Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
  • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/36—Blood coagulation or fibrinolysis factors
  • A61K38/37—Factors VIII
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1274—Non-vesicle bilayer structures, e.g. liquid crystals, tubules, cubic phases or cochleates; Sponge phases
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/04—Fusion polypeptide containing a localisation/targetting motif containing an ER retention signal such as a C-terminal HDEL motif

Definitions

• the present invention relates to protein complexes having low immunogenicity and a method of making same.
• Hemophilia is a bleeding disorder caused by the deficiency of factor VIII (anti hemophilic factor, AHF or FVIII).
• FVIII is a multi domain protein comprising of six domains Al, A2, A3, B, Cl and C2 and activation of this protein by thrombin results in heavy (Al and A2) and light chain (A3, Cl and C2) [1, 2].
• Replacement therapy using blood concentrate, recombinant factor VIII and variants of factor VIII is the first line therapy for hemophilia. However, 15-35% of patients develop neutralizing antibodies and such immune response compromises therapy for hemophilia. Current treatment regimens to overcome neutralizing antibody development exist but are not cost effective. Development of less immunogenic Factor VIII preparations could offer an alternate clinical approach.
• the C2 domain is also a membrane binding domain and it binds to phosphatidyl serine (PS) on platelet membranes as part of its coagulation cascade [6,7].
• PS phosphatidyl serine
• the anticoagulant action of antibodies to the C2 domain is due to inhibition of binding of factor VIII to phospholipid. It has been shown that the monoclonal antibodies against the C2 domain prevent the binding of Factor VIII to phospholipid containing membranes and based on these observations it was concluded that the epitope and membrane binding regions overlap [10, H].
• liposomes have been used as adjuvants to increase the immune response [26-31].
• development of lipid complex to reduce immune response and antigenicity has not been investigated and therefore, there continues to be a need to develop approaches for reducing the immunogenicity of therapeutic proteins.
• Another approach has been to modify the sequence of the specific epitopes to reduce immuno toxicity [32] .
• amino acid substitutions could lead to loss of biological activity.
• compositions having low antigenicity and immunogenicity and methods of making same comprising a therapeutic agent such as a protein, polypeptide or peptide and one or more molecules capable of binding to the protein (referred to herein as the binding agent) in such a way as to reduce its immunogenicity and antigenicity are disclosed.
• a therapeutic agent such as a protein, polypeptide or peptide and one or more molecules capable of binding to the protein (referred to herein as the binding agent) in such a way as to reduce its immunogenicity and antigenicity
• binding agents include serine compounds such as phosphoserine, phosphatidyl serine, or phospholipids comprising phosphatidyl serine (PS); phosphatidyl choline (PC), phospatidic acid (PA), or phosphoethanolamine (PE); or phospholipids containing PA, PC, or PE.
• the protein-binding agent complexes can be in the form of (1) liquid or freeze dried form of this liquid containing protein-binding agent complex (2) novel non- liposomal structures, (3) liposomes (4) micelles (5) cochleate (6) non-bilayer structures which reduce the immune response.
• the present invention also discloses a method for reducing the immunogenicity and/or antigenicity of a protein by forming a complex with a binding agent (such as a serine containing compound).
• a binding agent such as a serine containing compound.
• the protein-binding agent complex may be stabilized with suitable buffers.
• the dried lipid film containing dimyristyl phosphatidyl choline (DMPC) and brain phosphatidyl serine (bPS) is hydrated using protein (such as FVIII) in various buffer systems.
• Novel, non liposomal structures are formed using DMPC, bPS in 30OmM NaCl and 5mM CaCl 2 .
• Conventional liposomes are formed as the buffer system is changed to water or phosphate buffered saline. This can also be accomplished by reducing the calcium or PS concentrations.
• the removal of DMPC and using 100% PS and a sonication or extrusion step leads to cochleate structures and use of PS with intermediate acyl chain length results in micellar structures. Use of shorter acyl chain length at lower concentrations yield protein-lipid complexes in solution.
• Figure 1 is the melting profile of FVIII at different heating rates.
