Patent Application
20220016241
This disclosure relates generally to the field of immunogenicity of antigenic proteins. More particularly, this invention relates to compositions and methods for: (i) reducing the immunogenicity, (ii) improving the pharmacokinetics, and (iii) induction of tolerance, to biotherapeutics, including therapeutic proteins, peptides and antibodies.
Biotherapeutics, which includes recombinant proteins, peptides and antibodies, represent a growing class of pharmaceutical agents. This class is already being used to treat a broad range of diseases. While treatments using these agents can be highly effective, such treatments often encounter problems related to stability and immunogenicity. It is not uncommon for therapeutic proteins to form aggregates, wherein unfolded therapeutic proteins associate with each other and form complexes that can lead to undesired immune responses. Equally, if not more, concerning is the inherent immunogenicity of such therapies which often leads to antibody-based immune responses that impact their safety and efficacy. For example, antibody-based immune responses (e.g., anti-drug antibodies) can not only alter pharmacokinetics (e.g. increase the rate of clearance/reduced circulation time) but also abrogate the pharmacological activity of the protein. The latter occurs when the antibody-based immune response involves the development of neutralizing antibodies. Over time, as the titers of neutralizing antibodies increase, the therapy becomes less effective and can lead to additional morbidities and sometimes even mortality, especially in cases where no alternative therapies exist.
The “immunogenicity” of a biotherapeutic is influenced by various factors, including route of administration, concomitant medications, drug doses and treatment scheduled, genetics, age, gender, immune and nutritional status, disease characteristics, the size and structure of the drug, how “human” the drug is, the clearance rate of immune complexes, and drug solubility. Because there are so many factors which induce immunogenicity, there can be a broad range of “immunogenicities” for a particular biotherapeutic between different studies.
Numerous examples exist for the development of anti-drug antibodies against biotherapeutics. Many examples involve monoclonal antibodies, which can include chimeric, humanized and even fully human antibodies. A specific example is biologic anti-tumor necrosis factor (anti-TNF) therapies such as adalimumab (Humira®) and infliximab (Remicade®), which are used to treat autoimmune disease. The formation of anti-drug antibodies is recognized as the mechanism explaining the failure of anti-TNF biotherapeutics in the treatment of Rheumatoid Arthritis (“RA”). Anti-drug antibodies developed towards anti-TNF therapeutics reduce the clinical effectiveness of these therapeutics and necessitate higher dosing or discontinuation. Neutralizing antibodies cause 12% to 44% of RA patients to develop resistance or adverse reactions to anti-TNF agents. Because of anti-drug antibody formation, at least ⅓ of RA patients do not respond to treatment or lose initial responsiveness. Each of the currently marketed anti-TNF biotherapeutics (Remicade® (infliximab), Humira® (adalimumab), Simponi® (golimumab), Enbrel® (etanercept), and Cimzia® (PEGylated certolizumab)) has a respective range of “immunogenicity.”
Other examples involve protein-based replacement therapies, including but not limited to replacement therapies (e.g. Factor VIII) for coagulation disorders such as hemophilia and enzyme replacement therapies for lysosomal storage diseases such as Pompe disease. In a specific example, patients suffering from Pompe disease possess a deficiency in the enzyme acid alpha glucosidase (“GAA”). The current treatment for the disease is enzyme replacement therapy with alglucosidase alfa, which is marketed as Myozyme® and Lumizyme®. Like the anti-TNF biotherapeutics mentioned above, these drugs are immunogenic, often resulting in undesirable immune responses that ultimately render the therapy less-effective or even ineffective.
Several approaches have been used and/or investigated to minimize immunogenicity of biotherapeutics, including, but not limited to, development of less-immunogenic formulations, modifying treatment regimens, use of steroids (i.e. generalized immunosuppression), and various delivery approaches. Each approach has its own set of drawbacks and limitations.
In addition to immunogenicity, another concern facing subcutaneous routes of administration of biotherapeutics is their incomplete bioavailability, which is presumably due, in part, to the degradation of the biotherapeutic at or near the site of injection.
Therefore, a safe and effective approach to minimize immunogenicity, improve pharmacokinetics, and induce antigen-specific immune tolerance is desirable.
The present disclosure describes methods and compositions for reducing the immunogenicity of antigens (e.g. biotherapeutics), improving their pharmacokinetic properties, and inducing specific immunological tolerance to such antigens.
This present disclosure is based on the surprising observation that concomitant or sequential administration of certain lipidic particles or OPLS, and antigens, even in the absence of association of the antigen with OPLS or incorporation of the antigen into lipidic particles (and/or shielding of immunogenic epitopes) (i.e. formation of complexes), results in both immunological hypo-responsiveness against the antigen and an improvement in its pharmacokinetic properties. This is especially surprising considering that the reduction in immunogenicity is seen even when administration takes place via the subcutaneous route, which is widely believed to be highly immunogenic due to the presence of high numbers of dendritic cells (i.e. high immunologic exposure).
Immunological tolerance can be viewed as a continuum that ranges from immunological hypo-responsiveness to non-responsiveness to an antigen. For therapeutic purposes, it is generally preferred that immunological tolerance is specific to the antigen in question so as to avoid complications that arise from generalized immunosuppression. In the context of this disclosure “immunological tolerance” to an antigen is evidenced by any one or combination of the following: (a) increases in one or more of the following: (i) T regulatory cells, (ii) TGF-b, and/or (iii) IL-10, and/or (b) decreases in one or more of the following: (i) antigen-specific antibody titers, (ii) B cells, including antigen-specific memory B cells, (iii) IL-6, (iv) IL-17, (v) CD40, (vi) CD80, and/or (vii) CD86, and/or (c) hypo-responsiveness following re-challenge with the antigen. One or more of these identifiers can be evaluated in culture conditions. By “specific” it is meant that an immune response to non-relevant antigens (antigens that were not complexed to or co-administered, either concomitantly or sequentially, with the lipidic compositions of the present disclosure) is not affected.
In one aspect, this disclosure provides a method for inducing immune tolerance to agents (such as therapeutic agents). The agents are administered in conjunction with the administration of OPLS. In one embodiment, the agent and OPLS are administered as separate compositions. The administrations may be carried out at the same time (concomitantly) or may be carried out one after the other (sequentially). In one embodiment, the OPLS and the agent may be combined before administration, but the association of the OPLS and the agent is low or absent (i.e. undetectable).
In one embodiment, the bioavailability of the biotherapeutics is increased by improving lymphatic uptake after sc administration. While agents (e.g. albumin) that act as volume expanders have been explored in the past, these agents have no ability to reduce the immunogenicity of the co-administered biologic. In the present disclosure, it was observed that administration of a biologic in a hypertonic buffer could increase bioavailability as well as induce immune tolerance.
In one embodiment, the therapeutic agent is administered in conjunction with OPLS at a sub-therapeutic dose. At this dose, the therapeutic agent does not exert a clinically relevant effect for the indication for which it is intended. However, as described in this disclosure, surprisingly, it is able to induce specific immune tolerance toward the agent. The therapeutic and OPLS may be administered in the same composition or may be administered separately (concomitantly or sequentially).
In one embodiment, the antigen (such as a therapeutic agent) is administered in conjunction with OPLS (as a single formulation, or separate formulations—whether administered concomitantly or sequentially), wherein the agent is at a therapeutic dose. In one embodiment, if administered as a single formulation, the OPLS and the agent are loosely bound (Kd of higher than 10 μM and up and including 500 μM) or unbound (Kd of higher than 500 μM). In one embodiment, if administered as a single formulation, the agent and OPLS are combined immediately prior to administration so that there is no detectable complexation of the OPLS and the antigen.
In one embodiment, the administration of the agent and OPLS is done via a cutaneous route. The cutaneous route may be a sub-cutaneous injection, transdermal or intradermal delivery, topical application or any other delivery method via the skin.
In one aspect, this disclosure provides a method for inducing immune tolerance by ex vivo exposure to a therapeutic antigen and OPLS. The method comprises obtaining dendritic cells from an individual, contacting the dendritic cells in vitro with OPLS and the antigen, and returning the dendritic cells to the host.
In one aspect, the present disclosure provides kits for inducing immune tolerance. The kits comprise one or more of the following: OPLS or other serine containing lipid formulations, therapeutic agents, hypertonic buffer, administration devices and agents, instructions for administration of the various compositions.
The present disclosure provides compositions comprising OPLS or lipidic particles that, when administered in combination with or in a sequential manner with one or more antigens, provides for a reduction in immunogenicity of the antigen and an improvement in its pharmacokinetic properties and/or the induction of immune tolerance towards the antigen.
As used herein, the term biotherapeutics means biologically-based therapeutics, including recombinant proteins, peptides, therapeutic/prophylactic antibodies (e.g. monoclonal antibodies) and fragments thereof. Biotherapeutics, in this description, serve as and are sometimes referred to as an antigen.
The agent (such as a therapeutic agent) is delivered in conjunction with an immune tolerance inducing agent such as O-Phospho-L-Serine (“OPLS”); or lipidic particles which may be: (a) unilamellar or multilamellar liposomes comprised of phospatidylserine (“PS”) and one or more phospholipids (“PS-liposomes”); or (b) non-liposomal structures comprised of serine including micelles, condensed structures, and cochleate. OPLS is available commercially. The phospholipids, including PS, can be obtained from various sources both natural and synthetic. For example, PS can be obtained from porcine brain PS or plant-based soy (soya bean) PS. Egg PC and PS and synthetic PC are also available commercially. The term lipidic compositions includes OPLS as well as lipidic particles.
