematically represented in
Copolymer-1 (also known as Copaxone, glatiramer acetate, COP-1, or YEAK random copolymer), is used for the treatment of multiple sclerosis. Random copolymers are described in International PCT Publication Nos. WO 00/05250, WO 00/05249; WO 02/59143, WO 0027417, WO 96/32119, WO/2005/085323, in U.S. Patent Publication Nos. 2004/003888, 2002/005546, 2003/0004099, 2003/0064915 and 2002/0037848, in U.S. Pat. Nos. 6,514,938, 5,800,808 and 5,858,964, Copolymer-1 has been used in combination with a mucosal adjuvant and an A beta peptide for the development of an Alzheimer's vaccine (Frenkel, Dan et al., 2005, J Clin Invest., 115:2423), and has been described as a constituent in a method of vaccination designed to regenerate neuronal tissue (U.S. Pat. No. 6,844,314).
Tracing back steps to the defined peptide search, there have also been attempts to identify the active peptide(s) within the RSP mixture. The drawback of this technology lies in the very nature of the attempt to determine discrete substitutes for the randomness that COP-1 encompasses.
Effective as the random sequence polymer approach may be, even the improvements have not resolved the drawback and limitation of COP-1, which is, for example, the undefined nature of what is effective in each motif and the possibility of containing a large proportion of truly inactive peptides, lowering the concentration of the active components, or worse, adversely stimulating the immune system. Additionally, these compounds are difficult to manufacture and to obtain consistency from lot-to-lot.
Despite the modest success of the existing approaches, need remains for a composition and a method to create such composition that would serve effectively as a vaccine and immunogen by eliciting beneficial immune responses consistently and over time toward pathological proteins or peptides related to a PCD, for which existing vaccine compositions have failed to be effective.
SUMMARY OF THE INVENTIONThe instant invention comprises a process for the solid phase synthesis of directed epitope peptide mixtures useful in modulation of the immune system in the treatment of a protein conformational disorder, and the composition prepared by such process. The instant invention also comprises a process for producing antibodies that are therapeutically or prophylactically useful in treatment of protein conformational disorders, useful for use as research reagents, or useful as diagnostic tools for such disorders, by eliciting immune responses using a composition comprising directed epitope peptide mixtures, and the antibodies thus produced. An aspect of the invention is a method of prophylactic or therapeutic treatment for protein conformational disorders by administering the DSP composition of the present invention or by administering an antibody produced by the process of the present invention using a DSP composition. Another aspect is a method of diagnosis of a protein conformational disorder.
An aspect of the present invention is a process for manufacturing a composition comprising directed-sequence polymers (DSPs), and the further aspect of the present invention is a composition thus manufactured.
An embodiment of the invention is a process comprising the steps of (1) selecting a first base peptide sequence, wherein the sequence is an amino acid sequence of an epitope of an antigen associated with a protein conformational disorder; (2) synthesizing by solid phase peptide synthesis a first cassette of the DSPs according to a rule described below and particular input amino acid ratios; and (3) extending the length of the DSPs by repeating step (2) for 1 to 5 cycles, either under the same condition every cycle or using a different input ratio of amino acids in the mixture; or repeating steps (1) and (2) for 1 to 15 cycles and elongating the DSP using cassettes based on more than one base peptide; or assembling 1 to 15 cassettes synthesized in a single cycle of step (2), either all under the same condition in step (2) or in different conditions.
In the embodiment above, the cassette of the DSP is synthesized with one of several amino acids in each amino acid position. The amino acid to be incorporated to a particular position is randomly selected from (i) the original amino acid in that position; (ii) a replacement defined according to amino acid similarity shown in the similarity table of
The length of a DSP can be one of the original defined sequence peptide or 30 lengths of the original defined sequence peptide. The length of the combined sequence can be between about 10 and about 300 amino acids.
The percentage of alanine as compared to all of the other amino acids in the DSP combined is greater than 10%, and does not exceed 90%. In one embodiment, the alanine percentage is between 10% and 70%. In another embodiment, the alanine percentage is between 15% and 50%.
Thus, in one embodiment, the composition comprises DSP having a length of between about 10 to 300 amino acids, wherein each of such DSPs comprises between 1-15 cassettes, each block comprising between 8-100 amino acids; each cassette is derived from a base peptide sequence of an epitope of an antigen associated with a protein conformation disorder, and the amino acid residue of each amino acid position is selected from (i) the original amino acid in that position; (ii) a replacement defined according to amino acid similarity shown in the similarity table of
The complexity of the peptide mixture manufactured according to the process of the invention is greater than 5×102 different peptides. Preferably the complexity of the mixture is greater than 1×104 different peptides. More preferably the complexity of the mixture is greater than 1×106 different peptides.
In some embodiments, the base peptide sequence used for the process to manufacture the DSP composition is an epi tope relevant to the pathology of protein conformational disorders affecting the central and/or peripheral nervous system, selected from the group consisting of: Alzheimer's disease (AD), Dutch hereditary cerebral hemorrhage with amyloidosis (a.k.a cerebrovascular amyloidosis), congophilic angiopathy; Pick's disease, progressive supranuclear palsy; familial British dementia; Parkinson's disease (PD), Lewy-body related diseases, multiple system atrophy, Hallervorden-Spatz disease; amyotrophic lateral sclerosis (ALS); Huntington's disease (HD); spinocerebellar ataxia; neuronal intranuclear inclusion disease; hereditary dentatorubral-pallidoluysian atrophy; prion-related diseases such as scrapie, bovine spongiform encephalopathy, variant Creutzfeldt-Jakob disease, Gerstmann-Sträusser Scheinker syndrome, kuru, fatal familial insomnia, and related disorders; hereditary cystatin c amyloid angiopathy; dementia pugilistica; other neurodegenerative diseases and nerve atrophy; and other disorders characterized by cerebral atrophy and detection of intracellular and/or extracellular fibrillar aggregates as the disorder progresses.
In a particular embodiment, the protein conformational disorder is Parkinson's disease. In another embodiment, the protein conformational disorder is Alzheimer's disease. In another embodiment the conformational disorder is a priori-related disease. In another embodiment, the conformational disorder is amyotrophic lateral sclerosis. In a particular embodiment, the conformational disorder is Huntington's disease.
In other embodiments, the base peptide sequence used for the process to manufacture the DSP composition is an epitope relevant to the pathology of protein conformational disorders affecting multiple organs or organs other than the central nervous system, selected from the group consisting of: spinal and bulbar muscular atrophy; hereditary systemic and cerebral amyloidosis, Finnish-type familial amyloidosis; senile systemic amyloidosis (a.k.a, senile cardiac amyloidosis), familial amyloid polyneuropathy; Type-2 diabetes, in particular pancreatic islet amyloidosis; dialysis-related amyloidosis (DRA); inflammation-associated reactive systemic amyloidosis (a.k.a. AA amyloidosis); aortic medial amyloidosis; medulary carcinoma of the thyroid; hereditary renal amyloidosis; light chain associated amyloidosis, light chain deposition disease, light chain cast nephropathy, light chain cardiomyopathy; arial amyloidosis; injection-localized amyloidosis; cystic fibrosis (CF); sickle cell anemia, and other disorders wherein fibrillogenesis is observed in the affected organs or tissues.
In some embodiments, the base peptide sequence from which the DSP sequences are derived is selected from a group consisting of SEQ ID NO: 3 through 13.
In another embodiment, the protein conformational disorder is dialysis-related amyloidosis.
A particular embodiment of an aspect of the invention is a process of preparing a DSP composition as above, wherein the base sequence is selected from an epitope derived from proteins or peptides that do not strongly elicit immune reaction, and therefore is inadequate for preventing, ameliorating, or overcoming the pathology associated with the epitope. In a different embodiment, the epitope of the native sequence on its own as an antigen elicits an unwanted immune response, such as autoimmune-type of response, induction of anergy, or an agonistic or antagonistic stimulation that is contrary to a desired effect, each of which is detrimental to the improvement of the disease condition to which an immune reaction is sought. The unwanted immune response may be an autoimmune response against non-pathological tissue in vivo.
These DSP compositions may be used to elicit desired immune reactions. An embodiment of such use is to comprise therapeutic compositions useful for the treatment of PCDs as, for example, an active vaccine. Another embodiment of such use is to prepare antibodies against such DSPs, and to deliver said antibody as a passive vaccine. The immunizing composition may, but need not, include an adjuvant and other materials as immune boosters or stabilizers. The peptides comprising a DSP composition may be conjugated to a larger protein, such as keyhole limpet hemocyanin, bovine serum albumin, or ovalbumin for use as antigens, particularly for the preparation of antibodies. The peptides of a DSP may also be conjugated to a dendrimer, or synthesized as a dendrimer.
One aspect of the present invention is a pharmaceutical composition comprising a DSP composition as described herein, optionally as a pharmaceutically acceptable salt. In a preferred embodiment, such pharmaceutical composition comprising a DSP composition, when administered to a subject, causes a favorable modification of otherwise limited and inadequate immune response in the subject desirous of such a modification, such as an increase in appropriate immune responses, particularly to a protein associated with a pathological condition. The DSP may comprise one or more cassettes, such cassettes comprising the amino acid sequences that are derived from the first base peptide sequence. There may also be one or more cassettes having amino acid sequences that are derived from a second base peptide sequence of a second epitope.
Another aspect of the invention is the method of producing antibodies against DSP composition as described above, and the further aspect of the present invention is antibodies thus produced. In some embodiments, the method comprises (i) preparing a DSP composition according to the methods herein; (ii) administering said DSP composition to an animal; and (iii)(a) isolating antibodies immunoreactive with said DSP composition from said animal, or (iii)(b) isolating cells that produce antibodies immunoreactive with said DSP composition from said animal, and then isolating antibodies immunoreactive with said DSP composition from said isolated cells. The method of the instant invention encompasses producing and/or selecting an antibody which binds more specifically or which binds in a different conformation than those commonly obtained by selecting for a binding with a native epitope. A DSP composition is used to prepare antibodies that specifically bind to the base sequence, but including those that are different from antibodies elicited simply by the base peptide. The antibodies may bind to peptides or full length sequences corresponding to the native epitope. The antibodies may also bind to peptides or full length sequences corresponding to a disease-associated conformation of a specific epitope. In addition, the antibodies may bind to peptides or full length sequences that contain modifications such as post-translational modifications (for example, phosphorylation, acetylation, and methylation). The method is drawn to increasing the diversity of antibodies generated to react with a ligand. Further, the method is drawn to overcoming the problem of creating antibodies against ligands with low immunogenicity. Still further, the method is drawn to overcoming problems relating to generating antibodies having reactivities to only a single species. The method of the instant invention further encompasses the generation of novel functioning antibodies having antigen binding properties that elicit a varied amount of downstream consequences to the binding event. In some embodiments, the antibodies are immunoreactive with a protein comprising the base peptide, with or without post-translational modification, and wherein said protein is a full-length protein associated with the protein conformational disorder or a fragment of such full-length protein, and wherein said protein is in a pathological or non-pathological conformation.
In an embodiment of this aspect of the invention, the antibodies are modified antibodies having an engineered Fc region, wherein the engineered Fc region confers favorable pharmacodynamic profiles, in one embodiment, the Fc region enhances clearance of antibody-antigen complex. In another embodiment, the Fc region is not immunogenic to the subject. In certain embodiments, the Fc regions derive from an IgA, IgG, IgE, IgM, or IgD. The antibodies may be, for example, polyclonal or monoclonal antibodies. The antibody may also be a humanized antibody, an scFv antibody, or an antibody fragment such as a Fab fragment.
An aspect of the invention is a composition comprising a scaffold to which antibodies are attached, which antibodies are generated against a DSP composition as described above, wherein the base sequence is a sequence of a protein known to be associated with a protein conformational disorder. In one embodiment, the scaffold is a membrane compatible with haemodialysis. In a particular embodiment, the antibodies are conjugated to such membrane. In another embodiment, the antibodies are conjugated to a resin, such as CN—Br agarose resin (for example CN—Br Sepharose® (Pharmacia), to create an immunoaffinity resin.
The instant invention further comprises a method for the generation of antibodies useful as therapeutic agents for the treatment of disease,
In another embodiment, the instant invention comprises a method of creating antibody reagents for use in research studies. The instant invention also comprises a method of creating antibody reagents for use as diagnostic tools.