• Figure 2 is the antibody binding assay that shows the conformational changes in the C2 domain. Binding of monoclonal antibodies ESH 4 and ESH8 to rFVIII as determined by sandwich ELISA following the heating of rFVIII at 60°C and 15°C/hr to the indicated temperatures.
• Figure 3 is a representation of size exclusion chromatography (SEC) profiles of Factor VIII in the presence or absence of O-Phospho-L-Serine.
• Figure 4 is a representation of antigenicity of FVIII-O-Phospho-L-Serine studied by sandwich ELISA.
• Figure 5 A and 5B are representation of the effect of OPLS, phosphocholine and phosphatidic acid on the immunogenicity of rFVIII. Average total antibody titres (5A) and inhibitory titres (5B) are shown for the indicated binding agent-rFVIII complexes compared to rFVIII.
• Figure 6 is a representation of folding studies of FVIII in O phospho L-Serine.
• Figure 7 is a representation of antigenicity of FVIII-PS complex in liposomes studied by sandwich ELISA.
• FVIII Free FVIII
• Invention FVIII Composition used in the present invention
• DMPC+FVIII Physical Mixture of DMPC liposomes and FVIII lacking specific protein (FVIII) lipid (PS) complex.
• Figure 8 A is a representation of the immune response in animal models for free FVIII and FVIII-PS complex.
• FVIII Free FVIII
• Invention FVIII Composition used in the present invention.
• Figure 8B is a representation of FVIII and FVIII-PS complex liposomes in Factor VIII knockout Hemophilia A mice model.
• Figure 9 is the photograph of a Dextran density gradient showing the non- liposomal, low water volume fraction containing FVIII-PS complex.
• Figure 10 is a representation of the effect of DCPS on the antigenicity of rFVIII
• AHF AHF
• Factor VIII FVIII
• the present invention provides a method for reducing the antigenicity and immunogenicity of proteins. While the term "protein” is used throughout the application, it is intended to include peptides (generally considered to be 50 or less amino acids) as well as polypeptides (generally considered to be more than 50 amino acids).
• the method of the present invention comprises the steps of forming complexes of one or more proteins, polypeptides or peptides with a phospholipid, preferably a phospholipid containing serine.
• a phospholipid preferably a phospholipid containing serine.
• Various types of protein-lipid structures can be formed depending upon the particular phospholipid, concentration and combinations of phospholipids
• Liposome means a generally spherical or spheroidal cluster or aggregate of lipid compounds, typically in the form of one or more concentric layers, for example, monolayers, bilayers or multi-layers. They may also be referred to as lipid vesicles.
• the liposomes may be formulated, for example, from ionic lipids and/or non-ionic lipids.
• Cochleates or “cochleate sturcutres” generally refer to a multilamellar lipid vesicle that is generally in the shape of a spiral or a tubule.
• Micelles refers to colloidal entities formulated from lipids. Micelles may comprise a monolayer, bilayer, or hexagonal phase structure.
• phosphoserine, phosphatidyl serine, or PS containing phospholipids with short acyl chain length did not lead to the formation of lipid molecular assemblies.
• the structures formed are simple complexes. These simple complexes are characterized by mostly ionic bonding.
• Use of serine containing phospholipids having intermediate acyl chain length i.e., between 5- 12 acyl chain carbon atoms) above its critical micellar concentration, form micelles and below critical micellar concentration form simple complexes.
• the phospholipids having longer acyl chain length (12-18 carbon atoms) due to the molecular architecture, tend to form several molecular assembles such as liposomes, non-bilayer structures and cochleate structures.
• phosphatidiyl compounds such as PC, PG, PA and PE and phospholipids containing PC, PG, PA and PE results in the formation of micelles for intermediate length acyl chain carbon atoms and liposomes for longer length acyl chain carbon atoms.
• the acyl chain may be a diacyl chain or a single acyl chain.
• phosphatidyl serine, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol means there is no acyl chain in the compound.
• the lipid composition may be varied to prepare liposome, non-bilayer structures and cochleate phases.