The present disclosure provides compositions and methods for improving pharmacokinetic (PK) properties. In one embodiment, an “improvement” in pharmacokinetic properties means an improved half-life or bioavailability for the antigen. For example, when OPLS or lipidic compositions were subcutaneously administered with an antigen (such as Factor VIII), there was an improvement in the lymphatic uptake and the plasma PK profile relative to an administration of the free protein administered subcutaneously. Improvement in lymphatic uptake and plasma concentrations was also seen when using Rituximab as the antigen, providing further evidence that this is a broadly applicable approach.
In one embodiment, the immune tolerance inducing agent comprises, consists essentially of, or consists of OPLS. In one embodiment, the only immune tolerance inducing agent in the lipidic composition is OPLS. The OPLS concentration can be from 1 mM to 100 mM including all integers and ranges therebetween. In one embodiment, the OPLS amount is from 100 to 300 nM including all integers and ranges therebetween. In one embodiment, the mM concentration is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100. In another embodiment, the OPLS concentration is between 21 mM and 100 mM including all integers therebetween. In another embodiment, it is between 25 mM and 75 mM including all integers therebetween. In another embodiment, the OPLS concentration is between 35 mM and 65 mM including all integers therebetween. In yet another embodiment, the OPLS concentration is between 45 mM and 55 mM including all integers therebetween. In one example, the OPLS concentration is 50 mM. In one embodiment, the OPLS is 125, 150, 175, 200, 225, 250 or 275 mM. Alternatively, the amount of OPLS required to practice the methods disclosed herein can be described in terms of dose. The dose can be any amount above 1 mg. In one embodiment, the dose of OPLS can be from 1 mg to 1,000 mg including all integers therebetween. In a particular example, the dose is 5 mg.
In general, any suitable buffer may be used. For example, any phosphate buffer (including phosphate buffered saline) or TRIS buffer may be used. In one embodiment, the reconstitution medium described in U.S. Pat. No. 8,349,794 is used. The pH of the buffer may be from 5 to 8. In one embodiment, the pH of the buffer may be from 5.5 to 8 or 5.5 to 7.6, or 6.0 to 7.6. In another embodiment, the pH of the buffer may be from 6 to 8, or 7.2 to 7.6 or 7.4.
In one embodiment, the method for improving lymphatic uptake and/or plasma concentrations of a concomitantly or sequentially administered antigen is achieved through the use of a hypertonic solution comprising OPLS. In one embodiment, the compositions comprise, OPLS and NaCl. In one embodiment, the compositions comprise OPLS and mannitol, and optionally, NaCl. The increase in osmolality can be achieved by any combination of any amount of one or more of OPLS, mannitol and NaCl.
In another embodiment, the method for improving lymphatic uptake and/or plasma concentrations of a concomitantly or sequentially administered antigen is achieved through the use of a hypertonic solution (defined herein as having an osmolality of greater than 300 mmol/kg) comprising at least one of: OPLS and mannitol. In one embodiment, the osmolality of the solution ranges between 300 mmol/kg and 1,100 mmol/kg, including all integers and ranges therebetween. In one embodiment, the osmolality of the solution ranges between 300 mmol/kg and 800 mmol/kg, including all integers therebetween. In one embodiment, the osmolality is from 301 to 1,100 mmol/kg or from 500-600 mmol/kg. In one embodiment, the osmolality is from 290 to 300 mmol/kg (including all integers and ranges therebetween). In specific embodiments for hypertonic solutions comprising OPLS, wherein the osmolality of the solution ranges between 300 mmol/kg and 1,100 mmol/kg, higher concentrations (e.g. >100 mM) of OPLS result in greater lymphatic uptake and/or plasma concentrations that lower concentrations (e.g. <100 mM) of OPLS. Similarly, in specific embodiments for hypertonic solutions comprising mannitol, wherein the osmolality of the solution ranges between 300 mmol/kg and 1,100 mmol/kg, higher concentrations (e.g. >100 mM) of mannitol result in greater lymphatic uptake and/or plasma concentrations than lower concentrations (e.g. <100 mM) of mannitol.
Note that although lipidic compositions comprising, consisting essentially of or consisting of O-Phospho-D-Serine (“OPDS”) were used in certain examples disclosed herein, effects similar to those provided using OPLS were observed. While not intending to be bound by any particular theory, it is considered that the observed effects of OPDS, at least in part, are due to the fact that OPDS is very unstable and that a portion of OPDS is getting converted to OPLS in vivo.
In another embodiment, the lipidic particles are liposomes comprised of phosphatidylserine (“PS”) and phosphatidylcholine (“PC”) and, optionally, cholesterol (sometimes referred to as “PS/PC”). In one embodiment, the phospholipids in the lipidic compositions comprise or consist essentially of, or consist of PS and PC present in the ratio of 10:90 to 30:70 and all ratios therebetween. In one embodiment, the only phospholipids in the lipidic particles are PS and PC as described above. In some embodiments cholesterol may also be present, but is not necessary to practice the methods disclosed herein. However, in cases where the antigen is complexed (vs. co-administered) with the lipidic particle and release of the antigen is desired, it is preferable to include 1-33% cholesterol (percent of PS and PC together).
In other embodiments, the phospholipids in the lipidic compositions comprise or consist essentially of, or consist of PS, phosphatidylinositol (“PI”) and PC and, optionally, cholesterol (sometimes referred to as “PS/PI”). The combination of PS and PI is preferably not more than 60 mol % and PC is at least 40 mol %. The PI component of the PS/PI liposomes is from 1 mol % to 30 mol % and all integer mol % values there between. For PS liposomes (PS/PC and PS/PI liposomes), the PS should be at least 1 mol %. In one embodiment, PS is at least 10 mol %.
PS, PC and PI may have from 0-22 carbon atoms in the acyl chain. For example, when the carbon atom is 0 for PS, the molecule is O-Phospho-L-Serine (OPLS). In one embodiment, the acyl chains have 1 to 22, 2 to 22, or 6 to 22 carbons (and all integer number of carbons and ranges therebetween). The acyl chains can be saturated or unsaturated and may be same or different lengths. Some examples of acyl chains are: Lauric acid, Myristic acid, Palmitic acid, Stearic acid, Arachidic acid, Behenic acid, Oleic acid, Palmitoleic acid, Linoleic acid, and Arachidonic acid. The PC may have one or two acyl chains. For example, the PC may include lysophosphatidylcholine (LPC). For example, in one embodiment, the composition of the PS liposomes was PS:LPC:PC as 30:10:70 mol %. The LPC can be 0 mol % to 20 mol %. In one embodiment, it is 1 mol % to 20 mol % and all integer mol % values and ranges therebetween.
The lipidic particles of the present invention can be prepared by thin lipid film hydration using the appropriate molar ratios of PS, PC and, optionally, cholesterol; or appropriate ratio of PS, PC and PI, and optionally cholesterol, in a suitable buffer. The lipids are dissolved in chloroform and the solvent is dried. The resulting multilamellar vesicles (MLVs) are extruded through the desired size filters (sizing device) under high pressure to obtain lipidic structures of the present invention. In one embodiment, the size of 50, 60, 70, 80, 90, 95 or 100% (including all percentages between 50 and 100) of the lipidic particles is from 40 nm to 4 micron including all sizes therebetween in the nanometer and micrometer range. In another embodiment, size of the particles is from 60 to 140 nm. In another embodiment the particles are less than 140 nm (as calculated from micrographs and dynamic light scattering measurements) so that the particles are not filtered out in the Reticulo Endothelial System (RES) so as to become available for the immune system reaction. Thus, in one embodiment, at least 50% of the particles are less than 140 nm. For example, the particles are less than 120 nm such as from 40 and 100 nm. In various embodiments, 50, 60, 70, 80, 90, 95 or 100% of the particles are less than 140 nm such as from 40 and 100 nm or 60 to 100 nm.
Preparation of lipidic compositions comprising, consisting essentially of, or consisting of OPLS, can be as simple as a buffer preparation. The desired amount of OPLS can be weighed out and added to any buffer. The pH of such OPLS buffer solution can then be adjusted to a desired value (such as 7.4) using methods known in the art.
The antigen may be a peptide (generally 50 amino acids or less) a polypeptide (generally 100 amino acids or less) or proteins (larger than 100 amino acids). The agent may be a therapeutic antigen or may be an antigen against which an individual is already primed, but against which immune tolerance is desired (such as in allergic reactions or transplant situations). The proteins or peptides may be neutral or charged (negatively or positively). Such proteins include proteins involved in the blood coagulation cascade including Factor VIII (FVIII), Factor VII (FVII), Factor IX (FIX), Factor V (FV), and von Willebrand Factor (vWF), von Heldebrant Factor, tissue plasminogen activator, insulin, growth hormone, erythropoietin alpha, VEG-F, Thrombopoietin, lysozyme, acid alpha glucosidase (GAA), therapeutic antibodies, including monoclonal antibodies such as adalimumab, myelin sheath proteins, myelin oligodendrocyte glycoprotein (MOG), glutamic acid decarboxylase 65 (GAD), insulin receptor, and the like. In one embodiment, the antigen is a monoclonal antibody or fragment thereof (such as Fc portion).” In one embodiment, the antigen can be one that ordinarily provokes a relatively mild allergic reaction, such as would typically be caused by pollen, animal dander, house dust and the like, or an antigen that ordinarily would provoke in the individual a severe allergic reaction, such as components in venom from insect stings, nut allergens, certain antibiotics, and other compositions that can cause severe allergic responses in the particular individual in question or may be a transplant relevant antigen.