Another aspect of the present invention is a composition comprising antibodies generated against a DSP composition as described above, wherein the base sequence is a sequence of a protein known to be associated with a protein conformational disorder. More particularly, such protein is known to form an aggregate or fibril. In particular, antibodies thus generated are specific to the pathological conformation of such protein.
Another aspect of the present invention is a method of enhancing immune responses by administering a DSP composition to a subject in need thereof which subject is afflicted with a PCD. This application also provides a method for prophylactic or therapeutic treatment of a PCD comprising the steps of administering to a subject in need thereof an effective amount of a DSP composition, for the prevention or amelioration of symptoms of said disorder. Using the same principle as for the production of antibodies, antibodies may be produced in vivo, i.e., the compositions for stimulating antibody production may be used as active vaccines. Immunization steps of all the representative methods described below can be modified for in vivo use of the immunogens of the present invention as vaccines. In a particular embodiment, the subject exhibits only a limited and inadequate immune response to undesirable immunogens associated with a PCD.
An aspect of the present invention is a method of treating a subject afflicted with a protein con formational disorder, comprising the steps of administering an antibody prepared using a DSP composition as described, above. The treatment may be therapeutic, palliative, or prophylactic. In a particular embodiment, the protein conformational disorder is Parkinson's disease. In another embodiment, the protein conformational disorder is dialysis-related amyloidosis. In another embodiment, the protein conformational disorder is Alzheimer's disease.
Another aspect of the present invention is a method of treating a subject afflicted with a protein conformational disorder, comprising the steps of contacting under sterile conditions the blood of the subject to a membrane or a resin having conjugated with antibodies specific to a protein associated with a protein conformational disorder and prepared using a DSP composition, such antibodies described above, wherein the protein associated with a protein conformational disorder binds to such antibodies and is removed from the blood, and returning the blood to the subject. In a particular embodiment, the protein conformational disorder is dialysis-related amyloidosis. In one embodiment, the blood of the subject is contacted with the antibody as an additional step of therapeutic haemodialysis.
An embodiment of the invention is a method of prophylactic treatment of a subject at risk for developing a protein conformational disorder by contacting under sterile conditions the blood of the subject to a membrane or a resin having conjugated with antibodies specific to a protein associated with a protein conformational disorder and prepared using a DSP composition, such antibodies described above, wherein the protein associated with a protein conformational disorder binds to such antibodies and is removed from the blood, and returning the blood to the subject, whereby preventing the onset of such protein conformational disorder.
It has previously been shown that mixtures of related peptides may be therapeutically more effective than a single peptide. Lustgarten et al., J. Immunol. 2006, 176: 1796-1805; Quandt et al., Molec. Immunol. 2003, 40: 1075-1087. The effectiveness of a peptide mixture as opposed to a single peptide is the likelihood of interaction with the relevant epitopes that are not yet fully defined, particularly in terms of the conformationally specific interactions. Therefore, to increase and maintain the likelihood of long-term effectiveness, these previous treatment modalities have been modified. For example, a therapeutic composition based on an APL may include multiple peptides created by the APL method in combination with the original peptide, or other APLs. Fairchild et al., Curr. Topics Peptide & Protein Res. 6, 2004. Each APL would have a defined sequence, but the composition may be a mixture of APLs with more than one sequence. A reverse example involving conceptually similar altered peptide ligands involves an inventors attempt to reduce the amount of variation created by pathogens to avoid immune recognition (viral alteration of immunogenic epitopes over time, eg the creation of altered peptide ligands), by using the very changes created by the pathogen in an epitope sequence to create a limited diversity pool of peptides potentially useful in vaccinations (U.S. Pat. No. 7,118,874).
The instant invention draws out the most useful properties of the previous treatment modalities yet removes the limitations of each. The instant invention utilizes: (1) the specific immunologic relevance of a defined epitope peptide, (2) the modulatory properties of an APL, (3) the multivalency of MAPs, (4) and the alanine content from RSP to generate a directed expansion via alteration and degeneration of epitope variability that forms a complex yet directed peptide library useful for delivery as a vaccine. The approach is schematically represented in
The instant invention relates to a “Directed-Sequence Polymer” (DSP). A DSP is a library of related peptides having a sequence derived from a base peptide sequence, which may be but not limited to a native epitope associated with a desired immune response. Designing a DSP starts with a sequence of a known peptide epitope. A DSP has one or more amino acid residues that differ from that of the base peptide sequence, the substitution of which is determined by a defined rule. A DSP composition comprising multiple DSPs is synthesized by applying a set of synthesis rules that define the amino acid variations and the ratio of occurrence of introduction of such amino acid residues at any given position of the sequence to the base peptide sequence. Thus, a DSP is not synthesized as a single peptide, but is always synthesized as part of a composition comprising multiple related DSPs, the overall mixture of which is reproducible and consistent with the rules of synthesis that were applied. The overall composition of amino acids that make up the epitope is modified via the introduction of different, related amino acids to each residue position, such introduction made in accordance with a defined set of rules. The result is a mixture of related peptides useful in and of itself as a therapeutic, and which is useful to induce production of antibodies that react with specificity with the known sequence, but are not easily elicited by a simple immunization using the known sequence. The schematic for the steps for creating a DSP composition, starting from the choice of a base peptide, is shown in
The method of synthesizing a DSP composition utilizes and maintains the natural order of amino acid residues of a defined peptide sequence of a specified length. Each amino acid position is subjected to change based on a defined set of rules. In a preferred embodiment the amino acids are substituted according to the similarity depicted in
For the solid phase synthesis procedure of the instant invention, the mixture of amino acids for a given position in the peptide is defined by a ratio one to another. Prior to staring the synthesis, such ratio is determined for each position along the peptide. The resulting directed order peptide mixture comprises a multiplicity of related peptide sequences.
I. Base Peptide SequencesTo create a meaningful DSP composition, one first needs to define the base peptide sequence to derive the DSPs from. The base peptide sequences can be derived in many ways. A peptide sequence useful for this purpose is a peptide sequence related to immune response in a mammal. Alternatively, the base peptide sequence can be derived from the group consisting of a virus, bacteria or parasite. These peptide sequences are, for example, partial sequences of certain heat shock proteins as an epitope, HLA derived peptide ligand sequences, organ-derived peptide sequences, and empirically derived peptide sequences, such as through screening of a library created by combinatory chemistry.
Peptide Sequences Related to Protein Conformational DiseasesIn some embodiments, the base peptide sequence is an epitope relevant to the pathology of protein conformational disorders affecting the central aid/or peripheral nervous system, selected from the group consisting of: Alzheimer's disease (AD), Dutch hereditary cerebral hemorrhage with amyloidosis (a.k.a cerebrovascular amyloidosis), congophilic angiopathy; Pick's disease, progressive supranuclear palsy; familial British dementia; Parkinson's disease (PD), Lewy-body related diseases, multiple system atrophy, Hallervorden-Spatz disease; amyotrophic lateral sclerosis (ALS); Huntington's disease (HD); spinocerebellar ataxia; neuronal intranuclear inclusion disease; hereditary dentatorubral-pallidoluysian atrophy; prion-related diseases such as scrapie, bovine spongiform encephalopathy, variant Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker syndrome, kuru, fatal familial insomnia, and related disorders; hereditary cystatin c amyloid angiopathy; dementia pugilistica; and other neurodegenerative diseases characterized by cerebral and nerve atrophy and detection of intracellular and/or extracellular fibrillar aggregates as the disorder progresses.
In other embodiments, the base peptide sequence is an epitope relevant to the pathology of protein conformational disorders affecting multiple organs or organs other than the central nervous system, selected from the group consisting of: spinal and bulbar muscular atrophy; hereditary systemic and cerebral amyloidosis, Finnish-type familial amyloidosis; senile systemic amyloidosis (a.k.a. senile cardiac amyloidosis), familial amyloid polyneuropathy; Type-2 diabetes, in particular pancreatic islet amyloidosis; dialysis-related amyloidosis (DRA); inflammation-associated reactive systemic amyloidosis (a.k.a. AA amyloidosis); aortic medial amyloidosis; medulary carcinoma of the thyroid; hereditary renal amyloidosis; light chain associated amyloidosis, light chain deposition disease, light chain cast nephropathy, light chain cardiomyopathy; atrial amyloidosis; injection-localized amyloidosis; cystic fibrosis (CF); sickle cell anemia, and other disorders wherein fibrillogenesis is observed in the affected organs or tissues.
Examples of natively unfolded proteins and peptides, and those suspected to be natively unfolded, that undergo fibrillogenesis, and therefore associated with protein conformational disorders and may be use as the source sequences of the base peptides for the preparation of a DSP composition, include: prion protein and its fragments, amyloid beta protein and its fragments, abri protein, tau protein, alpha-synuclein and its central fragment, islet amyloid polypeptide (a.k.a. amylin), exon I of huntingtin, prothymosin alpha, amino-terminal domain of androgen receptor protein, ataxin-1, DRPLA protein (a.k.a. atrophin-1), and calcitonin.
Examples of globular proteins that undergo fibrillogenesis and therefore associated with protein conformational disorders and may be use as the source sequences of the base peptides for the preparation of a DSP composition, include: cystatin c, transthyretin, beta 2 microglobulin, serum amyloid A protein and its fragments, huntingtin and its fragments (including exon I of huntingin, immunoglobulin light chain variable domains, insulin, lysozyme (in particular human lysozyme), alpha lactalbumin, and monellin, ligand- and DNA-binding domains of androgen receptor protein, lactadherein and more specifically its fragments (for example, a.a. residue 245-294, a.k.a. medin), gelsolin, apolipoprotein A1, fibrinogen and its fragments, and atrial natriuretic factor. Fragments of all the proteins in this paragraph may also be used as source sequences.
As specific examples, in Alzheimer's disease, pathology correlates strongly with the presence of a 4 kDa amyloid beta (Aβ) peptide that is part of Aβ peptide precursor (APP), cleaved by enzyme presenilin 1 (PS1). Most Aβ are 40 amino acids long, and designated Aα40, Aβ40, Aβ1-40, or, having varied amino terminal, Aβx-40. Further, studies have indicated that the fibrillar form of Aβ1-40 stimulates the microglia, which cell type is currently thought to play an important role in the pathogenesis of Alzheimer's disease. (Jekabsone, A. et al. J. Neuroinflammation 3:24 (2006)). The peptide sequence of Aβ1-40 is shown as SEQ ID NO: 3 in Table I. On the other hand, Aβ1-42, which is a minor fraction of plaque-forming Aβ, is thought to contribute to the initiation of the formation of fibrillar Aβ. This “long form” of the peptide is described as SEQ ID NO:4 in Table I. Therefore, the base peptide sequence is such Aβ peptide, exemplified by SEQ ID NO: 4. The base peptide sequence may also be that of shorter peptide, i.e., Aβx-40, Aβ1-11, which has been reported in some cases to have clinical significance, Aβ14-23, or Aβ16-20. Tjernberg, L. O. et al., Biochem. J. 366:343-351 (2002),
A further specific example is Parkinson's Disease (PD). PD is a degenerative neurological disorder currently without a cure affecting 1-2% of the individuals over 50 years of age. The neuropathological hallmarks are characterized by progressive loss of neuromelanin containing dopaminergic neurons in the substantia nigra pars compacta (SNpc) with the presence of eosinophillic, intracytoplamic, proteinaceous inclusions termed Lewy Bodies (LB). α-Synuclein is the most abundant protein in Lewy Bodies, and appears to be an important mediator, perhaps even a causal factor, of toxicity in PD. Thus, reduction of toxic α-Synuclein is thought to be beneficial to PD patients. The sequence of one such mouse α-Syuclein peptide, derived from the C-terminal region of the full length protein, is shown as SEQ ID NO: 5 in Table I. (Benner, E. J. et al., PLoS ONE 3(1): e 376 (2008)). Further, elimination or sequestration of nitrated α-Synucein and fragments thereof, appear to have favorable effects on the patients suffering from PD. Therapeutically effective antibodies are said to be directed at the nitrated α-Synuclein but not native. Therefore, the base peptide sequence is, for example, SEQ ID NO: 5. In another embodiment of the instant invention, the base peptide sequence is a fragment comprising amino acids 121-137 of human α-Synuclein (DNEAYEMPSEEGYQDYE) (SEQ ID NO: 6). In yet other embodiments, the α-Synuclein fragment (121-137) sequence is substituted at positions 121 and 122 in different species, tri-nitrated at each Y (tyrosine) position, and/or phosphorylated at S129.