• a lipid composition of phosphatidyl choline (PC): phsophatidyl serine (PS) or phosphoserine with high PC content will form liposomes upon hydration with buffers containing Ca 2+ and Na + .
• the presence of Phosphatidyl Ethanolamine PE and PS promote the formation of non-bilayer structures.
• the formulation with PS (over 90mol%) in the presence of Ca 2+ with no or lower Na + (10OmM) promote the formation of cochleate cylinders.
• Conditions such as temperature, Ca 24 VNa 2+ can be altered to reduce the size of cochleate cylinders in the nano-particles containing protein-lipid complex.
• the phospholipids useful for the present invention include serine containing compounds including phospholipids.
• serine containing phospholipids are 0-Phospho-L-Serine (OPLS), Dicaproyl Phosphatidyl Serine, Dioctanoyl Phosphatidyl Serine, Dimyristoyl, Dipalmitoyl, Dioleoyl-, Disteroyl- Phosphatidyl Serine.
• the serine containing phsopholipids maybe used in combination with other molecules including other phospholipids.
• phosphatidyl serine can be used in combination with phosphatidyl chloline or Phosphatidyl ethanolamine.
• the PS, PC, PA and PE are acylatd or diacylated.
• the phosphatidyl serine may be obtained from any source such as natural (brain) or from synthetic origin. Phosphatidyl serine may be used in combination with phospatidyl choline (PC).
• the phosphotidyl choline may be dimyristoyl phophatidyl choline.
• the ratio of the PS and PC can be varied from 1 :9 to 9: 1. hi one embodiment, the ratio is 3:7.
• the protein-lipid compositions of the present invention are preferably stabilized and stored in suitable buffer systems.
• buffers include TRIS buffer and HEPES buffer and sodium and calcium salts.
• alcohol such as 10% ethanol may be added.
• the protein-lipid complexes of the present invention can be characterized by standard methods. For example, fluorescence studies can be carried out on a SLM AMINCO 8000 series instrument or PTI 380 instrument using 4nm as excitation and emission slits. The samples can be excited at 280 nm and the emission spectra scanned in the range of 300 to 400 nm. The emission spectra of free Factor VIII was observed around 335 nm and the addition of OPLS reduced the intensity of fluorescence emission indicating that the tertiary structure of the protein is altered slightly.
• the particle size of the lipid associated protein can be determined using a standard particle sizer (such as NICOMP 315 model).
• the particle size distribution can be analyzed using both Gaussian and NICOMP analysis for unimoidal and bimoidal distribution.
• the size of latex beads can be used as standard controls with each measurement.
• the lipid structures can also be analyzed by negative staining electron microscopy. Such methods are routine in the art and can be used to confirm that there are no aggregates and to classify the structures as liposomes, non-bilayers or cochleates.
• the formation of non-bilayer and cochleate structures can also be investigated using Laurdan fluorescence.
• the lipid structures can be labeled with the probe by mixing the lipid containing solution with aqueous solution of the probe (containing 0.01% ethanol).
• the samples can be excited at 340 nm and the emission spectra were monitored at 440 nm.
• the excitation spectra can be acquired in the range of 320 and 420 nm, with emission monochromator at 440 nm.
• the protein lipid complexes of the present invention can be delivered to an individual (such as an animal including a human being) by any standard means of administration such as intramuscular, intranasal, intraperitoneal, intravenous, oral, rectal, subcutaneous, topical and the like.
• the complexes may be delivered directly to or near the target site or may be delivered directly or indirectly into the circulation.
• the complexes may be delivered in pharmaceutically acceptable carriers which are well known in the art.
• compositions of the present invention exhibit reduced immunogenicity as well as reduced antigenicity. Accordingly, such compositions can be used for reducing immune response in an individual against a therapeutic agent.
• compositions of the present invention can also be used for delivery of a therapeutic agent to an individual in whom an immune reaction to the protein has already occurred. Thus, these composition can be used before or after the occurrence of an immune reaction.
• this invention provides specific FVIII-lipid complexes.