In further embodiments, examples of additional antigens that can be used in the methods described herein include, but are not limited to, monoclonal antibodies (e.g., Adalimumab, Rituximab, infliximab, trastuzumab, Tocilizumab, Daratumumab, Peginesatide, Abatacept, Mepolizumab, Pertuzumab, Golimumab, Vedolizumab, Daratumumab, PF-06480605, PF-06801591, and combinations thereof); antibody-drug conjugates (ADCs) (e.g., brentuximab vedotin, trastuzumab emtansine, polatuzumab vedotin-piiq, enfortumab vedotin, trastuzumab deruxtecan, sacituzumab govitecan, Gemtuzumab ozogamicin, Cantuzumab mertansine, Inotuzumab ozogamicin, Moxetumomab pasudotox, Belantamab mafodotin-blmf, Loncastuximab tesirine-lpyl, Trastuzumab duocarmazine, Mirvetuximab soravtansine, and combinations thereof); bispecific antibodies (e.g., Blinatumomab, Emicizumab, Amivantamab, Catumaxomab, and combinations thereof); interferons (IFNs) (e.g., IFN-beta); interleukins (ILs) (e.g., IL-2, IL-12); enzymes (e.g., acid alpha glucosidase (GAA) and hyaluronidase); hormones and growth factors (e.g., insulin, hGH); blood factors (e.g., FVIII, FVIIa); hematopoietic factors (e.g., Erythropoeitin (EPO), granulocyte colony-stimulating factor (GCSF), granulocyte-macrophage colony-stimulating factor (GMCSF), thrombopoietin); fusion proteins; gene therapy delivery vehicle-associated peptides/proteins (e.g., Cas9, AAV), which may be vector-associated peptides/proteins; and autoantigens (e.g., MoG, nicotinic acetylcholine receptor (nAChR), collagen, aquaporin-4).
In some embodiments it is desirable to associate (i.e. complex) the antigen and the lipidic composition. To effect association (i.e. form a complex) of the antigen with the lipidic structures, the antigen in a suitable buffer is added to the lipidic structures. The free antigen is then separated from the lipidic structures by centrifugation methods such as density gradient centrifugations. In various embodiments, the association efficiency of the antigen with the lipidic particles is at least 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90 and 95%. If desired, the lipidic particles with the associated antigen can be lyophilized for future use. In one embodiment, the lipidic structures of the present invention prior to association with the antigen can be lyophilized and stored. When needed, the lipidic structures can be reconstituted and then used for combination with antigen to effect association of the antigen with the lipidic structures prior to use.
In embodiments where the antigen is associated with the PS-liposomes, the association can be such that the molar ratio between the antigen to lipid is between 1:10 (antigen:lipid) to 1:30,000 (antigen:lipid) and all ratios therebetween. In one embodiment it is about 1:10,000 (antigen:lipid). As the therapeutic dose of an antigen increases, the ratio decreases. In other embodiments, the ratio is about 1:2,000 or 1:4,000 (antigen:lipid). The maximum antigen:lipid ratio of the present disclosure is 1:30,000. Since dosing for different proteins varies, the amount of dosing required will alter the ratio.
In some embodiments, the lipidic composition is not complexed with the antigen. In one embodiment, the lipidic particles of the present invention prior to administration can be lyophilized and stored. When needed, the lipidic particles can be reconstituted and then administered to an individual.
In embodiments where OPLS is used, the molar ratio between the antigen to OPLS can be from 1:1 to 1:1×107 and all ratios therebetween and all ranges therebetween. In one embodiment, it is from 1:1 to 1:1×106 all ratios therebetween. In one embodiment, the ratio of antigen to OPLS is between 1:1 to 1:1,000 and all ratios therebetween.
Where we describe a complex or that an antigen and a lipidic composition are associated (i.e., complexed), it is meant that the Kd for the antigen and the lipidic composition is less than 10 μM. In one embodiment, the Kd is 8.4 μM. Where we describe that an antigen and a lipidic composition are not complexed, we mean that the Kd for the antigen and the lipidic composition is greater than or equal to 10 μM. The range of bound to free is as follows: where the dissociation constant (Kd) is less than 10 μM the protein is considered bound or complexed; where the Kd is 10 μM to 500 μM, the protein is considered loosely bound with non-specific interactions; where the Kd is greater than or equal to 500 μM the protein is unbound (‘free”). In one embodiment, the Kd is greater than 10 μM. In one embodiment, the Kd can be from 10 μM to 5,000 μM including all integers therebetween. In various embodiments, the Kd is at least 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μM. In other embodiments, the Kd is10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 1000, 1500, 2000, 2500, or 3000 μM.
In some embodiments, at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of the antigen is present as unbound or loosely bound to the lipidic composition (such as OPLS). For example, in embodiments, a composition may be administered via a cutaneous route, wherein the composition comprises a therapeutic agent that is loosely bound or unbound to OPLS.
In one embodiment, the OPLS or other lipidic composition may be combined with the antigen just prior to administration (such as less than 1 minute or within 1 to 5 minutes of the time of administration). In one embodiment, the combining is done at room temperature (such as from 20 to 26° C. and all integer temperatures therebetween, or less than 20° C.), just prior to administration so that there is no, or, insignificant complexation of the OPLS and the antigen. In specific embodiments, the combined OPLS and antigen are not exposed to a temperature above 22, 23, 24, 25 or 26° C. so that there is no opportunity for complex formation.
The compositions described in this disclosure can be used for any individual (e.g., a human or a non-human mammal). For example, they can be administered to an individual who has previously been administered and/or exposed to the antigen but has not previously developed immune intolerance to said antigen. In yet another example, they can be administered to an individual who has previously been administered and/or exposed to the antigen and has developed immune intolerance to said antigen. The individual may or may not be showing indications of a recent immune intolerance. They are also useful for administration to naïve individuals (i.e., those individuals who have not been administered and/or exposed to the antigen previously). Immune intolerance as used herein means the individual should have measurable (by standard methods such as ELISA or activity assays) antibody production. Conversely, a lack of immune intolerance means the individual has no measureable antibodies. By “administered” or “administration” is meant that the antigen and the lipidic particles are delivered to the individual or introduced into the individual's body by any means or route of delivery such as intravenous, intramuscular, intraperitonial, mucosal, subcutaneous, transdermal, intradermal, oral or the like. In one embodiment, the antigen and the lipidic particles are administered via a cutaneous route such as subcutaneously.
In one aspect, the disclosure provides a method for reducing the immunogenicity and/or improving the pharmacokinetic properties of an antigen comprising the steps of: a) administering to an individual, a composition comprising the antigen; and b) sequentially or concomitantly with the administration of the composition in a), administering to the individual a composition comprising one or more lipidic compositions disclosed herein. In one embodiment, the one or more lipidic compositions are selected from the group consisting of OPLS and PS liposomes, as defined herein. In one embodiment the PS liposomes are selected from the group consisting of PS/PC and PS/PI, as described herein. In one embodiment, the Kd for the antigen and the lipidic composition is greater than or equal to 10 μM. In one embodiment, one or both of lipidic composition and the antigen are delivered via a cutaneous route.
In other embodiments the method for reducing the immunogenicity and/or improving the pharmacokinetic properties of an antigen may be further comprise of one or both of the following: (i) obtaining a biological sample from the individual and measuring one or more of the following: T regulatory cells, TGF-β, IL-10, antigen-specific antibody titers, B cells, including antigen-specific memory B cells, IL-6, IL-17, CD40, CD80, and/or CD86, and/or (ii) following the administration of the antigen and OPLS or another lipidic composition, obtaining a biological sample from the individual and measuring one or more of the following: T regulatory cells, TGF-β, IL-10, antigen-specific antibody titers, B cells, including antigen-specific memory B cells, IL-6, IL-17, CD40, CD80, and/or CD86. In embodiments where both (i) and (ii) are practiced, levels of the cells and/or biomarkers described above may be compared to measure the effectiveness of the method.
In still other embodiments, the method for reducing the immunogenicity and/or improving the pharmacokinetic properties of an antigen (such as FVIII, GAA and adalimumab) described above may be repeated 1 time per week to three or more times per week either indefinitely or for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or more weeks in an effort to achieve a greater degree of immune tolerance to develop. After a suitable period of time (such as at least 4 days after the last primer), the individual can be administered a second composition (one or more administrations) comprising the antigen in the free form, complexed to or in combination with lipidic particles having a different composition than the composition previously administered (the “priming composition”), or complexed (covalently or noncovalently) to PEG, or surfactants or microspheres and the like. The lipidic structures may include micelles, cochleates, lipidic molecular assemblies and the like. In one embodiment, the individual is administered the second composition (which may be a therapeutic composition) after 4 to 30 days, including all integer number of days and ranges therebetween, of the last priming composition administration. In one embodiment, the individual is administered the second composition after 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 days of the last of the last priming composition administration. Less immunogenic formulations are disclosed in U.S. Pat. Nos. 7,351,688; 7,875,288; 7,875,289; 7,625,584; and patent application nos. 20090053297; PCT/US2010/041196, the relevant disclosure of which is incorporated herein by reference.