Another embodiment of the invention is based on the base peptide sequence relevant to prion-diseases. SEQ ID NO: 10 is human prion protein sequence. A relevant peptide is selected from partial sequences of SEQ ID NO: 10. Various species' prion sequences are disclosed by Harmeyer, S. et al., J Gen Virol. 79(Pt 4):937-45 (1998), the entirety of which is incorporated herein by reference. The amino acid variations by species can be used to design the substituting amino acids.
Yet another embodiment of the invention is based on the base peptide sequence derived from superoxide dismutase I (SODI). SODI mutation is known to have causal relationship with the pathology of some forms of familial ALS. It has been reported that the antisera raised against a mutant form of SODI, human G93A SODI recombinant protein, had protective effect on a mouse model of ALS carrying G37R mutant SOD) (line 29), which overexpress human SODI protein by 4-fold higher than endogenous mouse SODI. Urushitani, M. et al., Proc. Nat. Acad. Sci. USA, 104(7): 2495-2500 (2007). An example of SODI protein sequence is SEQ ID NO: 11, Therefore a base peptide sequence is a partial sequence of SEQ ID NO: 9.
Misfolded protein also plays a role in Huntington's disease, a genetic disorder caused by the pathological expansion of a polyglutamine (polyQ) tract in the huntingtin (htt) protein (SEQ ID NO: 12), resulting in neurodegeneration and premature death of the afflicted individual. A single-chain antibody that binds to an epitope formed by the N-terminal 17 amino acids of htt (Lecerf, J.-M. et al., Proc Natl Acad Sci USA. 98(8): 4764-4769 (2001) SEQ ID NO: 7) has been shown to reduce symptoms in a Drosophila model of Huntington's disease. (Wolfgang, W. J. et al., Proc Natl Acad Sci USA. 102(32): 11563-11568 (2005)) Therefore, a base peptide sequence is SEQ ID NO: 7.
A further specific example is Dialysis-related Amyloidosis (DRA). DRA may be caused by different forms of blood filtration, such as haemodialysis, hemofiltration, or Continuous Ambulatory Peritoneal Dialysis (CAPD). DRA has an incidence of greater than 95% of patients on dialysis for more than 15 years with beta-2-microglobulin (B2M, SEQ ID NO: 9) amyloidosis being prevalent and predictably increasing over time. Conformational isomers of B2M have been observed in a clinical setting (Uji et of. Nephron Clin Pract 2009; 111:c173-c181). B2M is part of the human leukocyte antigen (HLA) class I molecule, and has a prominent beta-pleated structure characteristic of amyloid fibrils. B2M is known to circulate as an unbound monomer distributed in the extracellular space. B2M undergoes fibrillogenesis to form amyloid deposits in a variety of tissues. This deposition causes renal failure, which causes an increase in synthesis and release of B2M, exacerbating the condition. Thus, in an embodiment of the invention, a protein the base sequence of which is used for preparation of a DSP composition is beta 2 microglobulin (SEQ ID NO: 9) and fragments thereof. An exemplary fragment of B2M is that spanning amino acid residues 21-40, SEQ ID NO: 8 in Table I, useful as a base peptide for DRA.
In some embodiments, the DSP (for example, a DSP used to treat or diagnose DRA) is modified with advanced glycation end (AGE) products, useful to elicit immune responses and to generate antibodies against certain AGE products.
AGE products are a heterogeneous group of carbohydrate molecules formed by non-enzymatic glycation and oxidative reactions between reducing sugars and protein amino groups. As described in Niwa (Seminars in Dialysis, 14(2) (March-April) 2001 pp. 123-126), AGE-modification of B2M is often observed in DRA patients, and appears to contribute to the pathology of DRA. In particular, the author observed imidazolone, Nε-(carboxymethyl)lysine (CML), and pentosidine modifications. As AGE-modified B2M accumulates, chemotaxis is enhanced, stimulating macrophages to release pro-inflammatory cytokines and interfering with collagen synthesis. Furthermore, AGE-B2M interacts with mononuclear phagocytes (MPs), cells important in the pathogenesis of inflammatory arthropathy. This interaction prompts the MPs to secrete elevated levels of TNFα and interleukin-1, worsening inflammation (Rashid et al., IMAJ 2006; 8:36-39).
To date, both haemodialysis and peritoneal dialysis have been found unsatisfactory in removing AGE products from the bloodstream; thus, new methods are needed to lower the levels of AGE products in DRA patients. Advanced glycation end products may be formed primarily on B2M aggregates rather than monomers, and thus may be useful in producing antibodies with specificity to the pathogenic aggregate form of B2M. Alternatively, oxidation of B2M may enhance amyloid deposition.
Empirically Derived Base Peptide SequencesAs described in the above sections, peptide sequences with some significance to a disease state or an adverse reaction may be identified through experimental investigation of a relevant epitope. These sequences may include non-naturally occurring peptide sequences that proved to be useful in treating a disease or a condition, an example found in the international patent application publication WO 2006/031727, U.S. Pat. No. 6,930,168 and the related scientific publication Stern et al., Proc. Nat. Acad. Sci. USA, 2005, 102:1620-25.
Further, epitopes are empirically determined by identifying candidate sequences by positional scanning of synthetic combinatorial peptide libraries (see, for example, D. Wilson et al., above; R. Houghten et al., above; Hernandez et al., Eur J Immunol., 2004, 34:2331-41), or by making overlapping peptide sequences of the entire protein of interest, and testing those peptides for immune reactivity (using, for example, any readout assay useful for such purposes, described in Current Protocols in Immunology Edited by John E Coligan, Ada M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strober NIH, John Wiley & Sons) in an in vitro or in vivo assay system appropriate for the disease and species the epitope is sought for. For example, for the design of a multiple sclerosis drug, an example of an appropriate system uses cells that derive from human subjects with MS.
After identifying a candidate epitope, a probable set of additional related epitopes are generated using modeling and prediction algorithms described in readily available references, for example WO 2000/042559, align and analyze the predicted binding of these probable epitopes using available prediction methods described in, for example, WO 2005/103679, WO 2002/073193 and WO 99/45954. Selecting from the peptides having the highest predicted activity/binding, take 40% of the predicted sequences and acquire the percentage of any given amino acid at each position. Use those percentages to create the rules for amino acid incorporation into a DSP synthesis.
Other Sources of Base Peptide SequencesExamples of epitopes identified as part of a naturally occurring, full length protein or synthetic peptides that were identified to have similar activities as such epitopes are shown in the table below.
Steps in the creation of a DSP sequentially encompass the following:
(a) Identify a protein having known or believed association with a pathology.
(b) Select from within the protein a peptide or peptides, each having a fixed sequence, that are associated with the pathology and immunologically relevant. If no peptides have been described, then peptides useful in the treatment of the pathology of interest are created. One exemplary method is to create a library of peptides that collectively span the entire length of the protein of interest. This may be done by, for example, partial endopeptidase digestion or by peptide synthesis. The library is screened for immunologically relevant peptides using appropriate detection methods such as binding affinity determination using antibodies detected in the sera of patients with the target pathology. The peptides may be further examined for immunogenicity useful for the treatment of the pathology in an in vitro or in vivo experimental system.
(c) the amino acid substitutions are decided based on either of two sets of rules, defined or empirical and are set forth below;
(d) Solid phase synthesis of DSP according to the rules is performed, and pharmaceutically acceptable formulation the DSP is delivered as a therapeutic.
The rules of synthesis for a composition comprising DSPs are outlined below. Briefly, a DSP may be envisioned as a polypeptide having a defined length that is either the same length as or multiples of the length of the base peptide sequence. For each residue position of the base peptide sequence, one or more substitute residue is defined. The rule of synthesis defines the ratio among the original base peptide residue for that position, the first substitute residue, the second substitute residue, the third substitute residue, and an alanine, to occupy any given residue position.
The substitute residues are defined according either: (1) to a rational comparison and finding of similarities of relevant characteristics of the original residue with those of the substitute residue, (2) in accordance with the differences at a given position between species, (3) in accordance with the differences at a given position within individuals of the same species, or (4) to a comparison of reported experimental results on the relative activities of actual peptides having slight variations from the base sequence. The substitute residues defined in either of these two approaches are termed “conserved substitution” herein.
An example of a rational comparison and findings of similarity is the methods described by Kosiol et al., J. Theoretical Biol., 2004, 228:97-106. Amino acids are grouped together in a matrix, referred therein as PAM replacement matrix.
A comparison of experimental results showing the relative activities of peptides having slight variations from the base sequence can also be used as a basis for the rule for substitution. The sequences of the peptides responsible for observed changes are aligned and the type and percent presence of the new amino acid are noted. If there is more than one amino acid substitution at any given position of the peptide, the frequency of occurrence of an amino acid and the magnitude of activity change compared to the original sequence are taken into account to determine the order of prevalent substitution. Examples of the overall process leading up to the rule generation for DSP synthesis can be found using libraries (Molec. Immunol. 40:1047-1055; Molec. Immunol. 40:1063-74; J Autoimmunity 20:199-201; and J. Immunol 163:6424-34), by making altered peptide ligands of overlapping peptides representing the entire protein of interest (Atkinson et al., J. Clin. Invest. 94:2125-29; Meini et al., J. Clin. Invest. 92:2633-43) or de novo (U.S. Pat. Nos. 7,058,515; 6,376,246; 6,368,861; 7,024,312; 6,376,246; 7,024,312; 6,961,664; 6,917,882). Briefly, a cellular material of interest is chosen as the assay system to rank the immunoreactivity of the peptides to be interrogated. Such an assay system can be either an in vitro or in vivo system, and can comprise adaptive or innate immune reactivity. Readouts for the assay system can be the up- or down-regulation of the status of the activation state of a protein, a change in the localization of a protein, the expression of the mRNA encoding for the protein, the relative concentration of a protein, changes in the generation of specific cell types, changes in cellular phenotype, changes in cellular activation, changes in cell number, changes in organ size or function, changes in animal behavior or phenotype. Once the assay or assays are performed the results are analyzed to determine the prevalence of any particular amino acid as a conserved substitution. If more than three residues in a given position within the peptide sequence are identified as generating a change in immunologic function, the top three residues first by frequency of representation in the interrogated peptides, and second by the magnitude of changes elicited. Once chosen, the relative amounts of the residues are defined. As depicted in
As described in
A cassette may be repeated more than once. After a desired number of multiples of the cassette, if the desired length of the DSP is not yet reached, the DSP sequence is further defined by applying the same process, possibly using different ratio among the original, substitute, second substitute, and alanine residues.
N or C-terminal DSP modifiers may be added to the synthesis rules. The purpose of such modifiers include but are not limited to enhancing binding to specific proteins as in the case of RDG-based amino acid sequences (U.S. Pat. Nos. 5,773,412; 5,770,565) used as targeting moieties, or peptides that are known to bind to a wide array of HLA-DR species, such as AKAVAAWTLK AAA(SEQ ID NO: 14) (U.S. App. Pub. No. 2006/0018915) as a DR-targeting moiety. Such modifiers may include moieties which enhance complexation to delivery systems including sustained release delivery systems. Modifiers can be resorbable matrix constructs/synthesizable backbones such as PLGA. Modifiers can be protease resistant moieties such as D-amino acids.
Thus, for any given base peptide sequence, a set of synthesis rules is applied to yield a composition comprising reproducible, consistent mixture of DSPs.
In some embodiments, the DSP (for example, a DSP used to treat or diagnose DRA) is modified with advanced glycation end (AGE) products. AGE products are a heterogeneous group of carbohydrate molecules formed by non-enzymatic glycation and oxidative reactions between reducing sugars and protein amino groups.
III. Peptide Synthesis MethodsAny known solid phase synthesis appropriate for peptide synthesis may be used to synthesize a composition comprising DSPs, for example as originally described by Merrifield (J. Am. Chem. Soc., 1963, 85:2149) and any variation thereof. More specifically, the synthesis is done in multiple steps by the Solid Phase Peptide Synthesis (SPPS) approach using Fmoc protected amino acids. SPPS is based on sequential addition of protected amino acid derivatives, with side chain protection where appropriate, to a polymeric support (bead). The base-labile Fmoc group is used for N-protection. After removing the protecting group (via piperidine hydrolysis) the next amino acid mixture is added using a coupling reagent (TBTU). After the final amino acid is coupled, the N-terminus is acetylated.
The resulting peptides (attached to the polymeric support through its C-terminus) are cleaved with TFA to yield the crude peptide. During this cleavage step, all of the side chains protecting groups are also cleaved. After precipitation with diisopropyl ether, the solid is filtered and dried. The resulting peptides are analyzed and stored at 2-8° C.