• the protein-lipid complexes may form novel lipidic structures as well as structures such as liposomes, cochleate, micelles and non-bilayer structures to reduce the immune response and antigenicity.
• the method involves developing specific FVIII-lipid complex preferably stabilized by buffer conditions.
• carrier properties such as hydrophobic shielding and cellular (antigen presenting cells APCs) uptake of particulate matters.
• the present invention provides a method for reducing the immune response against FVIII.
• immunogenicity is reduced while the clotting activity is maintained. It is considered that the reduction in immunigenicity is accomplished by complexing of the phospholipids with the C2 and A2 domains.
• the improved pharmaceutical properties of the complex such as stability, altered clearance mechanism to increase circulation time and reduced antigenicity and immunogenicity is an unexpected observation.
• FVIII was used.
• the FVIII-O-phospho-L-serine (OPLS) complex was formed by mixing 20ug of the protein with 5 and 2OmM of the OPLS in 25mM TRIS, 30OmM NaCl and 5mM CaCl 2 .
• This example describes the stability of the protein-lipid complexes of the present invention (Example. 1).
• the unfolding of the protein results in the aggregation of the protein and this in turn leads to the irreversibility of unfolding.
• the aggregation is initiated by small conformational changes in C2 domain.
• the unfolding/refolding studies were carried out with free FVIII and FVIII complexed to PS as described in Example 1 to determine the stability of the formulation containing protein and O-Phospho-L-Serine that is believed to bind to the C2 domain of FVIII.
• CD Circular Dichroism
• SEC size exclusion chromatography
• rFVIII domain specific antibody binding and clotting activity studies were carried out to investigate the temperature dependent physical and functional changes of recombinant human FVIII (rFVIII).
• the activation energy associated with the above transition was calculated to be -127.98 Kcal/Mole (-534.97 KJ/Mole).
• Antibody binding studies indicated that conformational changes in the lipid-binding region (2303-2332) of the C2 domain may at least in part be responsible for the initiation of aggregation ( Figure. 2).
• Analysis of the SEC profile of FVIII in the presence and in the absence of OPLS clearly showed that the monomelic population is significantly higher than that of aggregated protein in the presence of PS, possibly due to the interference of OPLS in the aggregation kinetics of Factor VIII (Figure. 3).
• the data indicates that the complex improves the stability of FVIII and may help to reduce the immunogenecity by reducing the aggregates.
• O-phospho-L-serine was complexed with the protein (likely shielding the C2 domain). 20 ug/ml of the protein was mixed with 5 and 20 mM of phospho-L-serine in different buffers including 25 mM TRIS, 300 mM NaCl and 5 mM CaCl2. The binding to the monoclonal antibodies was studies by sandwich ELISA. As shown in Figure 4, the recovery of native like structures were not possible for excipient free protein due to aggregation whereas presence of O-Phospho- L-Serine resulted in substantial recovery of native structure.
• EXAMPLE 3 This example demonstrates that the protein-lipid composition of the present invention (Example. 1) reduces the immunogenicity against the protein in Sprague- Dawley rats.
• OPLS-Factor VIII complex was administered to Sprague-Dawley rats.
• This rat model has been shown to be suitable to study antibody development to FVIII.
• the antibody titer measured by ELISA for free FVIII and FVIII-PS complex.
• Two weeks after the administration the analysis of antibody titer for Factor VIII -OPLS complex was found to be non- immunotoxic.
• the antibody titers for free FVIII is 563.72 ⁇ 916.15 and no detectable antibody titers was observed for FVIII-OPLS complex.
• composition of the present invention can be made as small unilamellar vesicles.
• 0.3mg/ml of DMPC and 0.15mg/ml of bPS dissolved in a round bottomed flask and the solvent was evaporated to form a thin film.
• the film was then hydrated to form MLVs and the MLVs were extruded through 200nm polycarbonate filters to form SUV's in the size range of 160 nm.
• This example demonstrates the general methodology of the ELISA assay to investigate the antigenicity and the participation of particular epitope region of the protein in forming the complex.
• the antibody binding of the protein-lipid complexes was investigated by antibody capture ELISA and sandwich ELISA.