The phospholipid composition of the lipidic particles in the second composition (which may be a therapeutic composition) may be the same or different from the phospholipid composition of the lipidic particles of the first composition (which is a priming composition). The phospholipid composition change may include the type of phospholipid or the ratio of different phospholipids. In one embodiment, the therapeutic composition includes LPC and sphingosine.
In one embodiment, the frequency of administration of the priming composition is once-a-week for four weeks via the subcutaneous route. In one embodiment, at least four weekly administrations are used to induce tolerance. Administrations of the antigen and lipidic compositions may take place either concomitantly or sequentially, as described herein. More doses of antigen in combination with or complexed to either PS-liposomes or OPLS can be used if necessary.
Similar to the current practice of administering “booster” vaccines, this method can be repeated in the event said individual becomes immunologically responsive over time following future exposures to said antigen.
In another aspect, the present disclosure provides a method for administering an antigen (a) without inducing an increase in antigen-specific B memory cells or (b) which reduces the number of antigen-specific B memory cells in an individual comprising the step of administering to the individual an antigen and one or more lipidic compositions. In one embodiment, the antigen and/or lipidic composition are administered via cutaneous route. For example, one or both of OPLS and the antigen compositions may be administered subcutaneously, intra- or trans-dermally, and/or via topical route. In one embodiment the one or more lipidic compositions are selected from the group consisting of OPLS and PS liposomes. In one embodiment the PS liposomes are selected from the group consisting of PS/PC and PS/PI, as described herein. In some embodiments, the antigen and the one or more lipidic compositions are present in the same formulation. In other embodiments, the antigen and the one or more lipidic compositions are present in different formulation. In embodiments where the antigen and the one or more lipidic compositions are present in the same formulation, the Kd for the antigen and the one or more lipidic compositions is greater than or equal to 10 μM. Administration of the lipidic composition and the antigen may take place either concomitantly or separately.
In embodiments where the antigen and lipidic compositions are administered concomitantly, the lipidic composition and the antigen may be present within the same formulation or within different formulations.
In embodiments where the antigen and lipidic composition are present within the same formulation, they may be either complexed or not complexed. In one embodiment, the antigen and the lipidic component (such as OPLS) are loosely bound or are unbound).
In embodiments where the antigen and lipidic compositions are administered sequentially, either the antigen or the lipidic composition may be administered first provided that whichever component is administered second is administered within close proximity to the first, both in terms of space and time, so as to allow them to be presented together. The sequence of administration is irrelevant. In one embodiment, the antigen and the lipidic composition are administered at anatomical sites that are less than 2 inches apart from one another. Administration of the second component can be carried out within 0.5, 1, 1.5, or 2 inches from the site of administration of the first component. In one embodiment, the antigen and the lipidic composition are administered within 24 hours from one another. Administration of the second component can be carried out within 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 120, 240, 300, 480, 600, 720, 1080 or 1440 minutes from the time the first component is administered. In a preferred embodiment, they are administered via the same or similar cutaneous routes. For example, both administered subcutaneously or the antigen administered subcutaneously and the lipidic composition administered transdermally.
In one embodiment, the antigen (e.g. protein, peptide or polypeptide) is administered subcutaneously (such as by subcutaneous injection) and OPLS is delivered to the same or nearby site by topical route. For topical or transdermal delivery, OPLS may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with suitable preservatives, buffers, or propellants. The topical formulations may also contain additional excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. Topical formulations may be liquids, emulsions, sprays, or foams. The formulations may also contain additional excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays or foams can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. In one embodiment, dermal patches may be used. These have the added advantage of providing controlled delivery to the site. Such dosage forms can be made by dissolving or dispersing OPLS in a suitable medium and applying the formulation to a portion of the patch. Absorption enhancers can be used to increase the flux of OPLS into and across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active ingredient in a polymer matrix or gel. In one embodiment, the OPLS compositions are applied to dermal patches, bandages, adhesive bandages, gauzes or other similar materials that can be directly applied to the desired area. A portion of the dermal patch (such as an adhesive bandage) on which OPLS composition is present may be protected by a sealed, removable covering until use. The adhesive surfaces may also be protected by the same or different removable covering. At the time of use, the covering or coverings can be removed and the strip or patch applied to the area where delivery of the OPLS to or through the skin in desired.
In one aspect of the disclosure are provided kits useful for reducing immunogenicity of an antigen, or improving the pharmacokinetic properties of an antigen, or reducing the number of antigen-specific B memory cells. In one embodiment, the kit comprises OPLS and instructions for administration of OPLS in conjunction with an antigen, and optionally, administration devices for administration of the OPLS and/or the antigen. The kit may also optionally comprise one or more antigens. The instructions for administration of OPLS and the antigen may indicate that these should be administered via a cutaneous route. The instructions may also indicate the site of administration and temporal or spatial separation of the administrations. In one embodiment, the instructions may also indicate that the OPLS and the antigen may be combined just prior to administration (such as within 1 minute or within 1 to 5 minutes of the administration), and the temperature range at which the OPLS and the antigen may be combined (such as from 20 to 26° C.).
In one embodiment, a kit comprises the antigen (e.g. protein, peptide or polypeptide) component and the lipidic composition component. The kit may also comprise a set of instructions for preparation and use of the components. In one embodiment, the lipidic composition is selected from the group consisting of OPLS and PS liposomes. In one embodiment, the PS liposomes are selected from the group consisting of PS/PC (with optionally PE, where the PC concentration is 1-10 mol % and PE mole % displaces PC amount) and PS/PI, as described herein. In one embodiment, the kit comprises an antigen (such as Factor VIII) in a lyophilized form, a vial of reconstitution medium for preparing the antigen and/or the lipidic composition (such as OPLS) for administration (such as subcutaneous administration), a lipidic composition delivery article, and a set of instructions for storage, preparation and/or use of the various components. In some embodiments, the reconstitution medium is comprised of lipids selected from the group consisting of phosphatidylserine, phosphatidylcholine, and combinations thereof. In other embodiments, the reconstitution medium is an OPLS solution. The reconstitution medium is a buffer solution, however the reconstitution medium as defined in this disclosure may or may not contain lipids. If the reconstitution medium does not contain lipids, then the other vial in the kit will provide the requisite lipid. If this is the case, it can be any phosphatidylserine-containing solution, including OPLS. The lipidic composition delivery article may be a container with a liquid or lyophilized formulation comprised of the lipidic composition (such as OPLS and PS liposomes), a syringe prepackaged with the lipidic composition, a spray or foaming can containing the formulation comprised of the lipidic composition for topical application, or a topical patch comprising the formulation comprised of the lipidic formulation. In embodiments where topical application is to be practiced, OPLS is the preferred lipidic composition due to its ability to penetrate the skin. The set of instructions may be printed materials or electronic information storage medium such as thumb drives, electronic cards, and the like. The set of instructions may provide instructions regarding site, sequence and frequency of administration of the components and any other information relevant to the intended use. The various components of the kit may be provided in separate vials or containers within the kit, or as one unit (such as in separate compartments in a one blister packaging).
In one embodiment, this disclosure provides a method for inducing immune tolerance toward an antigen comprising: a) providing a first composition comprising OPLS, which is substantially free of the antigen, and a second composition comprising the antigen, which is substantially free of OPLS, and b) administering the first and the second compositions separately through a cutaneous route to an individual; wherein the two compositions are administered at sites that are within 2 inches of each other. The antigen and OPLS may be administered in isotonic solution (270 to 300 mmole/kg) or in hypertonic solutions (greater than 300 mmol/kg). In various embodiments, the agent may be a therapeutic, which may be present at therapeutic or sub-therapeutic dose. One or both compositions may be administered once or more weekly for 1 to 12 weeks or more. The term “substantially free” as used in this disclosure means that the amount of the particular materials present in the composition is not sufficient to have any detectable clinically relevant biological effect. For example, a composition of OPLS is substantially free of an antigen if less than 0.5 mole % of the antigen is present. Similarly, a composition of the antigen is substantially free of OPLS (or other lipidic components) if less than 0.5 mole % of OPLS (or a lipidic component) is present. In some embodiments, less than 0.4, 0.3, 0.2, 0.1, 0.05 mole % of OPLS or antigen are present in the compositions of the antigen or OPLS respectively.
In one embodiment, this disclosure provides a method for inducing immune tolerance toward an antigen comprising administering to an individual a composition comprising OPLS and a sub-therapeutic amount of antigen. The OPLS and the sub-therapeutic dose of antigen may be in separate compositions or the same composition. When present in the same composition, the OPLS and the antigen are such that they are loosely bound or unbound. The compositions may be delivered via the same or different cutaneous routes. A sub-therapeutic dose of a therapeutic antigen is defined as a dose that is at least 50% less than (i.e., it may be ½ of) the lowest acceptable therapeutic dose. The therapeutic doses of various therapeutics are well known to clinicians and those skilled in the art. In various embodiments, the sub-therapeutic dose may be any fraction from ½ to 1/100th of the therapeutic dose. In one embodiment, it may be from 1/100th to 1/500th of a therapeutic dose. As an example, a therapeutic dose for FVIII is considered to be 40 IU/kg to 400 IU/kg, and a sub-therapeutic dose is 20 IU/kg and lower. In one embodiment, a sub-therapeutic dose is about 10 IU/kg. As another example, the clinical or therapeutic dose of GAA is 20 mg/kg, while a sub-therapeutic dose is 10 mg/kg. In one embodiment, the sub-therapeutic dose is 1 μg to 1 mg/kg.