Additionally, any peptide synthesis method that allows synthesis incorporating more than one amino acid species at a controlled ratio in any given position of the peptide sequence is suitable for use with this invention. Further, as described below, DSPs may be peptidomimetics or include unnatural or modified amino acid, necessitating the adaptation to allow addition of such chemical species to the polymers synthesized up to that point.
The synthesis may include unnatural amino acids, or amino acid analogs. In some embodiments, the DSPs are comprised of naturally occurring and synthetic derivatives, for example, selenocysteine. Amino acids further include amino acid analogs. An amino acid “analog” is a chemically related form of the amino acid having a different configuration, for example, an isomer, or a D-configuration rather than an L-configuration, or an organic molecule with the approximate size and shape of the amino acid, or an amino acid with modification to the atoms that are involved in the peptide bond, so as to be protease resistant when polymerized in a polypeptide.
The DSPs for use in the present invention can be composed of L- or D-amino acids or mixtures thereof. As is known by those of skill in the art, L-amino acids occur in most natural proteins. However, D-amino acids are commercially available and can be substituted for some or all of the amino acids used to make DSPs of the present invention. The present invention contemplates DSPs containing both D- and L-amino acids, as well as DSPs consisting essentially of either L- or D-amino acids.
In certain embodiments, the DSPs of the present invention include such linear DSPs that are further modified by substituting or appending different chemical moieties. In one embodiment, such modification is at a residue location and in an amount sufficient to inhibit proteolytic degradation of the DSPs in a subject. For example, the amino acid modification may be the presence of at least one proline residue in the sequence; the residue is present in at least one of carboxy- and amino termini; further, the proline can be present within four residues of at least one of the carboxy- and amino-termini. Further, the amino acid modification may be the presence of a D-amino acid.
In certain embodiments, the subject DSPs is a peptidomimetic. Peptidomimetics are compounds based on, or derived from, peptides and proteins. The DSP peptidomimetics of the present invention typically can be obtained by structural modification of one or more native amino acid residues, e.g., using one or more unnatural amino acids, conformational restraints, isosteric replacement, and the like. The subject peptidomimetics constitute the continuum of structural space between peptides and non-peptide synthetic structures.
Such peptidomimetics can have such attributes as being non-hydrolyzable (e.g., increased stability against proteases or other physiological conditions which degrade the corresponding peptide DSPs), increased specificity and/or potency. For illustrative purposes, peptide analogs of the present invention can be generated using, for example, benzodiazepines (e.g., see Freidinger et al. in “Peptides: Chemistry and Biology,” G. R. Marshall ed., ESCOM Publisher. Leiden, Netherlands, 1988), substituted gamma lactam rings (Garvey et al. in “Peptides: Chemistry and Biology,” G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, p 123), C-7 mimics (Huffman et al. in “Peptides: Chemistry and Biology,” G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, p. 105), keto-methylene pseudopeptides (Ewenson et al. J. Med. Chem., 1986, 29:295; and Ewenson et al. in “Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium),” Pierce Chemical Co. Rockland, Ill., 1985), β-turn dipeptide cores (Nagai et al., Tetrahedron Lett., 1985 26:647; and Sato et al. J. Chem. Soc. Perkin Trans., 1986, 1:1231), β-aminoalcohols (Gordon et al. Biochem. Biophys. Res. Commun., 1985, 126:419; and Dann et al. Biochem. Biophys. Res. Commun., 1986, 134:71), diaminoketones (Natarajan et al. Biochem. Biophys. Res. Commun., 1984, 124:141), and methyleneamino-modified (Roark et al. in “Peptides: Chemistry and Biology,” G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, p 134). Also, see generally, Session III: Analytic and synthetic methods, in “Peptides: Chemistry and Biology,” G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988.
The molecular weight of a DSP composition can be adjusted during polypeptide synthesis or after the DSPs have been synthesized. To adjust the molecular weight during polypeptide synthesis, the synthetic conditions or the amounts of amino acids are adjusted so that synthesis stops when the polypeptide reaches the approximate length which is desired. After synthesis, polypeptides with the desired molecular weight can be obtained by any available size selection procedure, such as chromatography of the polypeptides on a molecular weight sizing column or gel, and collection of the molecular weight ranges desired. The present polypeptides can also be partially hydrolyzed to remove high molecular weight species, for example, by acid or enzymatic hydrolysis, and then purified to remove the acid or enzymes.
In one embodiment, the DSPs with a desired molecular weight may be prepared by a process which includes reacting a protected polypeptide with hydrobromic acid to form a trifluoroacetyl-polypeptide having the desired molecular weight profile. The reaction is performed for a time and at a temperature which is predetermined by one or more test reactions. During the test reaction, the time and temperature are varied and the molecular weight range of a given batch of test polypeptides is determined. The test conditions which provide the optimal molecular weight range for that batch of polypeptides are used for the batch. Thus, a trifluoroacetyl-polypeptide having the desired molecular weight profile can be produced by a process which includes reacting the protected polypeptide with hydrobromic acid for a time and at a temperature predetermined by test reaction. The trifluoroacetyl-polypeptide with the desired molecular weight profile is then further treated with an aqueous piperidine solution to form a low toxicity polypeptide having the desired molecular weight.
In one particular embodiment, a test sample of protected polypeptide from a given batch is reacted with hydrobromic acid for about 10-50 hours at a temperature of about 20-28° C. The best conditions for that batch are determined by running several test reactions. For example, in one embodiment, the protected polypeptide is reacted with hydrobromic acid for about 17 hours at a temperature of about 26° C.
A further embodiment of the instant invention is the use of specific gylogenated forms of a DSP to create antibodies against such a form of a ligand. In one embodiment the ligand itself is an antibody. In one embodiment of the instant invention, the post-translational modification of a DSP is performed using glycogen synthase, or alternatively using chemical complexation techniques well known in the art.
IV. Antibody ProductionThe method is drawn to increasing the diversity of antibodies generated to react with a ligand. Further, the method is drawn to overcoming the problem of creating antibodies against ligands with low immunogenicity. Still further, the method is drawn to overcoming problems relating to generating antibodies having reactivities to only a single species. The instant invention comprises a method of creating antibody reagents for use in research studies. The instant invention comprises a method of creating antibody reagents for use as diagnostic tools. The instant invention further comprises a method for the generation of antibodies useful as therapeutic agents for the treatment of disease. Using the same principle, antibodies may be produced in vivo, i.e., the compositions for stimulating antibody production may be used as vaccines. Immunization steps of all the representative methods described below can be modified for in vivo use of the immunogens of the present invention as vaccines.
A method of preparing antibodies using a known antigen or a mixture of antigens is well known in the art. The method of preparing antibodies using a DSP composition is described in U.S. App. Publ. No. 2009-0036653. Briefly, antibodies are produced by designing and synthesizing the peptides comprising a DSP composition as described above, creating antibodies by introducing the DSP into an in vivo setting, or alternatively introducing the DSP into an in vitro setting, or still alternatively contacting the DSP with a system of maintaining the connection between antibody phenotype and genotype such as phage display, determining the activity of the generated antibodies by contacting the antibodies with the native molecule of interest, selecting antibodies having desired activity, such activity being either of a higher affinity antibody, or alternatively a lower affinity antibody, a single species reactivity, or alternatively a multi-species reactivity, a single-molecule of interest reactivity or alternatively a multi-molecule reactivity.
The instant invention also comprises a process for producing antibodies that are therapeutically or prophylactically useful in treatment of protein conformational disorders, or useful for use as research reagents, and as diagnostic tools for such disorders, by eliciting immune responses using a composition comprising directed epitope peptide mixtures. The invention also encompasses composition comprising antibodies thus produced.
The method of the instant invention also encompasses an augmentation of the paratopes associated with an antibody response to an antigen of interest. The method of the instant invention further encompasses the generation of novel functioning antibodies having antigen binding properties that elicit a varied amount of downstream consequences to the binding event.
Briefly, the method comprises the steps of selecting a protein relevant to a protein conformational disorder, determining relevant epitopes within the protein known or suspected to be closely associated with the disorder, selecting the relevant epitope, performing directed permutations of the epitope so as to create an expanded yet related series of antigens, performing solid phase synthesis thus creating a directed sequence polymer (DSP), using the DSP collectively as a set of antigens by placing the DSP in contact with a means of antibody generation, determining the activity of the generated antibodies, selecting antibodies having the desired activity, and utilizing the antibody as a single species reagent, multi-species reagent, single species diagnostic, multi-species diagnostic, or alternatively as a therapeutic. The means of antibody generation is, for example, an animal to be immunized by the DSP and cells from such an animal (e.g. spleen cells from a mouse for monoclonal antibody production), a phage display library, or a B cell library.
Alternatively, the instant invention encompasses methods of producing antibodies, the methods comprising: selecting a protein of interest, selecting the amino acids that make up the epitope, combining the amino acids into a linear peptide, performing directed permutations, synthesizing the DSP using solid phase chemistry, preparing the DSP as a pharmaceutically acceptable salt, introducing the DSP into a host, harvesting primary tissue containing antibody from the host after one week, alternatively harvesting primary tissue containing antibody from the host after a time greater one week, determining the activity of the generated antibodies, selecting, and utilizing the antibody as a reagent, diagnostic, or alternatively as a therapeutic.
The peptides comprising a DSP composition may be conjugated to a larger protein, such as keyhole limpet hemocyanin, bovine serum albumin, or ovalbumin for use as antigens. The peptides of a DSP may also be conjugated to a dendrimer, or synthesized as a dendrimer. The immunizing composition may include an adjuvant and other materials as immune boosters or stabilizers. Peptide dendrimers solve certain manufacturing issue of soluble peptide mixtures, in part by the promise of delivering to a patient a consistent ratio and quantity of each of the peptides in the mixture. This approach is schematically represented in
Dendrimers have been made using amino, hydroxyl, carboxy, poly(propylenimine), silicone and polyamino amine cores (G. M. Dykes et al., J. Chem. Technol. Biotechnol., 2001, 76:903-918, P. Sadler and J. Jezek, Rev. Mol. Biotechnol., 2002, 80:195-229, and J. P. Tam, Methods Org. Chemistry, 2004, Vol E22d 129-168. Peptide dendrimers can be divided into three types: grafted peptide dendrimers, branching polyamino acids and multiple antigen peptides (MAPs).
The branching strategies in MAPs vary widely. The majority of first generation branches have used lysine. Second generation solid phase synthesis of MAPs has seen an interest in proline. The interest is said to come from both the properties of its secondary amine which decreases the reactivity during production, as well as its role in many cellular functions.
Simple MAPs have been synthesized using solid phase chemistry, with this type of synthesis strategy called divergent. Synthesis methods have been described which involves a two-step iterative reaction sequence producing concentric shells of dendritic beta-alanine units covalently linked in the second step to various functional groups (Kojima et al., Bioconjugate Chem., 2000, 11:910-17). These types of MAPs, which are synthesized using the divergent strategy, by necessity have simple branching schemes with few distinct members, as the purification and characterization are untenable with more complex MAPs. The end-product needs to be purified away from deletion compounds having similar characteristics to the end-product. Purifications have been described using gel filtration chromatography, reverse phase high-performance liquid chromatography (HPLC), or electromigration methods.
For complex MAPs, for example, those having a multiplicity of branching moieties, convergent synthesis is the preferred synthesis strategy. Convergent synthesis can be performed using either fragment condensation or ligation of the pre-purified fragments. There are many types of ligations: natural (true peptide bond created), thiol, hydrazone, or other. MAPs prepared using convergent synthesis strategies are easier to purify, as the end-product will look distinctly different from the reaction byproducts. HPLC was first used to purify convergent MAPs (J. C. Spetzler et al., Int. J. Pept. Protein Res., 1995, 45:78-85).
Thus produced, another aspect of the present invention is a composition comprising antibodies generated against a DSP composition as described above, wherein the base sequence is a sequence of a protein known to be associated with a protein conformational disorder. More particularly, such protein is known to form an aggregate or fibril. In particular, antibodies thus generated are specific to the pathological conformation of such protein.
In an embodiment of this aspect of the invention, the antibodies are modified antibodies having an engineered Fc region, wherein the engineered Fc region confers favorable pharmacodynamic profiles. In one embodiment, the Fc region enhances clearance of antibody-antigen complex. In another embodiment, the Fc region is not immunogenic to the subject. Such modified antibodies may be created after antibodies with certain desired (complementarity determining regions) are identified, by replacing chemically or by molecular biological means the Fc region with an IgA, IgG, IgE, IgM, or IgD region.