• sandwich ELISA 96 well plates (Nunc-Maxisorb) were coated with an anti-C2 domain antibody (ESH4) by incubating 50 ⁇ l/well solution of the antibody at a concentration of 5 ⁇ g/ml in carbonate buffer (0.2 M, pH 9.4) overnight at 4 °C.
• the plate was then washed 10 times with 100 ⁇ l of Phosphate buffer containing 0.05% Tween 20 (PBT consisting of 1OmM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , 0.14 mM NaCl, 2.7mM KCl, and 0.02% NaN 3 ).
• PBT Phosphate buffer containing 0.05% Tween 20
• the remaining nonspecific protein binding sites on the plastic's adsorptive surface were blocked by incubating 200 ⁇ l of blocking buffer consisting of 1% bovine serum albumin in phosphate buffer (PB consisting of 1 OmM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , 0.14 mM NaCl, and 2.7mM KCl) for 2 hours at room temperature.
• PB bovine serum albumin
• the plates were washed 10 times with PBT and 50 ⁇ l of 100 ng/ml of rFVIII or rFVIII/OPLS (Examples 1 and 3) or Liposome associated rFVIII (Examples 5) in blocking buffer was added and incubated at 37°C for 1 hour.
• the plates were washed 10 times with PBT and incubated with 50 ⁇ l of biotinylated ESH8 - another anti-C2 antibody, at 1 ⁇ g/ml concentration and 50 ⁇ l of a 1 : 1000 dilution of avidin-alkaline phosphatase conjugate, both in blocking buffer at room temperature for 1 hour.
• the plates were washed 10 times with PBT and 100 ⁇ l of 1 mg/ml p-nitrophenyl phosphate solution in diethanolamine buffer (consisting of IM diethanolamine, 0.5 mM MgCl 2 and 0.02% NaN 3 ). The plates were incubated at room temperature for 30 minutes and the reaction was quenched by adding 100 ⁇ l of 3 N NaOH. Absorbance was read by a plate reader at 405 nm. The ELISA studies indicated that less C2 domain specific antibodies, ESH8 or ESH4 bound to the protein in the presence of PS (OPLS or liposomes Figure. 6).
• Example 5 demonstrates that the protein-lipid composition of the present invention (Example. 5) reduces the immunogenicity against the protein in animal models, Sprague-Dawley rats and Factor VIII knock out mice, Hemophilia A model.
• the antibody titres evaluated in Sprague-Dawley rats at 4 th and 6 th weeks post administration of the protein-liposomes complex was found to be lower for FVIII-PS complex compared to free FVIII ( Figure. 8A).
• the following examples illustrate the formation of protein-lipid complexes in micelles, non-bilayered structures, cochleate structures and in novel non-liposomal lipid particles.
• This example describes the formation of micelles by the compositions described herein.
• the protein solution was mixed with a shorter acyl chain lipids (Dihexanoyl phosphatidyl Serine (below and above 0.3mM) at lower and higher concentration (below and above critical micellar concentrations) and the resulting micellar particles were characterized.
• the structure of the micelles were characterized by light scattering, circular dichroism and fluorescence studies.
• the functional assays such as activity and antibody binding were carried out.
• EXAMPLE 9 This example describes the formation of cochleate structures by the compositions described herein. 0.15mg/ml of bPS was dissolved in chloroform and the solvent was evaporated to form a thin lipid film. The film was then hydrated in several buffer system at pH 7.0 and the MLVs were either extruded or sonicated to form SUVs. The resulting SUVs were mixed with protein in buffer system containing 5mM CaCl 2 to form cochleate structure. The lipid structures were analyzed by light scattering, differential interference microscopy, negative stain electron microscopy and by fluorescence studies. These studies showed that the lipid structures formed by this procedure were cochleate in nature.
• This example is another illustration of the preparation of protein-lipid complexes of the present invention.
• 0.15mg/ml of bPS and 0.3mg.ml of dioleoyl phosphatidylethanolamine (DOPE) was dissolved in chloroform and the solvent was evaporated to form a thin lipid film.