In one embodiment, this disclosure provides a method for inducing immune tolerance toward an antigen comprising administering to an individual a composition comprising OPLS and a therapeutic amount of antigen. The OPLS and the therapeutic dose of antigen may be in separate compositions or the same composition. When present in the same composition, the OPLS and the antigen are such that they are loosely bound (Kd of 10 μM to 500 μM or unbound (Kd of greater than 500 μM). The compositions may be delivered via the same or different cutaneous routes. In one embodiment, the agent is an antibody, which may be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a fragment of an antibody, or combinations thereof.
In one embodiment, the present disclosure provides a method for inducing immune tolerance toward an antigen in an individual comprising a) obtaining dendritic cells from the individual; b) contacting the dendritic cells with OPLS and the antigen in vitro; and c) returning the dendritic cells to the individual. The dendritic cells may be exposed to OPLS and the antigen at the same time or sequentially. The antigen may be used at a therapeutic dose or a sub-therapeutic dose. In one embodiment, the dendritic cells may be exposed to more than one antigens.
In one aspect, the present disclosure provides kits for inducing immune tolerance. The kits may comprise one or more of the following: a composition comprising OPLS (which is substantially free or completely free of the antigen); a composition comprising one or more antigens (which is substantially free or completely free of OPLS or other lipid components); instructions for administration via a cutaneous route, or instructions for exposing cells in vitro; optionally, one or more administration devices for administration of the compositions. The administration devices may be syringes, injection pens, dermal patches and the like.
The following examples are presented to illustrate the present invention. They are not intended to be limiting in any manner.
This example provides characterization of administered OPLS antigen compositions.
Materials: Full length recombinant human FVIII (Baxter Biosciences, Carlsbad, Calif.) was obtained as a gift from the Western New York Hemophilia Foundation. Ova and human IgG were from Sigam (St. Louis, Mo.) Humira and Rituximab are commercially available. For the present disclosure, Humira was obtained commercially, from a pharmacy. O-Phosphso-L-Serine (OPLS) was purchased from Sigma (Sr. Louis, Mo.). All solvents and buffer salts were obtained from Fisher Scientific (Fairlawn, N.J.) or from Sigma (St. Louis, Mo.). Anti-FVIII monoclonal antibodies ESH8 were obtained from American Diagnostica Inc. (Greenwich, Conn.). Goat anti-mouse IgG was purchased from Southern Biotechnology Associates, Inc. (Birmingham, Ala.). Bovine serum albumin (BSA) was purchased from Sigma (St. Louis, Mo.) while the phosphatase substrate system (diethanolamine buffer and p-nitrophenyl phosphate) was purchased from KPL Inc. (Gaithersburg, Md.). Chromogenix Coamatic FVIII assay kit was purchased from DiaPharma Group (West Chester, Ohio) for the detection of FVIII in plasma. anti-Ova IgG titier analysis kit was from alphadiagnostic international.
Effect of OPLS on the immunogenicity of FVIII after SC administration in-vivo: 21 HA mice between 6 and 8 weeks of age were divided into 3 groups: group one received 1 ug FVIII SC in the abdominal region. Group two received 1 μg FVIII plus 0.4 mg OPLS SC in the abdominal region and group 3 received 1 μg FVIII plus 5 mg OPLS sc in the abdominal region. Incubation of FVIII with OPLS at 37° C. for 30 minutes ensures complexation, but for this example, OPLS and FVIII were combined at the desired concentrations at room temperature (22° C.) and immediately administered (within 1 minute). This treatment was given one day 1, 8, 10 and 12. Three days after the last injection the animals were sacrificed. Blood was centrifuged at 7500 rpm for 5 min at 4 degrees Celsius. Plasma was then removed and stored at −80 degrees Celsius until analysis. The results are shown in
Total titers were determined by classical standard ELISA. Anti-FVIII antibody (ESH8) was used as a control. Statistics was done on the arithmetic mean of the log transformed data, graphs are reported as geometric means. Non-responders were given the value of the plate specific factor for uniformity.
PK of FVIII in HA mice in the presence and absence of O-Phospho-L-Serine: 60 HA mice between 6 and 8 weeks of age where divided into 2 groups of 30 animals each. First group received 5 IU/g FVIII in the abdominal region with massaging for 60 second after each injection. The second group received FVIII plus OPLS (5 IU/gFVIII plus 0.4 mgOPLS) in the abdominal region followed by massaging as above. 3 animals from each group were sacrificed at the following pre-set time point 4, 6, 9, 12, 15, 18, 24, 33, and 44 Hours. Total blood as well as the inguinal lymph nodes were collected. Blood was centrifuged at 7500 rpm for 5 min at 4° C. All samples were stored at −80° C. until analysis. Samples were analyzed using Coamatic Factor VIII kit (DiaPharma West Chester, Ohio, USA) assay according to manufacturer's instruction.
Intravenous co-administration of OPLS and biotherapeutic (e.g. FVIII) has little effect on the pharmacokinetics: only a slight increase in plasma exposure was seen (
Effect of OPLS on the cytokine profile of Bone Marrow Derived Dendritic Cells (BMDC) in presence and absence of protein antigen (FVIII): Bone marrow was isolated from 2 HA mice between 6 and 8 weeks of age. Briefly, bone marrow was cultured in RPMI media supplemented with Fetal Bovine Albumin, 2-Macraptoethanol, Pen/Strip and GMCSF. Media was changed on days 3,6 and 8 of culture and DC's were used on day 9 or 10. This prolonged culturing method results in 70-80% immature DC's on day 9 or 10. Once the DC are harvested they are exposed to one of the following treatment groups: Naïve (negative control), FVIII alone (positive control), FVIIII with increasing dose of OPLS 10 mM, 50 mM and 100 mM OPLS, OPLS alone (100 nM), and Lipopolysacarides (positive control for inflammation response). Cells were platted in triplicate and 2 sets were made. One was used for cytokine analysis and was allowed to remain in culture for 72 hours before media was collected for cytokine analysis. TGF-β, TNF-α and IL-6 concentrations were determined using standard ELISA (DuoSet ELISA kit, R&D System Inc. Minneapolis, Minn., USA). The other set was allowed to stay in culture for 24 house before media was removed and the DC's were co-cultured with CD4+ T-cells isolated from spleens of HA mice exposed to an aggressive immunization schedule with FVIII as follow: week 1: Monday 1 μg of FVIII given SC. Week 2: Monday, Wednesday and Friday 1 μg of FVIII given SC on each of those days. 72 hours after the last immunization the animals are sacrificed and spleens are collected, crushed and CD4+ T-cell were isolated using a negative isolation kit (Miltenyi Biotec, Auburn, Calif., USA) as per the manufacturer's protocol. After 72 hours of co-culturing with DC, the supernatant is removed and cytokine analysis was done using standard ELISA to determine TFG-I3 and IL-17 production by T-cells. (DuoSet ELISA kit, R&D System Inc. Minneapolis, Minn., USA). The results are shown in
The binding of OPLS to several therapeutic proteins was evaluated using fluorescence spectroscopy (
This example describes the effect of OPLS on the immunogenicity of ovalbumin (Ova) after SC administration in-vivo:
Methods: 14 SW mice between 6 and 8 weeks of age were divided into 2 groups: group one received 1 μg OVA SC in the abdominal region. Group two received 1 μg OVA/5 mg OPLS SC in the abdominal region. This treatment was given one day 1, 8, 10 and 12. Three days after the last injection the animals were sacrificed. Blood was centrifuged at 7500 rpm for 5 min at 4° C. Plasma was then removed and stored at −80° C. until analysis Titers values were obtained using an anti-Ova IgG titier analysis kit as per the manufacturers instruction (alphadiagnostic international). Statistics was done on the arithmetic mean of the log transformed data, graphs are reported as geometric means. Non-responders were given the value of the plate specific factor for uniformity.
As shown in
This example describes the effect of OPLS on the Immunogenicity of Acid Alpha Glucosidase Protein (GAA)
Materials and Methods: GAA Source: Full length recombinant rhGAA was produced in Human Kidney Embryo Cells for our use by Creative Biomart (Shirley, N.Y.); OPLS Source: Sigma-Aldrich (St. Louis, Mo.). n=8 animals/group were immunized with 4 weekly s.c. injections of 1 μg of GAA in 100 μl of either sterile TRIS for the free group or in 5 mg OPLS in sterile TRIS for the OPLS-GAA group. A group of 8 mice will receive the same dose of CBrhGAA in 5 mg O-phospho-D-serine (OPDS) solution as a negative control. After a 2 week rest period, a blood sample from the saphenous vein will be collected. Samples were stored at −80° C. until analysis. These samples were then analyzed for anti-GAA Ab titers. Anti-GAA antibody titers were measured as follows: ELISA plate (maxisorp, NUNC) was coated with 50 μl of 5 μg/ml of sterile rhGAA in sodium carbonate-bicarbonate buffer pH =9.6. The plate was incubated overnight at 4° C. The plate was washed six times with PBS containing 0.05% Tween 20 (PBS-T, 250 μl/well) using an automated plate washer. The plate was blocked with PBS containing 1% BSA (300 μl/well) as the block buffer for 2 hr at 37° C. The plate was washed six times with PBS-T. A serial dilution of samples (100 ul/well) were then added to the plate in duplicate. The samples incubated for 1 hr at 37° C. The plate was washed six times with PBS-T. Detection antibody (1:10000 dilution and 100 μl/well) in block buffer was added to the wells and incubated for 1 hr at room temperature. The plate was washed six times with PBS-T. 3,3′,5,5′-Tetramethylbenzidine substrate (100 μl/well) was added to the wells and incubated for 30 min at room temperature. The reaction was stopped with 2 N sulfuric acid and read immediately at 450 nm using a micro-plate reader. The titer values are quantitated using a statistical endpoint method as described by Frey et al. Control plasma samples were run on each plate. Statistical Analysis: Statistical difference (p<0.05) one-way ANOVA followed by Dunnette's post-hoc multiple comparison test using GraphPad Prism (Frey et al., J. Immunol. Methods, 1998. 221(1-2): p. 35-41).