In another embodiment, the antibodies are humanized antibodies having desired CDRs (complementarity determining regions), such CDRs having been identified using DSP compositions or antibodies having such CDRs having been generated using DSP compositions. Humanized antibodies may be made according to any means known in the art, including CDR grafting and the introduction of point mutations to reduce immunogenicity. In yet another embodiment, the antibodies are single chain variable fragment (scFv), either engineered from an identified antibody, or generated using a phage display library and other means and screened for desired antibodies using DSP compositions. Methods of scFv production and phage display are known in the art.
The antibodies may also have a detectable label, such as a radiolabel, an enzymatic label, or a fluorescent label. In some embodiments, the fluorescent label is selected from the group consisting of Texas Red, phycoerythrin (PE), cytochrome c, and fluorescent isothiocyanate (FITC). In addition, labels such as biotin followed by streptavidin-alkaline phosphatase (AP), horseradish peroxidase (HRP) are contemplated.
This disclosure also provides antibodies with at least 70%, 80%, 90%, 95%, or 99% amino acid sequence identity to the anti-DSP antibodies described above. Antibodies in general have well characterized structure-activity relationships, and one of skill in the art would be well aware that certain mutations would be unlikely to disrupt the antigen-binding function of an antibody. For example, conservative substitutions in the constant region would be unlikely to disrupt antigen binding, while substitutions in the CDRs would be more likely to disrupt antigen binding.
An aspect of the invention is a composition comprising a scaffold or support material to which antibodies are attached, which antibodies are generated against a DSP composition as described above, wherein the base sequence is a sequence of a protein known to be associated with a protein conformational disorder. In one embodiment, the scaffold is a membrane compatible with haemodialysis. Membranes for haemodialysis are typically semi-permeable, allowing for water and some dissolved solutes to pass through. The membranes can have different pore sizes and are thus categorized as low-flux or high-flux. Membranes can be made from a variety of materials, including cellulose acetate, polyarylethersulfone, polyamide, polyvinylpyrrolidone, polycarbonate, and polyacrylonitrile. In a particular embodiment, the antibodies are conjugated to such membrane. This will allow for removal of specified proteins at while haemodialysis is carried out. This process is useful, inter alia, for treating removing amyloid forms of B2M and treating DRA. In another embodiment, the antibodies are conjugated to a resin, such as CN—Br agarose resin (for example CN—Br Sepharose (Pharmacia), to create an immunoaffinity resin.
V. Pharmaceutical CompositionOne aspect of the present invention is a pharmaceutical composition comprising a DSP composition. As described below in the method of treatment as an aspect of this invention, the DSP composition produced by the process of the invention is useful in treatment of a protein conformational disorder in a subject.
The DSPs of the present invention may be administered to the subject as a composition which comprises a pharmaceutically effective amount of DSPs and an acceptable carrier and/or excipients. A pharmaceutically acceptable carrier includes any solvents, dispersion media, or coatings that are physiologically compatible. Preferably, the carrier is suitable for oral, rectal, transmucosal (including by inhalation), parenteral, intravenous, intramuscular, intraperitoneal, intradermal, transdermal, topical, or subcutaneous administration. One exemplary pharmaceutically acceptable carrier is physiological saline. Other pharmaceutically acceptable carriers and their formulations are well-known and generally described in, for example, Remington 's Pharmaceutical Science (18th Ed., ed. Gennaro, Mack Publishing Co., Easton, Pa., 1990). Various pharmaceutically acceptable excipients are well-known in the art and can be found in, for example, Handbook of Pharmaceutical Excipients (4th ed., Ed. Rowe et al Pharmaceutical Press, Washington, D.C.). The composition can be formulated as a solution, microemulsion, liposome, capsule, tablet, or other suitable forms. The active component which comprises the copolymer may be coated in a material to protect it from inactivation by the environment prior to reaching the target site of action. The pharmaceutical compositions of the present invention are preferably sterile and non-pyrogenic at the time of delivery, and are preferably stable under the conditions of manufacture and storage. When desirable, the composition further comprises components to enhance stability, permeability, and/or bioavailability, such as particulate forms protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal and oral.
For oral administration, the pharmaceutical preparation may be in liquid form, for example, solutions, syrups or suspensions, or may be presented as a drug product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pre-gelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods well-known in the art.
In one embodiment, the oral composition is enterically-coated. Use of enteric coatings is well known in the art. For example, Lehman (1971) teaches enteric coatings such as Eudragit S and Eudragit L. The Handbook of Pharmaceutical Excipients, 2nd Ed., also teaches Eudragit S and Eudragit L applications. One Eudragit which may be used in the present invention is L30D55. Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
The compositions may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
For administration by inhalation, the compositions for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin, for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The compositions may be formulated for administration by injection, e.g., by bolus injection or continuous infusion in a parenteral, intravenous, intraperitoneal, intramuscular, or subcutaneous manner. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen free water, before use.
In a preferred embodiment, compositions comprising DSP compositions are formulated in accordance with routine procedures as pharmaceutical compositions adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline, with the intervals between administrations being greater than 24 hours, 32 hours, or more preferably greater than 36 or 48 hours. Where the composition is administered by injection, an ampoule of sterile water or saline for injection can be provided so that the ingredients may be mixed prior to administration.
In other embodiments of the present invention, the pharmaceutical compositions are regulated-release or sustained release formulations. DSP compositions of the present invention may be admixed with biologically compatible polymers or matrices which control the release rate of the copolymers into the immediate environment. Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). One embodiment of sustained release formulations is transdermal patches.
In some embodiments of the present invention, pharmaceutical compositions comprise DSPs formulated with oil and emulsifier to form water-in-oil microparticles and/or emulsions. The oil may be any non-toxic hydrophobic material liquid at ambient temperature to about body temperature, such as edible vegetable oils including safflower oil, soybean oil, corn oil, and canola oil; or mineral oil. Chemically defined oil substance such as lauryl glycol may also be used. The emulsifier useful for this embodiment includes Span 20 (sorbitan monolaurate) and phosphatidylcholine. In some embodiments, a DSP composition is prepared as an aqueous solution and is prepared into an water-in-oil emulsion dispersed in 95 to 65% oil such as mineral oil, and 5 to 35% emulsifier such as Span 20. In another embodiment of the invention, the emulsion is formed with alum rather than with oil and emulsifier. These emulsions and microparticles reduce the speed of uptake of DSPs, and achieve controlled delivery.
In another embodiment, the controlled and/or sustained delivery is achieved by implantable medical devices coated with sustained-release formulations, or implantable pharmaceutical formulation suitable for sustained-release of the active components.
Some embodiments of the invention are pharmaceutical compositions for targeted delivery of the DSP composition of the invention. In such embodiments, a pharmaceutical composition comprises a DSP composition that is complexed with a targeting moiety. The targeting moiety allows localized delivery of the DSP composition to a desired location or microenvironment within the subject. A targeting moiety include, and may be selected from, the group comprising a chemical group or functionality such as biotin or simple sugars, a single or double stranded DNA sequence of various lengths, a single or double stranded RNA sequence of various lengths, a peptide of various lengths, an antibody including single chain antibodies, Fab′, or modified antibodies, a lipid, or a glycolipid. More than one of such moiety may be used at the same time in combination. For examples of targeting moieties, see U.S. Pat. No. 6,268,488; U.S. Appl. Pub. No. 2003/0190676; and see, for example, www.covx.com/tech_creating.html.
In one embodiment of the invention, the complex has characteristics of a prodrug, causing the DSP composition to exhibit no pharmaceutical activity of the present invention until the dissolution of the complex in the subject. In another embodiment, the complex does not affect the activity of the DSP composition.
Any methods generally known to one skilled in the art may be used to produce a complex of the instant invention and a targeting moiety. The target moiety may be complexed to the DSPs by a chemical bond, which may be covalent, ionic, hydrophobic, or van der Waals force, directly or through another chemical entity. Alternatively, the target moiety may be co-localized with the DSPs through common medium such as a biocompatible resin within which the DSP composition is included. The manner of forming a complex is chosen also based on the active state of the instant invention while existing in the combination and whether a permanent complex or a transitory complex is desired.
In some embodiments, the pharmaceutical compositions also include additional therapeutically active agents. Such additional ingredient can be one or more of: an additional DSP composition that binds to a different target, an antibody which activates inflammatory molecules, or cytokines. Further additional ingredient can be activating cytokines and chemokines (as described in Shaw, Jennifer, Infection and Immunity, 69:4667-4672, 2001) taken from the group consisting of Mip1β, Mip1α, Mip-2, Mip3α, IP-10, MCP-1, TCA-3, IL-1, IL-18, IL-6, IFNγ, MIF, IL-12, CCR7.
Further, a form of vitamin D that is or becomes biologically active within the body of the subject receiving such form of vitamin D may also be used as an additional ingredient. The two main forms of vitamin D are: vitamin D3 or cholecalciferol, which is formed in the skin after exposure to sunlight or ultraviolet light, and ergocalciferol or vitamin D2 which is obtained by irradiation of plants or plant materials or foods. The differences are situated in the side chain. Vitamin D3 may be obtained from natural sources such as fatty fish such as herring and mackerel. In the body, two other forms of vitamin D3 can be found. Vitamin D3 is hydroxylated in the liver into 25-hydroxyvitamin D3 (25(OH)D), and subsequently in the kidney into 1,25-dihydroxyvitamin D3 (1,25(OH)2D), which is the active metabolite that stimulates the calcium absorption from the gut (Feldman et al., 2005). When 1,25(OH)2D is sufficiently available, 24,25-dihydroxyvitamin D (24,25(OH)2D) is formed in the kidney, which is further catabolized.
In certain embodiments, the composition is capable of raising an immune response without an adjuvant.
Another class of therapeutically active agents useful as an additional agent is immune boosters which increases the production of common lymphoid precursors (CLPs) from the multilineage potential cells. An example of such agent is PBI-1402 developed by ProMetic in Quebec, Canada.
The invention further provides a kit comprising (i) a composition comprising a DSP composition and (ii) instructions for administering the composition to a subject in need thereof at intervals greater than 24 hours, more preferably greater than 36 hours, for the treatment of a disease, such as a protein conformational disorder. In one embodiment, the PCD is DRA. In a preferred embodiment, the DSP composition is formulated in dosages for administration of greater than about 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, or 240 hours, or any intervening interval thereof. In another embodiment of the kits described herein, the instructions indicate that the DSP is to be administered every about 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, or 240 hours, or any interval in between. Kits may comprise additional components, such as packaging, instructions, and one or more apparatuses for the administration of the copolymer, such as a hypodermic syringe.
Other embodiments of the invention are kits that comprise a scaffold containing one or more DSP-generated antibody clones and the corresponding instructions on combining with haemodialysis.
Another aspect of the invention is a pharmaceutical composition comprising one or more antibodies generated and produced using the process described herein elsewhere. An antibody or antibodies that react to a protein conformational disease can be used to neutralize pathological proteins that such antibodies specifically bind, or to facilitate clearing from the body of a patient afflicted with such disease.
Pharmaceutically acceptable carriers and their formulations are well-known and generally described in, for example, Remington's Pharmaceutical Science (18th Ed., ed. Gennaro, Mack Publishing Co., Easton, Pa., 1990). Various pharmaceutically acceptable excipients are well-known in the art and can be found in, for example, Handbook of Pharmaceutical Excipients (4th ed., Ed. Rowe et al Pharmaceutical Press, Washington, D.C.). Further, formulations suitable for antibodies are generally known in the art, including buffers and excipients, and preservative agents such as protease inhibitors that are suitable for pharmaceutical use. The pharmaceutical compositions of the present invention are preferably sterile and non-pyrogenic at the time of delivery.
The compositions may be formulated for administration by injection, e.g., by bolus injection or continuous infusion in a parenteral, intravenous, intraperitoneal, intramuscular, or subcutaneous manner. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen free water, before use. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
In certain embodiments, antibodies are single chain variable fragments, to facilitate transport into the tissues due to its smaller size compared to naturally occurring antibodies. Such antibodies may further be associated with a carrier or agent to cross the blood brain barrier, for example, an anti-transferring antibody. See, for example, Friden et al., Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier, PNAS Jun. 1, 1991 vol. 88 no. 11 4771-4775.