• the resulting lipid film was hydrated with phosphate buffered saline to form hexagonal phases.
• the non-bilayer structures were characterized by fluorescence studies.
• EXAMPLE Il This example is another illustration of the preparation of protein-lipid complexes of the present invention in a novel non-liposomal structures.
• 0.3mg/ml of DMPC, 0.15 mg/ml of bPS were dissolved in chloroform and the solvent was evaporated to form a thin lipid film in a round bottom flask.
• the lipid was hydrated using a buffer system containing FVIII, 25mM TRIS, 30OmM NaCl and 5mM CaCl 2 and the solution was gently swirled either at room temperature at 37°C.
• the film was then hydrated in appropriate buffer (25mM TRIS, 30OmM NaCl and 5mM CaCl 2 ), with gentle swirling.
• the MLVs thus formed were subjected to dextran centrifugation gradient to separate the free protein from protein associated with MLVs.
• 0.5ml of the lipid associated protein was mixed with ImI of 20% w/v of dextran and a 3ml of 10% w/v dextran was layered over the above solution.
• 0.5ml of buffer layered on top The gradient was centrifuged for 35 rnin at 45K RPM using Beckman SW50.1 rotor.
• the results of the centrifugation study is shown in Figure 9. As is clear from the figure, there are some lipidic fractions that could not be floated and are denoted as fraction 3 in the figure. This fraction was observed at the interface of 14% and 10% dextran.
• this fraction did not have enough buyoancy or encapsulated water.
• Conventional liposomes generally float to the top of the gradient because of their entrapped water . Therefore, the fraction that does not float may be a non-liposomal protein containing lipidic particles.
• the fraction was collected and tested for lipid content by mass spectrometry and for protein content by activity. The mass spectroscopy studies showed that this fraction contained lipids including high PC content suggesting that it is not just PS-Ca+ complex.
• the activity assay showed approximately 40% of the initial protein was encapsulated in this fraction. There are several possible explanations for the dense fraction 3 which has no or little water content.
• This fraction may represent: (1) very small unilamellar vesicles with less encapsulated water volume.
• pre sized liposomes were prepared by extruding through polycarbonate filters. The extrusion was repeated 3 times and the size of the particles was determined to be around 160nm.
• the resulting SUVs were mixed with FVIII and were subjected to Dextran centrifugation gradient. This control study showed that these SUV's did not show fraction 3 band indicating that the observation of such bands is not due to the formation of small liposomes. Further, this experiment was performed under identical buffer and experimental conditions to rule out any artifacts in the dextran gradient.
• fraction 3 may represent the formation of cochleate structures, which have less water content [26].
• the formation of cochleate structures needs a very high PS content (>50%).
• the PS content used was around 30% and under these conditions the formation of cochleate structures has not been shown.
• the formation of collapsed Ca(PS)2 complex that has a dehydrated structure [27] may not float and can form a dense band. The formation of such collapsed structure requires very high PS content (>50%) but in the absence of Na+.
• the composition contains low PS content and a very high concentration of Na+ i.e., 30% PS and 30OmM NaCl is used and therefore, the possibility of Ca(PS)2 formation is ruled out.
• the PS and calcium system has been shown to promote vesicle fusion. However, the fusion of vesicles by divalent cations such as Ca 2+ is inhibited by the presence of Na+ as it competes with calcium for the lipid binding site.
• the estimated amount of Calcium bound per PS in this PC/PS ratio of 7:3 and in the presence of 30OmM NaCl and 5mM CaCl 2 is between 0.22 (50OmM NaCl) to 0.35 (10OmM NaCl) [28, 29].
• This estimated bound calcium per PS is less than the critical ratio of 0.35 to 0.39 required for fusion in a small unilamellar PS/PC vesicle system.
• the fusion of vesicles does not appear to be represent the dense band. This is because the larger PC fraction (>50%) may result in less PS-divalent cation complex and its ability to cluster into large domains to induce fusion)[28, 29].
• the dense band may be due to the formation of novel, non- liposomal lipid particles. This band is non-liposomal because of less encapsulated water volume.