As shown in
This example describes the effect of OPLS on the immunogenicity of Adalimumab.
Methods: n=8 animals/group were immunized with 4 weekly s.c. injections of 10 μg of adalimumab in 100 μl of either sterile TRIS for the free group or in 5 mg OPLS in sterile TRIS for the OPLS-Adalimumab group. After a 2 week rest period, a blood sample from the saphenous vein was collected for titer analysis. Titer values were obtained by classical sandwitch ELISA. Briefly, Nunc-Maxisorb 96-well plates were coated overnight at 4° C. with 50 μl/well of 2.5 μg/ml Humira. Plates were then washed and blocked with 1% bovine serum albumin (blocking buffer). 50 μl/well of various dilutions of the sample were added to the plate and incubated at room temperature for 2 h. Plasma samples from animals exposes to a sham treatment (buffer only) served as controls. The plates were then washed and incubated with 50 μl of goat anti-mouse anti-body-HRP conjugate at a 1:50000 dilution in blocking buffer at room temperature for 1 h. Color was developed for 20 min with 100 μl of TMB solution. 100 μl of 2 N H2SO4 was used to stop the reaction. Optical density at 405 nm was measured using a plate reader. Non-responders were given the value of the plate specific factor for uniformity.
As shown in
This example describes the effect of OPLS on Human IgG Immunogenicity.
Materials and Methods: 8 animals were used for this pilot study, 4 animals were injected with Human-IgG (Sigma) alone (1 μg) or with Human IgG plus OPLS (1 μg human IgG plus 0.4 mg OPLS). The study was done with 4 injections over 2 weeks period as follows: day 1, day 8, day 10 and day 12 followed by a 3 days washout period. ELISA was done to measure total titers. Briefly, Nunc-Maxisorb 96-well plates were coated overnight at 4° C. with 50 μl/well of 2.5 μg/ml Human IgG. Plates were then washed and blocked with 1% bovine serum albumin (blocking buffer). 50 μl/well of various dilutions of the sample were added to the plate and incubated at room temperature for 2 h. Plasma samples from animals exposes to a sham treatment (buffer only) served as controls. The plates were then washed and incubated with 50 μl of goat anti-mouse anti-body-HRP conjugate at a 1:50000 dilution in blocking buffer at room temperature for 1 h. Color was developed for 20 min with 100 μl of TMB solution. 100 μl of 2 N H2SO4 was used to stop the reaction. Optical density at 405 nm was measured using a plate reader. Non-responders were given the value of the plate specific factor for uniformity.
In this pilot study, animals were injected with Human-IgG (Sigma) alone (1 μg) or with OPLS (5mg). The study was done with 4 injections over 2 weeks period as follows: day 1, day 8, day 10 and day 12 followed by a 3 days washout period. Titers were obtained by classical ELISA as mentioned above. As shown in
This example describes the effect of OPLS on Rituximab Immunogenicity (RXT).
Materials and Methods: 30 SW mice between 6 and 8 weeks of age were divided into 3 groups: group one received 1 ug RXT SC in the abdominal region. Group two received 1 ug RXT/0.4 mg OPLS SC in the abdominal region and group 3 received 1 μg RXT/5 mg OPLS sc in the abdominal region. This treatment was given one day 1, 8, 10 and 12. Three days after the last injection the animals were sacrificed. Blood was centrifuged at 7500 rpm for 5 min at 4 degrees Celsius. Plasma was then removed and stored at −80 degrees Celsius until analysis Total titers were determined by classical standard ELISA. Briefly, Nunc-Maxisorb 96-well plates were coated overnight at 4° C. with 50 μl/well of 2.5 μg/ml RXT. Plates were then washed and blocked with 1% bovine serum albumin (blocking buffer). 50 μl/well of various dilutions of the sample were added to the plate and incubated at room temperature for 2 h. Plasma samples from animals exposes to a sham treatment (buffer only) served as controls. The plates were then washed and incubated with 50 μl of goat anti-mouse FC-specific anti-body-HRP conjugate at a 1:50000 dilution in blocking buffer at room temperature for 1 h. Color was developed for 20 min with 100 μl of TMB solution. 100 μl of 2 N H2SO4 was used to stop the reaction. Optical density at 405 nm was measured using a plate reader. Non-responders were given the value of the plate specific factor for uniformity. Statistics was done on the arithmetic mean of the log transformed data, graphs are reported as geometric means. Non-responders were given the value of the plate specific factor for uniformity.
As shown in
This example describes the effect of OPLS and PS-Liposomes on Acid Alpha Glucosidase (GAA) Peptide Immunogenicity
Materials and Methods. GAA peptide Source: This GAA peptide was produced by American Peptide Company (Sunnyvale, Calif.). They produced for us a peptide 13 amino acids long containing amino acids 339-352 of the full length GAA sequence, YIFLGPEPKSVVQ (SEQ ID NO:1). OPLS Source: Sigma-Aldrich (St. Louis, Mo.) Peptide concentration dose: 10 μg/injection. Free GAA peptide (n=4), PS-GAA peptide (n=4), and OPLS-GAA peptide (n=5). Each mouse received 4 weekly subcutaneous (s.c.) injections near the base of the tail of 100 μl/injection of their treatment. Two weeks after the last injection, a retro-orbital blood sample was taken from each mouse. Samples were stored at −80° C. until analysis. These samples were then analyzed for anti-GAA peptide Ab titers. Anti-GAA antibody titers were measured as follows: ELISA plate (maxisorp, NUNC) was coated with 100 μl of 10 μg/ml of sterile rhGAA in sodium carbonate-bicarbonate buffer pH=9.6. The plate was incubated overnight at 4° C. The plate was washed six times with PBS containing 0.05% Tween 20 (PBS-T, 250 μl/well) using an automated plate washer. The plate was blocked with PBS containing 1% BSA (300 μl/well) as the block buffer for 2 hr at 37° C. The plate was washed six times with PBS-T. A serial dilution of samples (100 μl/well) were then added to the plate in duplicate. The samples incubated for 1 hr at 37° C. The plate was washed six times with PBS-T. Detection antibody (1:10000 dilution and 100 μl/well) in block buffer was added to the wells and incubated for 1 hr at room temperature. The plate was washed six times with PBS-T. 3,3′,5,5′-Tetramethylbenzidine substrate (100 μl/well) was added to the wells and incubated for 30 min at room temperature. The reaction was stopped with 2 N sulfuric acid and read immediately at 450 nm using a micro-plate reader. The titer values are quantitated using a statistical endpoint method as described by Frey et al. Control plasma samples were run on each plate. Statistical Analysis: Statistical difference (p<0.05) one-way ANOVA followed by Dunnette's post-hoc multiple comparison test using GraphPad Prism.
As shown in
The GAA peptide did not bind to OPLS. Since the GAA peptide does not have internal fluorescence we needed to use a different method compared to the other protein-OPLS binding studies. Instead of using fluorescence to detect binding, ultraviolet/visible spectroscopy was used.
This example describes tolerance Induction towards GAA Protein using OPLS and PS-Liposomes.
Materials: Full length recombinant rhGAA was produced in Human Kidney Embryo Cells for our use by Creative Biomart (Shirley, N.Y.). Dimyristoyl phosphatidylcholine (DMPC), phosphatidylglycerol (PG) and brain phosphatidylserine (PS) were purchased from Avanti Polar Lipids (Alabaster, Ala.) O-phospho-L-serine (OPLS), Goat-anti-mouse IgG horseradish peroxidase, 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate were purchased from Sigma-Aldrich (St. Louis, Mo.). Animals: Breeding pairs of Pompe disease mice were purchased from a Jackson Laboratory (Bar Harbor, Me.). A colony homozygous for the knockout gene was established and animals aged from 8-12 weeks were used for the in vivo studies.
GAA Tolerance with OPLS. Methods: n=8 animals/group were immunized with 4 weekly sc injections of 1 μg of GAA in 100 μl of either sterile TRIS for the free group or in 5 mg OPLS in sterile TRIS for the OPLS-GAA group. A group of 8 mice will receive the same dose of GAA in 5 mg O-phospho-D-serine (OPDS) solution as a negative control. After a 2 week rest period, a blood sample from the saphenous vein was collected. The mice were then re-challenged with 1 μg of free GAA in Sterile TRIS for 4 weeks. The mice were sacrificed after another 2 week rest period and plasma was collected than stored at −80° C. until analysis by ELISA for anti-GAA Ab Titers.