VI. Methods of Treatment—Administration of DSPThe instant invention provides for a further improvement on the need to improve the effectiveness of peptide immunotherapies. The improvement takes form in an ability to dynamically administer the compound based on the ability of the compound to achieve an increased immune activation, while generating either a TH1 immune posture, or a TH2 immune posture, and while producing anti-compound antibodies at either a low or a high level. Dynamic administration of random sequence copolymer is comprised of any combination of dose, regimen, route of administration, and/or formulation. This dynamic immunomodulation provides for increased effectiveness at any of the multiple stages of a disease within a particular patient, as well as the ability to treat multiple, pathogenic antigenic-determinant unrelated diseases more effectively.
The invention provides methods for the treatment or prevention of a disease in a subject, preferably in a human, which subject is afflicted with or is suspected to be afflicted with the disease. Another embodiment of the present invention is a method for prophylactically treating a subject at risk of developing a protein conformational disorder by administering a DSP composition. A subject at risk is identified by, for example, determining the genetic susceptibility to a protein conformational disorder by testing for alleles of HLA that are associated with such disorder, and/or based on familial history, or other genetic markers that correlate with such disorder. In addition, many patients receiving dialysis or other form of blood filtration are at risk for developing DRA, especially if the blood filtration is performed over a long period of time, such as more than 3, 5, 7, or 10 years. Further, subjects that are asymptomatic but show biochemical markers of a protein conformational disorder are at risk of developing such disorder.
One aspect of the invention provides methods of treating or preventing a disease, the method comprising administering to said subject a dosing regimen of an effective amount of a DSP composition for the amelioration of a disease treatable with the DSP composition, said effective amount delivered to said subject at time intervals greater than 24 hours, 36 hours, or more preferably greater than 48 hours. A related aspect of the invention provides a method for the treatment of a subject in need thereof, comprising administering to said subject a dosing regimen of an effective amount of a DSP composition for the amelioration of a disease treatable with the DSP composition, said effective amount delivered to the subject using a sustained-release formulation which administers the DSP composition over a period of at least 2 days, at least 4 days, or at least 6 days, wherein the effective amount is an amount that is effective if delivered daily.
One aspect of the invention is the administration of a DSP composition to a subject in need there of, as described above, in combination with other therapeutic agents that are effective in treating the conditions that are treated by administration of the DSP, or conditions that accompany or occur concurrently with the conditions that are treated by administration of the DSP. The additional therapeutically active agents may treat the same or related disease as the DSP composition, or may be intended to treat an undesirable side effect of administration of the DSP composition, such as to reduce swelling at a site of intradermal injection. Alternatively, the other therapeutic agents enhance the activity of DSP compositions. Such additional therapeutic agents are, by way of example, antibodies, cytokines, growth factors, enzyme inhibitors, antibiotics, antiviral agents, anti-inflammatory including steroids, immune boosters, animetabolites, soluble cytokine receptors, and vitamin D or agents that increase the level of circulating vitamin D, toll-like receptor agonists, CpG oligodeoxynucleotides, surface charged poly(lactide-co-glycolide) microparticles, any of the above encapsulated into liposomes, archaeosome adjuvants, mucosal adjuvants, polyphosphazenes. Additional therapeutically active agents also include copolymers which bind to a HLA molecule associated with the disease such as another DSP composition. The HLA molecule may be an HLA-DQ molecule or an HLA-DR molecule. The enzyme inhibitor may be a protease inhibitor or a cyclooxygenase inhibitor. Examples of the therapeutically active agents to be administered in conjunction with the DSP composition are recited in Section IV, “Pharmaceutical Composition” section, though the administration of these agents are not limited to co-administration as a single composition. The additional therapeutic agents may be administered before, concomitantly with, or after the administration of the DSP composition, at such time that the effect of the additional therapeutic agents and the effect of the DSP composition overlap at some time point.
Alternatively, antigen/epitope non-specific treatments and therapies directly targeted at controlling T lymphocytes or their functions may be administered in conjunction with the DSP composition. The therapeutic agents useful for such treatment include Muromonab-CD3 (OKT3), antilymphocyte globulin (ALG), antithymocyte globulin (ATG), or interleukin-2 receptor monoclonal antibody (“mAb”) daclizumab or basiliximab. Other agents include soluble CTLA-4, an anti-CD154 mAb; anti-CD11a; a humanized mAb which inhibits VLA-4; anti-CD2, 3, or 4 antibodies; and anti-CD152 antibodies (J. B. Matthews et al., Amer. J. Transplantation, 2003, 3: 794-80).
When treating protein conformation diseases, such as DRA, it may be advantageous to administer the DSP therapeutic or DSP-specific antibody therapeutic in combination with one or more additional therapy. In certain embodiments, the additional therapy lowers B2M levels in the patient. In some embodiments, the additional therapy is a form of dialysis such as haemodialysis or CAPD. The efficacy of CAPD in removing B2M from the bloodstream is discussed in Lysaght et al. (Peritoneal Dialysis International, Vol. 9, pp 29-35, 1989). In various embodiments, B2M is removed from a patient's bloodstream using a direct hemoperfusion column comprising porous cellulose beads to which hydrophobic hexadecyl alkyl chain is covalently bound, as discussed in Kutsuki H (Biochimica et Biophysica Acta 1753 (2005) 141-145). In certain embodiments, the additional therapy removes unwanted products of haemodialysis. For instance, dialysis patients often have elevated levels of parathyroid hormone, advanced glycation end products, advanced lipoxidation end products, advanced oxidation protein products, granulocyte inhibitory proteins, or leptin (Horl, J Am Soc Nephrol 13: S62-S71, 2002). Levels of these products may be reduced by using a biocompatible membrane for haemodialysis. In addition, levels of these products may be reduced by passing the patient's blood over a substrate that contains a molecule (e.g. an antibody) specific to one or more of these products.
As discussed earlier, elevated levels of Cu++ promote the assembly of B2M monomers into amyloid aggregates. For this reason, it may be desirable to administer the therapies herein together with an agent that reduces Cu++ levels in a patient. For example, a copper chelator such as vitamin C, molybdenum, tetrathiomolybdate (e.g. Coprexa), penicillamine, trientine, or sulfur-containing amino acids may be used.
On the same principle, Cu++ may be added to a DSP peptide to promote its assumption of the disease-specific conformation. This may occur during the manufacturing process. After the peptide has taken on the disease-specific conformation, the copper may be removed, for instance with a copper chelator. The DSP peptide may then be used as a therapeutic or to generate an antibody.
In some embodiments, the therapeutic is co-administered with an inflammation-reducing agent. Inflammation-reducing agents are well known in the art and include steroids and NSAIDs (which typically inhibit COX enzymes). Classes of steroids include glucocorticoids and corticosteroids; examples include Hydrocortisone (Cortisol), Cortisone acetate, Prednisone, Prednisolone, Methylprednisolone, Dexamethasone, Betamethasone, Triamcinolone, Beclometasone, Fludrocortisone acetate, Deoxycorticosterone acetate (DOCA), and Aldosterone, NSAIDS include aminoarylcarboxylic acid derivatives such as enfenamic acid, etofenamate, flufenamic acid, isonixin, meclofenamic acid, mefanamic acid, niflumic acid, talniflumate, terofenamate and tolfenamic acid; arylacetic acid derivatives such as acemetacin, alclofenac, amfenac, bufexamac, cinmetacin, clopirac, diclofenac sodium, etodolac, felbinac, fenclofenac, fenclorac, fenclozic acid, fentiazac, glucametacin, ibufenac, indomethacin, isofezolac, isoxepac, lonazolac, metiazinic acid, oxametacine, proglumetacin, sulindac, tiaramide, tolmetin and zomepirac; arylbutyric acid derivatives such as bumadizon, butibufen, fenbufen and xenbucin; arylcarboxylic acids such as clidanac, ketorolac and tinoridine; arylpropionic acid derivatives such as alminoprofen, benoxaprofen, bucloxic acid; carprofen, fenoprofen, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indoprofen, ketoprofen, loxoprofen, miroprofen, naproxen, oxaprozin, piketoprofen, pirprofen, pranoprofen, protizinic acid, suprofen and tiaprofenic acid; pyrazoles such as difenamizole and epirizole; pyrazolones such as apazone, benzpiperylon, feprazone, mofebutazone, morazone, oxyphenbutazone, phenybutazone, pipebuzone, propyphenazone, ramifenazone, suxibuzone and thiazolinobutazone; salicylic acid derivatives such as acetaminosalol, aspirin, benorylate, bromosaligenin, calcium acetylsalicylate, diflunisal, etersalate, fendosal, gentisic acid, glycol salicylate, imidazole salicylate, lysine acetylsalicylate, mesalamine, morpholine salicylate, 1-naphthyl salicylate, olsalazine, parsalmide, phenyl acetylsalicylate, phenyl salicylate, salacetamide, salicylamine o-acetic acid, salicylsulfuric acid, salsalate and sulfasalazine; thiazinecarboxamides such as droxicam, isoxicam, piroxicam and tenoxicam; others such as {umlaut over (γ)}-acetamidocaproic acid, s-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole and tenidap; and pharmaceutically acceptable salts thereof; and other analgesics, such as acetaminophen. The dosage of analgesic and/or antipyretic such as aspirin, acetaminophen, etc. will be known to those skilled in the art and can be in the range of 80 mg to 250 mg. The dosage of NSAID will be known to those skilled in the art and can be in the range of 80 mg to 500 mg.
In one embodiment of the methods described herein, the route of administration can be oral, intraperitoneal, transdermal, subcutaneous, by intravenous or intramuscular injection, by inhalation, topical, intralesional, or by infusion; liposome-mediated delivery; intrathecal, gingival pocket, rectal, intravaginal, intrabronchial, nasal, transmucosal, intestinal, ocular or otic delivery, or any other methods known in the art as one skilled in the art may easily perceive. Administration can be systemic or local. In the event more than one DSP composition is being administered to a subject during the same or overlapping time period, such additional therapeutic agent may be administered by a route different from that for the administration of the DSP composition.
In general, an embodiment of the invention is to administer a suitable dose of a therapeutic DSP composition that will be the lowest effective dose to produce a therapeutic effect, for example, mitigating symptoms. The therapeutic DSP compositions are preferably administered at a dose per subject, which corresponds to a dose per day of at least about 2 mg, at least about 5 mg, at least about 10 mg, or at least about 20 mg as appropriate minimal starting dosages, or about x mg, wherein x is an integer between 1 and 20. In one embodiment of the methods described herein, a dose of about 0.01 to about 500 mg/kg can be administered. In general, the effective dosage of the DSP composition of the present invention is about 50 to about 400 micrograms of the composition per kilogram of the subject per day. In one specific embodiment, the equivalent dosage per day, regardless of the frequency with which the doses are administered, is from about 5 to 100, or more preferably, from about 10 to 40, or more preferably about 20 mg/day. In another specific embodiment, each individual dosage in the treatment regimen is from about 5 to 100, or more preferably from about 10 to 40, or more preferably about 20 mg/dose.
However, it is understood by one skilled in the art that the dose of the DSP composition of the invention will vary depending on the subject and upon the particular route of administration used. It is routine in the art to adjust the dosage to suit the individual subjects. Additionally, the effective amount may be based upon, among other things, the size of the DSPs, the biodegradability of the DSPs, the bioactivity of the DSPs and the bioavailability of the DSPs. If the DSPs does not degrade quickly, such as is expected when the DSPs comprise unnatural amino acids or are peptidomimetics, is bioavailable and highly active, a smaller amount will be required to be effective. The actual dosage suitable for a subject can easily be determined as a routine practice by one skilled in the art, for example a physician or a veterinarian given a general starting point. For example, the physician or veterinarian could start doses of the DSP composition of the invention employed in the pharmaceutical composition at a level lower than that required in order to achieve the desired therapeutic effect, and increase the dosage with time until the desired effect is achieved. The dosage of the DSP composition may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disorder or disease state is observed, or if the disorder or disease state has been ablated, or if an unacceptable side effects are seen with the starting dosage.
In one embodiment, a therapeutically effective amount of the DSP composition is administered to the subject in a treatment regimen comprising intervals of at least 36 hours, or more preferably 48 hours, between dosages. In another embodiment, the DSP composition is administered at intervals of at least 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, or 240 hours, or the equivalent amount of days. In some embodiments, the DSP composition is administered every other day, while in other embodiments it is administered weekly. If two different DSP compositions, or DSP composition with another therapeutic agent, are administered to the subject, such administration may take place at the same time, such as simultaneously, or essentially at the same time, such as in succession. Alternatively, their administration may be staggered. For example, two DSP compositions which are each administered every 48 hours may both be administered on the same days, or one may be administered one day and the other on the next day and so on in an alternating fashion.