• negative stain electron microscopy, differential contrast interference optical microscopy, light scattering, circular dichroism and fluorescence measurements were performed.
• rFVIII clotting activity was determined by one-stage activated partial thromboplastin time (APTT) assay using micronized silica as activator and FVIII deficient plasma as the substrate.
• the APTT assay was performed using a COAG-A- MATE model coagulation analyzer (Organon Teknika Corporation, Durham, NC). Briefly, rFVIII was added to FVIII deficient plasma and the clotting time was monitored.
• the activity of the rFVIII was then obtained from calibration curve constructed using the clotting times determined from various dilutions of a lyophilized reference concentrate of known activity.
• concentration of the protein was determined independently using Bicinchoninic acid (BCA) assay and compared with activity. For example, all the 20-22 ⁇ g/ml of the protein corresponds to specific activity of 87 - 95.6 IU.
• BCA Bicinchoninic acid
• the stock solution used to prepare the samples had a specific activity of2174 IU/0.5 mg/ml.
• DCPS dicaproyl phopatidylserine
• the FVIII- (DCPS) complex was formed by mixing 2ug of the protein with 0.5, 2 and 5 mM of the DCPS in 25mM TRIS, 30OmM NaCl and 5mM CaC12.
• the antigenicity of the complex was investigated using ELISA.
• DCPS inhibited the binding of rFVIII to ESH4 antibody at the concentrations tested i.e., 0.5, 2 and 5.0 uM. These concentrations are above and below the CMC of this phospholipid.
• EXAMPLE 14 This example describes the effect of PS containing phospholipids on the immunogenicity of proteins.
• the FVIII- (DCPS) complex was formed by mixing 2ug of the protein with 5 mM of DCPS in 25mM TRIS, 30OmM NaCl and ImM CaC12. This composition forms micellar structure and the immunogenicity of the complex was investigated in Hemophilia A mice.
• the FVIII- Dicaproyl Phosphatidyl Choline (DCPC) complex was formed by mixing 2ug of the protein with 2OmM of DCPC in 25mM TRIS, 30OmM NaCl and ImM CaCl2. This composition forms micellar structure and the immunogenicity of the complex was investigated in Hemophilia A mice.
• composition of the present invention can be made as liposomal vesicles.
• 11.25 ⁇ mol of DMPC and 4.83 ⁇ mol of BPS dissolved in choloroform were taken in a round-bottomed flask and the solvent was evaporated using a rota-evaporator to form a thin film on the walls of the flask.
• Lipid recovery was estimated by determination of phosphorous content by the method of Bartlett.
• the protein was associated with liposomes by incubating at 37°C for 30 minutes.
• the liposomes were then be used in immunizations as follows. Immunization of FVIII knockout mice consisted of four subcutaneous (s.c.) injections of rFVIII or rFVIII-liposomes (2 ⁇ g in 100 ⁇ l of Tris buffer) at weekly intervals. The molar ratio between the protein and lipid was maintained at 1 : 10,000.
• the protein was associated with liposomes by incubating at 37°C for 30 minutes. Immunization of FVIII knockout mice consisted of four subcutaneous (s.c.) injections of rFVIII or rFVIII-liposomes (2 ⁇ g in 100 ⁇ l of Tris buffer) at weekly intervals. The molar ratio between the protein and lipid was maintained at 1 : 10,000. DSPC having a high phase transition temperature (solid state) was used a negative control.
• the protein was associated with liposomes by incubating at 37°C for 30 minutes. Immunization of FVIII knockout mice consisted of four subcutaneous (s.c.) injections of rFVIII or rFVIII-liposomes (2 ⁇ g in 100 ⁇ l of Tris buffer) at weekly intervals. The molar ratio between the protein and lipid was maintained at 1 : 10,000. In another sample, 7.5 ⁇ mol of DMPC and 3.22 ⁇ mol of DOPG dissolved in choloroform were taken in a round-bottomed flask and the solvent was evaporated using a rota-evaporator to form a thin film on the walls of the flask.

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