GAA Tolerance with PS-liposomes. Methods: n=8 animals/group were immunized with 4 weekly sc injections of 1 μg of GAA in 100 μl of either sterile TRIS for the free group or PS liposomal formulation for PS group (30% PC, 70% DMPC). A group of 8 mice received the same dose of GAA in PG30 liposomal formulation(30% PG, 70% DMPC) as a negative, charge-matched control. After a 2 week rest period, a blood sample from the saphenous vein was collected. The mice were then re-challenged with 1 μg of free GAA in Sterile TRIS for 4 weeks. The mice were sacrificed after another 2 week rest period and plasma was collected than stored at −80° C. until analysis by ELISA for anti-GAA Ab titers.
Anti-GAA antibody titers. ELISA plate (maxisorp, NUNC) was coated with 50 μl of 5 μg/ml of sterile GAA in sodium carbonate-bicarbonate buffer pH=9.6. The plate was incubated overnight at 4° C. The plate was washed six times with PBS containing 0.05% Tween 20 (PBS-T, 250 μl/well) using an automated plate washer. The plate was blocked with PBS containing 1% BSA (300 μl/well) as the block buffer for 2 hr at 37° C. The plate was washed six times with PBS-T. A serial dilution of samples (100 μl/well) were then added to the plate in duplicate. The samples incubated for 1 hr at 37° C. The plate was washed six times with PBS-T. Detection antibody (1:10000 dilution and 100 μl/well) in block buffer was added to the wells and incubated for 1 hr at room temperature. The plate was washed six times with PBS-T. TMB substrate (100 μl/well) was added to the wells and incubated for 30 min at room temperature. The reaction was stopped with 2 N sulfuric acid and read immediately at 450 nm using a micro-plate reader. The titer values are quantitated using a statistical endpoint method as described by Frey et al. (Journal of Immunological Methods, 1998. 221(1-2): p. 35-41). Control plasma samples were run on each plate. Statistical Analysis: Statistical difference (p<0.05) one-way ANOVA followed by Dunnette's post-hoc multiple comparison test using GraphPad Prism.
Results: The anti-GAA antibody titers after a GAA rechallenge were found to be significantly lower for the animals treated with OPLS-GAA compared to those treated with free GAA as seen in
This example describes Tregs in PS mediated hypo-responsiveness.
Methodology: Immunization studies were conducted in hemophilia A mice model. The animals (n=3/treatment group) received S.C. injections of either free FVIII or FVIII-PS (2 μg of FVIII) every week for 12 weeks. Two weeks after the last injection, the CD4+ T cells were isolated from spleen of the immunized animal and were stained with FITC conjugated to CD4 antibody and PE conjugated to anti FoxP3 antibody. The double positive cells were analyzed using flow cytometry. In order to account for the spectral overlap of FITC and PE, compensation using singly labeled FITC and PE controls were acquired and compensation was carried out using FlowJO software. Statistical analysis was carried out using 1-tail paired t-test using Minitab software.
Results: The FoxP3 expression was analyzed as a biomarker for Treg. The CD4+FoxP3+ double positive cells were significantly higher for FVIII-PS treatment group compared to FVIII alone treatment group (
This example describes the role of Memory B cells in PS-mediated hyporesponsiveness.
Methodology: Immunization studies were conducted in GFP-FoxP3 knock-in hemophilia A mice model. The animals (n=7 or 8/treatment group) received S.C. injections of either free FVIII or FVIII-PS or FVIII-PG30 (2 μg of FVIII) every week for 12 weeks. One group of animals (n=5) was left untreated and served as the naïve control. Two weeks after the last injection, all animals were sacrificed and the memory B cells isolated from their spleens. Approximately 0.1×106 memory B cells from all mice were adoptively transferred via penile vein to a corresponding individual naïve GFP-FoxP3 hemophilic mice. After a 48 h wait period, all the recipient animals were immunized aggressively with four weekly s.c. injections of 1 μg of free FVIII/injection. Two weeks after the last injection, all animals were sacrificed and their plasma samples collected for analysis of antibody titer levels.
Results: The plasma samples were analyzed for the presence of total anti-FVIII antibody titer levels. Mice that received memory B cell from a FVIII treated or from a naïve (untreated) mice generated robust FVIII immune response. In comparison, mice that received memory B cell from a PS-FVIII treated donor mice exhibited significantly reduced anti-FVIII antibody titers. However, memory B cells transferred from mice treated with FVIII associated with another negative charge PG lipid (FVIII-PG) failed to significantly reduce the antibody titers (
This example describes the requirement for proximity for inducing immune tolerance.
Mice were injected with unloaded PS liposomes in one flank and free FVIII in the other flank for four weeks, and then re-challenged with free FVIII for four more weeks before sacrifice. If spatial proximity is not critical, then the treatment with PS and FVIII spatially separated should yield a reduction in titers. However, the data showed that both neutralizing (425±100 SEM, n=5) and total anti-FVIII (8197±1235 SEM, n=5) titers were not significantly different than free FVIII control, which had neutralizing titers of 469±63.3 SEM (n=5), and total anti-FVIII titers of 5153±893 SEM (n=5) (
The development of an immune response against exogenous FVIII administration is of major clinical concern. Neutralizing antibodies develop in 15-30% of patients undergoing replacement therapy, reducing the efficacy of this treatment for patients with hemophilia A. Current strategies to reverse this immunogenicity require the administration of large doses of FVIII with immunosuppressants for an extended period of time, frequently up to one year. This strategy is both costly and carries with it considerable risk for the patient who will have their immune system compromised during therapy. Thus, developing new ways to reduce the immunogenicity to FVIII is extremely important.
Previous studies have shown that the complexation of FVIII with PS reduces the immune response against FVIII in hemophiliac mice. The induction of hyporesponsiveness to FVIII by the PS-FVIII formulation can be thought of as a ‘reverse vaccination’ strategy. Rather than attempting to establish an immune response against an antigen, the goal of therapy is to desensitize the patient against an antigen. If the mechanism in which this is achieved resembles the way in which apoptotic cells induce tolerance to their intracellular proteins, then this hyporesponsiveness should be antigen specific. A nonspecific response would also be clinically undesirable, as it would lead to immunosuppression.
In order for this hyporesponsiveness to develop, spatial proximity should be required between FVIII and PS, as apoptosis is a local event which occurs on the cellular level. To test this, mice received unloaded PS liposomes in one flank and free FVIII in the opposite flank, and were re-challenged with free FVIII. These mice showed no difference in immune response to FVIII when compared to free FVIII control. This data suggests that close anatomical proximity is required in order for hyporesponsiveness to develop. This observation lends support to the theory that PS-FVIII uses apoptotic mimicry to induce tolerance to FVIII.
In conclusion, it is clear from this data that the immunologic effects of PS-FVIII liposome formulations are specific to both FVIII as an antigen as well as the anatomical proximity of FVIII to the PS containing liposomes. These conclusions are consistent with the hypothesis that tolerance induction via apoptotic mimicry is the mechanism for the observed reduction in immune response as a result of reverse vaccination PS-FVIII therapy. Future studies should address the possibility of PS containing liposome formulations to reverse established titers, as well as induce hyporesponsiveness to an antigen other than FVIII.
This example describes apoptotic induction in Dendritic Cells
Phosphatidylserine is a naturally occurring lipid and is part of vesicles released by apoptotic and other cell types and includes exosomes, ectosomes, microvesicles, membrane particles and apoptotic bodies. In one embodiment, proteins could be loaded into these particles after isolation and could be used to reduce immunogenicity and/or induction of tolerance. Dendritic cells were isolated from bone marrow progenitors of factor VIII deficient mice. Briefly, bone marrow cells were collected from femur and tibia. The cells were cultured for 8 days with change of media every two days. On day 8, cells from the suspension RPMI medium were collected, centrifuged, and plated in fresh plates. Immature dendritic cells were then used for apoptotic induction.
Apoptotic induction in dendritic cells was tested in several ways. Some of the methods tested were UV irradiation using 8W UV transilluminator for 1 hr/5 mins time points, by adding 5 mM sodium butyrate total concentration, heat shock at 45° C. for 1 hr, and serum depletion for 24 hrs. Cells were pelleted down after 48 hrs of induction at 300 g for 10 mins to remove cell debris followed by 2000-10,000 g for 20 mins and 100,000 g is required for exosomes. The apoptotic induction efficiency was later tested using Annexin V/Propidium Iodide kit from Sigma using fluorescent microscope. These apoptotic bodies and vesicles can be loaded with proteins (FVIII, GAA, Humira etc) to reduce immunogenicity and induction of tolerance.
This example describes characterization of safety of OPLS.
Given that the methods disclosed herein appear to work best with doses of OPLS that equal or exceed 5 mg, it is important to understand the safety and toxicity of high doses of OPLS. As shown in
This examples describes OPLS-GAA sequential administration study. To determine if OPLS can exert its immunomodulatory effects on the immune response to GAA when the two are administered in separate spaced injections, at the same injection site the following experiments were carried out. Mice (n=6 animals/group) were immunized with 4 weekly sc injections as follows. One group of animals received one single combined injection of 5 mg OPLS and 1 μg rhGAA(100 μl volume). Recombinant human GAA was produced by Creative Biomart. The second group received one injection of 5 mg OPLS (100 μl volume) then after a 5 minute wait they received a second injection of 1 μg rhGAA in sterile TRIS (100 μl volume) in the same injection site. The final control group received one injection of sterile TRIS (100 μl volume) then after a 5 minute wait they received a second injection of 1 μg rhGAA in sterile TRIS (100 μl volume) in the same injection site. After a two week rest period a saphenous blood sample was collected. All of the mice were then rechallenged with 4 weekly injections of 1 μg of rhGAA in 100 μl sterile TRIS (one single injection). Analysis was done by ELISA for total titers. The results are shown in
In this example, the effects of hypertonic buffers were tested. Hypertonic buffer systems were generating using three different excipients: NaCl, mannitol and O-Phospho-L-Serine (OPLS). NaCl was chosen as a generic salt to manipulate osmolarity. Mannitol is a common GRAS (Generally Regarded as Safe) excipient used in protein formulations. OPLS is the head group of the immune-modulatory lipid Phosphatidylserine (PS). All three excipients are highly soluble in aqueous media. The effects of these buffers was tested on rituximab pharmacokinetics after sc administration in Swiss Webster mice.