Treatment regimens with longer dosing intervals, consequently often with lower total exposure of DSPs, are expected to induce lower titers of antibodies against DSPs themselves, while still inducing desired protective effects. Such reduction of neutralizing antibodies are desirable because it is considered likely to help DSP compositions to retain its effectiveness without being neutralized, and it is associated with reduced risk of anaphylactic shocks, providing safer treatments of diseases. Longer interval regimens are also desirable in treatment of some of the diseases, because they strengthen the bias for T
In other embodiments, the DSP composition is administered in a treatment regimen which comprises at least one uneven time interval, wherein at least one of the time intervals is at least 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, or 240 hours, or the equivalent amount of days.
In one embodiment, the DSP composition is administered to be subject at least three times during a treatment regimen, such that there are at least two time intervals between administrations. These intervals may be denoted I1 and I2. If the DSP composition is administered four times, then there would be an additional interval between the third and fourth administrations, 13, such that the number of intervals for a given number “n” of administrations is n−1. Accordingly, in one embodiment, at least one of the time intervals between administrations is greater than about 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, or 240 hours. In another embodiment, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the total number n−1 of time intervals are at least about 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, or 240 hours.
In yet another embodiment, the average time interval between administrations ((I1+I2+ . . . +In-1)/n−1) is at least 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222, 228, 234, or 240 hours, or at least two weeks.
In another embodiment, the dosage regimen consists of two or more different interval sets. For example, a first part of the dosage regimen is administered to a subject daily, every other day, or every third day, for example, at about 22 mg copolymer/m2 body surface area of the subject, wherein the subject is a human. In some embodiment of the invention, the dosing regimen starts with dosing the subject every other day, every third day, weekly, biweekly, or monthly. The dosage for administration every other day or every third day may be up to about 65 mg/m2 and 110 mg/m2 respectively. For a dosing regimen comprising dosing of the random copolymer every week, the dose comprises up to about 500 mg/m2, and for a dosing regimen comprising dosing of the random copolymer every two weeks or every month, up to 1.5 g/m2 may be administered. The first part of the dosing regimen may be administered for up to 30 days, for example, 7, 14, 21, or 30 days. A subsequent second part of the dosing regimen with a different, longer interval administration with usually lower exposure (step-down dosage), administered weekly, every 14 days, or monthly may optionally follow, for example, at 500 mg/m2 body surface area weekly, up to maximum of about 1.5 g/m2 body surface area, continuing for 4 weeks up to two years, for example, 4, 6, 8, 12, 16, 26, 32, 40, 52, 63, 68, 78, or 104 weeks. Alternatively, if the disease goes into remission or generally improves, the dosage may be maintained or kept at lower than maximum amount, for example, at 140 mg/m2 body surface area weekly. If, during the step-down dosage regimen, the disease condition relapses, the first dosage regimen may be resumed until effect is seen, and the second dosing regimen may be implemented. This cycle may be repeated multiple times as necessary.
In other embodiments of the invention, any of the methods of the invention may be practiced using sustained release formulation comprising a DSP composition. When administering a DSP composition of the invention using a sustained release formula, the overall exposure to the DSP is generally lower than in bolus administration. For example, a first part of the dosage regimen is administered to a subject daily, every other day, or every third day, for example, at about 22 mg DSP/m2 body surface area of the subject, wherein the subject is a human. In some embodiment of the invention, the dosing regimen uses sustained release formula, dosing the subject every other day, every third day, weekly, biweekly, or monthly so that the copolymer is released during the interval. The dosage for administration every other day or every third day may be up to about 35 mg/m2 and 65 mg/m2 respectively. For a dosing regimen comprising dosing of the DSP composition every week, the dose comprises up to about 140 mg/m2, and for a dosing regimen comprising dosing of the DSP composition every two weeks or every month, up to 750 mg/m2 may be administered. The first part of the dosing regimen may be administered for up to 30 days, for example, 7, 14, 21, or 30 days. A subsequent second part of the dosing regimen with a different, longer interval administration with usually lower exposure (step-down dosage), administered weekly, every 14 days, or monthly may optionally follow, for example, at 140 mg/m2 body surface area weekly, up to maximum of about 1.5 g/m2 body surface area, continuing for 4 weeks up to two years, for example, 4, 6, 8, 12, 16, 26, 32, 40, 52, 63, 68, 78, or 104 weeks. Alternatively, if the disease goes into remission or generally improves, the dosage may be maintained or kept at lower than maximum amount, for example, at 140 mg/m2 body surface area weekly. If, during the step-down dosage regimen, the disease condition relapses, the first dosage regimen may be resumed until effect is seen, and the second dosing regimen may be implemented. This cycle may be repeated multiple times as necessary.
For such sustained release administration, such method comprises applying a sustained-release transdermal patch or implanting a sustained-release capsule or a coated implantable medical device so that a therapeutically effective dose of the copolymer of the present invention is delivered at defined time intervals to a subject of such a method. The DSP composition of the subject invention may be delivered via a capsule which allows regulated-release of the DSPs over a period of time. Controlled or sustained-release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Also comprehended by the invention are particulate compositions coated with polymers (e.g., poloxamers or poloxamines). In certain embodiments, a source of a DSP composition is stereotactically provided within or proximate to the area where pathology is observed or suspected.
An improvement in the symptoms of a subject afflicted with a disease as a result of administration of the DSP composition may be noted by a decrease in frequency of recurrences of episodes of the disease symptoms, by decrease in severity of symptoms, and by elimination of recurrent episodes for a period of time after the start of administration. A therapeutically effective dosage preferably reduces symptoms and frequency of recurrences by at least about 20%, for example, by at least about 40%, by at least about 60%, and by at least about 80%, or by about 100% elimination of one or more symptoms, or elimination of recurrences of the autoimmune disease, relative to untreated subjects. The period of time can be at least about one month, at least about six months, or at least about one year.
VI. Methods of Treatment—Administration of Antibodies Generated Using DSPAn aspect of the present invention is a method of treating a subject afflicted with a protein conformational disorder, comprising the steps of administering an antibody prepared using a DSP composition as described above. In a particular embodiment, the protein conformational disorder is Parkinson's disease. In another embodiment, the protein conformational disorder is dialysis-related amyloidosis. In another embodiment, the protein conformational disorder is Alzheimer's disease.
Another aspect of the present invention is a method of treatment using antibodies against a DSP composition related to a disease, in particular, a protein conformational disease. The antibodies useful for such method of treatment are antibodies generated against a DSP composition as described above, wherein the base sequence is a sequence of a protein known to be associated with a protein conformational disorder. More particularly, such protein is known to form an aggregate or fibril. In particular, antibodies thus generated are specific to the pathological conformation of such protein. The list of relevant diseases is recited above in this specification.
In an embodiment of this aspect of the invention, the antibodies for the use in the method of treatment are modified antibodies having an engineered Fc region, wherein the engineered Fc region confers favorable pharmacodynamic profiles. In one embodiment, the Fc region enhances clearance of antibody-antigen complex. In another embodiment, the Fc region is not immunogenic to the subject.
An aspect of the present invention is a method of treating a subject afflicted with a protein conformational disorder, comprising the steps of administering an antibody prepared using a DSP composition as described above. In a particular embodiment, the protein conformational disorder is Parkinson's disease. In another embodiment, the protein conformational disorder is dialysis-related amyloidosis. In another embodiment, the protein conformational disorder is Alzheimer's disease.
In an aspect of the invention, an antibody or antibodies identified by the method to generate antibodies against antigens associated with a protein conformational disease is cloned. The nucleic acids encoding such antibodies are cloned into an appropriate expression vector and delivered to a cellular site where such antibodies are desirable, at which site the antibodies are expressed.
Another aspect of the present invention is a method of treating a subject afflicted with a protein conformational disorder, comprising the steps of contacting under sterile conditions the blood of the subject to a membrane or a resin having conjugated with antibodies specific to a protein associated with a protein conformational disorder and prepared using a DSP composition, such antibodies described above, wherein the protein associated with a protein conformational disorder binds to such antibodies and is removed from the blood, and returning the blood to the subject. In a particular embodiment, the protein conformational disorder is dialysis-related amyloidosis. In one embodiment, the blood of the subject is contacted with the antibody as an additional step of therapeutic haemodialysis.
An embodiment of the invention is a method of prophylactic treatment of a subject at risk for developing a protein conformational disorder by contacting under sterile conditions the blood of the subject to a membrane or a resin having conjugated with antibodies specific to a protein associated with a protein conformational disorder and prepared using a DSP composition, such antibodies described above, wherein the protein associated with a protein conformational disorder binds to such antibodies and is removed from the blood, and returning the blood to the subject, whereby preventing the onset of such protein conformational disorder.
An aspect of the invention is a composition comprising a scaffold to which antibodies are attached, which antibodies are generated against a DSP composition as described above, wherein the base sequence is a sequence of a protein known to be associated with a protein conformational disorder. In one embodiment, the scaffold is a membrane compatible with haemodialysis. In a particular embodiment, the antibodies are conjugated to such membrane. In another embodiment, the antibodies are conjugated to a resin, such as CN—Br agarose resin (for example CN—Br Sepharose® (Pharmacia), to create an immunoaffinity resin.
VIII. Other MethodsThe instant invention also comprises a method of creating antibody reagents for use in research studies, and such antibodies useful for research. Certain antibodies generated or selected by their specific binding to a DSP composition is useful to identify specific conformation of a protein in its pathological and non-pathological state. Such antibodies, when conjugated to scaffolds, are further useful for isolating and purifying the target proteins and peptides. Such antibodies are also useful in preclinical investigations of candidate pharmaceutical agents, wherein such agent may disrupt or disturb the binding of such antibodies to the target proteins. The antibodies can also be used to detect certain pathological antibodies and to measure the effects of such candidate pharmaceutical agents.
The instant invention also comprises a method of creating antibody reagents for use as diagnostic tools.
An embodiment of the invention, a method of diagnosing a protein conformational disorder, comprising: (i) contacting a biological sample from a subject with an antibody the invention; (ii) contacting a control sample with the antibody; and (iii) measuring specific binding of the antibody to an antigen in the sample; wherein specific binding of the antibodies to the antigen is indicative of the subject being afflicted with the disorder. A number of methods for measuring antibody-protein binding are known in the art, including ELISA, Western blotting, and spot-blot. The control sample may be a standard sample, a sample from a second subject known to be free of the pathology that is being investigated, or a sample from the same subject at a different time point to determine the chronological changes of the disease conditions. In some embodiments, tests are performed simultaneously using the antibody of the invention and a positive control antibody that confirms that the biological sample contains sufficient material. The positive control antibody may recognize any protein that is reasonably expected to be present in all samples (i.e. from both healthy and diseased patients), and may recognize a housekeeping enzyme (for example). In some embodiments, the binding of the antibody of the invention is quantified; in other embodiments, the binding is evaluated qualitatively.
More particularly, such disorder to be detected is one of the disorders enumerated elsewhere in this application.
In some embodiments, the diagnostic test may be performed in vivo, identifying the affected locations within the body. The antibody may be labeled in such a manner that it can be detected within a patient's body, e.g. with MRI. This label may be an iron-containing compound, such as ferrous and ferric-containing compounds, e.g. ferric-oxides. Specific examples include Fe2O3 and Fe3O4. Antibodies labeled with iron-containing compounds may also be used for in vitro diagnosis, e.g. when MRI is performed on a biological sample.
DEFINITIONSThe term “associated with” means “coexistent with” or “in correlation with.” The term does not necessarily indicate causal relationship, though such relationship may exist.
The term “binding” refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, ionic and/or hydrogen-bond interactions under physiological conditions, and including interactions such as salt bridges and water bridges.
The term “HLA molecule” means any class II major histocompatibility complex glycoproteins.
The term “immunomodulation” means the process of increasing or decreasing the immune system's ability to mount a response against a particular antigenic determinant through the T-cell receptor (“TCR”)'s recognition of complexes formed by major histocompatibility complex (“MHC”) and antigens.
The term “MHC activity” refers to the ability of an MHC molecule to stimulate an immune response, e.g., by activating T cells. An inhibitor of MHC activity is capable of suppressing this activity, and thus inhibits the activation of T cells by MHC. In preferred embodiments, a subject inhibitor selectively inhibits activation by a particular class II MHC isotype or allotype. Such inhibitors may be capable of suppressing a particular undesirable MHC activity without interfering with all MHC activity in an organism, thereby selectively treating an unwanted immune response in an animal, such as a mammal, preferably a human, without compromising the animal's immune response in general.
The term “patient” refers to an animal, preferably a mammal, including humans as well as livestock and other veterinary subjects.
The terms “peptide”, “polypeptide” and “protein” are used interchangeably herein. These terms refer to unmodified amino acid chains, and also include minor modifications, such as phosphorylations, glycosylations and lipid modifications. The terms “peptide” and “peptidomimetic” are not mutually exclusive and include substantial overlap.
A “peptidomimetic” includes any modified form of an amino acid chain, such as a phosphorylation, capping, fatty acid modification and including unnatural backbone and/or side chain structures. As described below, a peptidomimetic comprises the structural continuum between an amino acid chain and a non-peptide small molecule. Peptidomimetics generally retain a recognizable peptide-like polymer unit structure. Thus, a peptidomimetic may retain the function of binding to a HLA protein forming a complex which activates autoreactive T cells in a patient suffering from an autoimmune disease.
The term “amino acid residue” is known in the art. In general the abbreviations used herein for designating the amino acids and the protective groups are based on recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochemistry (1972) 11:1726-1732). In certain embodiments, the amino acids used in the application of this invention are those naturally occurring amino acids found in proteins, or the naturally occurring anabolic or catabolic products of such amino acids which contain amino and carboxyl groups. Particularly suitable amino acid side chains include side chains selected from those of the following amino acids: glycine, alanine, valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine, and tryptophan.
The term “amino acid residue” further includes analogs, derivatives and congeners of any specific amino acid referred to herein, as well as C-terminal or N-terminal protected amino acid derivatives (e.g. modified with an N-terminal or C-terminal protecting group). For example, the present invention contemplates the use of amino acid analogs wherein a side chain is lengthened or shortened while still providing a carboxyl, amino or other reactive precursor functional group for cyclization, as well as amino acid analogs having variant side chains with appropriate functional groups). For instance, the subject compound can include an amino acid analog such as, for example, cyanoalanine, canavanine, djenkolic acid, norleucine, 3-phosphoserine, homoserine, dihydroxy-phenylalanine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, diaminopimelic acid, ornithine, or diaminobutyric acid. Other naturally occurring amino acid metabolites or precursors having side chains which are suitable herein will be recognized by those skilled in the art and are included in the scope of the present invention.
Most of the amino acids used in the DSPs of the present invention may exist in particular geometric or stereoisomeric forms. In preferred embodiments, the amino acids used to form the subject DSPs are (
“Amino acid similarity”, as used herein, means the relationship of those amino acids grouped together in
“Naturally occurring variations”, as used herein in reference to amino acids are allelic variations, isomeric and species differences of functionally equivalent proteins, naturally occurring amino acid modifications, whether or not incorporated while synthesis or post-synthesis (i.e. post-translation modification in vivo and post-synthesis modification in vitro) such as preformed phosphoylations, preformed nitrations, preformed glycosylations, modification by fatty acids (such as myristoylation), modified amino acid side chains including modification to produce amino acid analogs as described in paragraph defining “amino acid residue”, cross-linking such as disulfide bonds, and other known modifications.
“Prevent”, as used herein, means to delay or preclude the onset of, for example, one or more symptoms, of a disorder or condition.
“Treat”, as used herein, means at least lessening the severity or ameliorating the effects of, for example, one or more symptoms, of a disorder or condition.
“Treatment regimen” as used herein, encompasses therapeutic, palliative and prophylactic modalities of administration of one or more compositions comprising one or more DSP compositions. A particular treatment regimen may last for a period of time at a particular dosing pattern, which will vary depending upon the nature of the particular disease or disorder, its severity and the overall condition of the patient, and may extend from once daily, or more preferably once every 36 hours or 48 hours or longer, to once every month or several months.
The terms “structure-activity relationship” or “SAR” refer to the way in which altering the molecular structure of drugs alters their interaction with a receptor, enzyme, etc.
The practice of the present invention will employ, where appropriate and unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, virology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning: A Laboratory Manual, 3rd Ed., ed. by Sambrook and Russell (Cold Spring Harbor Laboratory Press: 2001); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Using Antibodies, Second Edition by Harlow and Lane, Cold Spring Harbor Press, New York, 1999; Current Protocols in Cell Biology, ed. by Bonifacino, Dasso, Lippincott-Schwartz, Harford, and Yamada, John Wiley and Sons, Inc., New York, 1999; and PCR Protocols, ed. by Bartlett et al., Humana Press, 2003; PHARMACOLOGY A Pathophysiologic Approach Edited by Joseph T. DiPiro, Robert Talbert, Gary, Yee, Gary Matzke, Barbara Wells, and L. Michael Posey. 5th edition 2002 McGraw Hill; Pathologic Basis of Disease. Ramzi Cotran, Vinay Kumar, Tucker Collins. 6th Edition 1999. Saunders.
EXAMPLES Example 1 Preparation of a DSP Composition from Fictitious Base PeptidesFor ease of understanding, as an illustration, preparation of a DSP composition deriving from two fictitious peptide sequences, representing a known epitope, is described and shown in the table depicted in
The DSP composition as described above is prepared using a solid phase peptide synthesis method as described elsewhere in this disclosure.
Example 2 Preparation of a DSP Composition from Alpha-Synuclein PeptideA DSP composition is designed from alpha-synuclein peptide (amino acid residues 121-137) using the preferred method of amino acid substitution described by Kosiol et al., J. Theoretical Biol. 228:97-106, 2004, as shown in
The indicated serine may be phosphorylated. The indicated tyrosine residues may be tri-nitrated. Thus, the synthesized DSP composition, prepared using a solid phase peptide synthesis method as described elsewhere in this disclosure, may be postsynthetically modified by phosphorylating certain of the serines and/or tri-nitrating the tyrosine. The modification is carried out under such condition that allows for partial population of the DSP peptide to be modified, resulting a further diversified library of peptides.
Example 3 Preparation of a DSP Composition from Aβ1-42 Base PeptideA DSP composition is designed from Aβ1-42 peptide using the preferred method of determining, for each amino acid position, the amino acid substitution described by Kosiol et al., J. Theoretical Biol. 228:97-106, 2004, as shown in
A DSP composition is designed from huntingtin peptide using the preferred method of determining, for each amino acid position, the amino acid substitution described by Kosiol et al., J. Theoretical Biol. 228:97-106, 2004, as shown in
The contents of any patents, patent applications, patent publications, or scientific articles referenced anywhere in this application are herein incorporated in their entirety. Additional references related to the disclosed invention is as follows.
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Sequence Listings in Addition to Table I
Claims
1-34. (canceled)
35. A process for generating antibodies comprising the steps of:
- (i) preparing a composition comprising directed-sequence polymers (DSPs), wherein the DSP composition has a complexity of greater than 5×102 different DSPs, comprising the steps of: (1) selecting a base peptide sequence, wherein the base peptide sequence is derived from an epitope of an antigen associated with a protein conformational disorder, (2) synthesizing by solid phase peptide synthesis a first cassette of the DSPs, the cassette having a sequence of amino acid positions corresponding to each amino acid of the base peptide sequence, wherein, for at least one amino acid position of the first cassette of the DSPs, an amino acid is added, said amino acid randomly selected from a mixture of amino acids consisting of the original amino acid found at the corresponding amino acid position of the base peptide sequence, optionally alanine (A), and, optionally, at least one conserved substitution, wherein the amino acids in the mixture are present in a fixed molar input ratio relative to each other, determined prior to starting synthesis, wherein the relative molar amount of A is more than 10% and less than 90% of the total amino acid concentration of the DSPs; (3) optionally extending the length of the DSPs by (a) repeating step (2) for 1 to 15 cycles and elongating the DSPs under the same condition including the input ratio of amino acids in the mixture; (b) repeating step (2) for 1 to 15 cycles and elongating the DSPs, for each cycle, using a different input ratio of amino acids in the mixture; (c) repeating steps (1) and (2) for 1 to 15 cycles and elongating the DSPs using cassettes based on more than one base peptide; (d) assembling 1 to 15 cassettes synthesized in a single cycle of step (2); or (e) assembling 1 to 15 cassettes, the first cassette synthesized under one condition of step (2), and second and more cassettes synthesized under one or more different conditions of step (2);
- wherein the number of cycles selected in step (3) is selected so that the final length of the DSPs is 10 to 300 amino acid residues;
- (ii) administering said DSP composition to an animal; and
- (iii)(a) isolating antibodies immunoreactive with said DSP composition from said animal, or
- (iii)(b) isolating cells that produce antibodies immunoreactive with said DSP composition from said animal, and then isolating antibodies immunoreactive with said DSP composition from said isolated cells.
36. The process according to claim 35, wherein the antibodies are immunoreactive with a protein comprising the base peptide, with or without post-translational modification, and wherein said protein is a full-length protein associated with the protein conformational disorder or a fragment of such full-length protein, and wherein said protein is in a pathological or non-pathological conformation.
37-56. (canceled)
57. A process for manufacturing a composition comprising directed-sequence polymers (DSPs), wherein the DSP composition has a complexity of greater than 5×102 different DSPs, comprising the steps of: wherein the number of cycles selected in step (3) is selected so that the final length of the DSPs is 10 to 300 amino acid residues.
- (1) selecting a base peptide sequence, wherein the base peptide sequence is derived from an epitope of an antigen associated with a protein conformational disorder,
- (2) synthesizing by solid phase peptide synthesis a first cassette of the DSPs, the cassette having a sequence of amino acid positions corresponding to each amino acid of the base peptide sequence, wherein, for at least one amino acid position of the first cassette of the DSPs, an amino acid is added, said amino acid randomly selected from a mixture of amino acids consisting of the original amino acid found at the corresponding amino acid position of the base peptide sequence, optionally alanine (A), and, optionally, at least one conserved substitution, wherein the amino acids in the mixture are present in a fixed molar input ratio relative to each other, determined prior to starting synthesis, wherein the relative molar amount of A is more than 10% and less than 90% of the total amino acid concentration of the DSPs;
- (3) optionally extending the length of the DSPs by (a) repeating step (2) for 1 to 15 cycles and elongating the DSPs under the same condition including the input ratio of amino acids in the mixture; (b) repeating step (2) for 1 to 15 cycles and elongating the DSPs, for each cycle, using a different input ratio of amino acids in the mixture; (c) repeating steps (1) and (2) for 1 to 15 cycles and elongating the DSPs using cassettes based on more than one base peptide; (d) assembling 1 to 15 cassettes synthesized in a single cycle of step (2); or (e) assembling 1 to 15 cassettes, the first cassette synthesized under one condition of step (2), and second and more cassettes synthesized under one or more different conditions of step (2);
58. The process according to claim 57, wherein the antigen is associated with a protein conformational disorder affecting the central and/or peripheral nervous system.
59. The process according to claim 57, wherein the protein conformational disorder is Parkinson's disease.
60. The process according to claim 57, wherein the protein conformational disease is dialysis-related amyloidosis.
61. The process according to claim 57, wherein the first base peptide sequence is selected from the group consisting of SEQ ID NO: 3 through 13.
62. The process according to claim 57, wherein A is more than about 10% and less than about 70% of the total amino acid concentration of the DSPs.
63. The process according to claim 57, wherein A is more than about 15% and less than about 50% of the total amino acid concentration of the DSPs.
64. The process according to claim 57, wherein the conserved substitution is defined according to the amino acid similarity table shown in FIG. 4.
65. The process according to claim 57, wherein the conserved substitution is determined based on empirical data of known naturally occurring variants of the epitope.
66. The process according to claim 57 wherein, for at least one amino acid position of the first cassette of the DSPs, an amino acid is added, said amino acid randomly selected from a mixture of amino acids comprising an original amino acid found at the corresponding amino acid position of the base peptide sequence, alanine (A), and, optionally, at least one amino acid serving as a conserved substitution.
Type: Application
Filed: Sep 12, 2013
Publication Date: Sep 18, 2014
Applicant: DECLION PHARMACEUTICALS, INC. (Boxford, MA)
Inventors: Dustan Bonnin (Belmont, MA), Eric Zanelli (Sudbury, MA), Thomas P. Mathers (Boxford, MA)
Application Number: 14/025,223
International Classification: C07K 1/04 (20060101); C07K 16/18 (20060101);