Material and Methods: Swiss Webster mice (19-22 g) (SW) were from Charles River Laboratories (Wilmington, Mass.). All animal experiments were conducted in accordance with guidelines established by Institutional Animal Use and Care Committee at the University at Buffalo, State University of New York.
Commercial preparation of rituximab (RXT) was used. Rat anti-rituximab antibody was purchased from AbD Serotec (Raleigh, N.C.). Goat anti-mouse FC-specific HRP conjugated antibody, 3,3′5,5′ tetramethyl benzidine (TMB) substrate system, Bovine serum albumin (BSA), O-Phospho-L-Serine (OPLS) and mannitol were purchased from Sigma (St. Louis, Mo.). All other solvents and buffer salts were obtained from Fisher Scientific (Fairlawn, N.J.) or from Sigma (St. Louis, Mo.).
Preparation and characterization of injection buffers: One isotonic and six different hypertonic TRIS buffers were prepared to investigate the effects of hypertonicity and buffer composition on rituximab lymphatic uptake and plasma exposure (Table 1.).
Isotonic TRIS buffer was prepared using 25 mM TRIS and 150 mM NaCl (Buffer A). Hypertonic (600 mmol/kg) TRIS buffers were prepared with 25 mM of TRIS containing 300 mM NaCl (Buffer B). Buffers “C” and “E” contained NaCl as well as 20 mM of OPLS or Mannitol. To further delineate the effects of buffer composition on lymphatic uptake, we prepared two buffers at 600 mmol/kg with a 270 mM of OPLS (Buffer D) or mannitol (Buffer F). Since osmolarity of these buffers is the same as buffers “C” and “E”, any changes in lymphatic uptake will be attributed to increase in OPLS and mannitol concentrations. pH was adjusted to 7.5. Osmolarity was measured using a 5500 Vapor Pressure Osmometer (Wescore Inc. Logen, Utah, USA) according to manufacturer's instruction.
Rituximab pharmacokinetics studies: 126 male SW mice were divided into 7 groups. Each group consisted of 18 animals, three for each time point of the PK profile. Each animal was given 1 ug/g RXT formulated in one of the formulations described above (table 1). All sc injections were in the abdominal region equidistance from the inguinal lymph node. Since absorption is expected to be complete by 5 day, the following preset time points 1, 5, 15, 24, 48, and 120 hr were chosen for sacrifice and sample collection. Total blood and both inguinal lymph nodes were collected. The disposition of RXT will be determined from the iv PK profile and convoluted with the absorption data generated from the sc studies. For iv PK study, animals were given 1 μg/g RXT in base buffer (buffer A) via the penile vein. Total blood was collected at the following times points: 0.5, 2, 15, 24, 48, and 120 hr. Blood was centrifuged at 7500 rpm for 5 min at 4° C. All samples were stored at −80° C. until analysis. Immediately before analysis; the inguinal lymph nodes were homogenized in 300 μl of PBS on ice and then analyzed.
Analysis of RXT in plasma and lymph node samples: Analysis of plasma and lymph node samples from PK studies was done with a standard ELISA. Briefly Nunc-Maxisorb 96-well plates were coated overnight at 4° C. with 100 μl/well of 1 μg/ml anti-RXT antibody (AbD Serotec Raleigh, N.C.). Plates were then washed and blocked with 1% bovine serum albumin (blocking buffer). Samples were diluted as needed to a final dilution of 1:100 or 1:1000 in blocking buffer and added to the plate. A serial dilution of RXT in blocking buffer also added to the plate for the standard curve. 100 μl of goat anti-human-HRP conjugate anti-body at a 5 μg/ml was used as a detection anti-body. Color was developed for 20 min with 100 μl of TMB solution. 50 μl of 2 N H2SO4 was used to stop the reaction. Optical density at 450 nm was measured using a plate reader.
Results: pharmacokinetics of rituximab in SW mice after iv and sc administration: The plasma concentration-time profile of rituximab after iv and sc administration of a 1 μg/g dose in isotonic buffer A is shown in
Effect of hypertonic buffer composition on lymphatic uptake of rituximab: To elucidate the effects of hypertonic buffer on lymphatic uptake we compared the concentration of rituximab in excised inguinal lymph nodes after sc administration of 1 μg/g of rituximab in isotonic buffer (Buffer A) and hypertonic buffer (Buffer B) in SW mice. As shown in
To further delineate the effect of hypertonic buffer composition on lymphatic uptake, lymph-node concentration of rituximab administered in the remaining 5 buffers in table 1 was compared to the results obtained from buffer B. As shown in
Interestingly, when mannitol was used (buffers D and F), an increase in lymph node concentration of rituximab administered in either mannitol-containing buffers was observed as compared to mannitol-free buffer B (
Effect of hypertonic buffer composition on rituximab plasma concentrations and bioavailability: To investigate whether improved lymphatic uptake would translate to improved bioavailability of rituximab, plasma samples were analyzed to determine rituximab concentrations and then modeled as above. As shown in
Overall, hypertonic buffers resulted in higher rituximab bioavailability as compared to the isotonic buffer. Furthermore, the buffer composition affected the bioavailability.
These data show that lymphatic uptake was improved when rituximab was administered in hypertonic (600 mmol/Kg). Furthermore, the composition of the buffer played a role in lymphatic uptake, mannitol and OPLS performed better than osmolarity-matched buffers containing NaCl alone. The improved lymphatic uptake was closely correlated with improved plasma exposure and improved bioavailability of rituximab.
While not intending to be bound by any particular theory, it is considered that hypertonic buffers in the sc space alter the environment in the interstitial milieu. This environment is the driving force behind the formation of interstitial fluid. Under normal conditions the blood filters through the arteriolar end of the capillary bed, a process driven by the increased hydrostatic pressure at that end as compared to the venular end of the capillaries. Downstream of the capillaries, the oncotic pressure increase within the blood capillaries, favoring the re-absorption of the filtrate back into blood carrying with it waste and byproducts from the tissue. Hyper-osmolarity in the interstitium, resulting from hypertonic buffers alters the osmotic conditions in the interstitial milieu increasing the interstitial volume and resulting in increased lymphatic drainage. The effects of volume expansion by hypertonic buffers in the sc space can be expected to last until osmolarity is restored. This may explain the advantage of mannitol and high dose of OPLS over NaCl alone.
This example demonstrates that OPLS reduces immunogenicity by inducing tolerogenic properties in dendritic cells.
Materials and Methods: Full length recombinant human FVIII (Baxter Biosciences, Carlsbad, Calif.) was used. O-Phospho-L-Serine (OPLS) was purchased from Sigma (St. Louis, Mo.). All solvents and buffer salts were obtained from Fisher Scientific (Fairlawn, N.J.) or from Sigma (St. Louis, Mo.). Anti-FVIII monoclonal antibody ESH8 was obtained from American Diagnostica Inc. (Greenwich, Conn.). Heat inactivated premium Bovine Serum Albumin (BSA) from Lonza Walkersville Inc. (Walkersville, Md.). RPMI-1640 media was from Gibco Life Technologies (Carlsbad, Calif.). Endosafe Endochrome-K® kit was purchased from Charles River Laboratories, (Charleston, S.C.). NUNC MaxiSorp 96 well plates were from Thermoscientific (Waltham, Mass.). Cytokine ELISA kits were from R&D System Inc. (DuoSet) (Minneapolis, Minn., USA) or from Invitrogen (Camarillo, Calif.). DilC18(5) was from Sigma (St. Louis, Mo.). BD FC-block, anti-CD40-PE, and anti-MHCII-FITC antibodies and respective isotypes were purchased from BD Biosciences (San Jose, Calif.) or eBiosciences (San Jose, Calif.)
Tolerance induction studies in HA mice: Breeding pairs of exon 16 knockout transgenic hemophilic mice (HA) were used. All animal studies were conducted in accordance with an approved protocol by the Institutional Animal Use and Care Committee at the University at Buffalo, State University of New York.
Immunization and re-challenge schedule: To delineate whether OPLS mediated reduction of antibody titer is due to immune tolerance or immune suppression, we performed re-challenge experiments. Forty HA mice were pre-treated with FVIII, FVIII plus OPLS or FVIII plus Dexamethasone once-a-week for four consecutive weeks by sc injections of endotoxin free formulation of 1 μg of FVIII alone or with 5 mg OPLS or 200 ng Dexamethasone (Dex) as shown in
Characterization of BMDC exposed to FVIII in the presence and absence of OPLS: In this set of experiments we examined the effects of OPLS on phenotypic maturation, cytokine profile and migratory potential of bone marrow derived dendritic cells (BMDC) exposed to FVIII in the presence and absence of OPLS.
Generation of bone marrow derived dendritic cells (BMDC): Bone marrow was isolated from 6 to 8 week old naïve HA. Briefly, bone marrow was cultured in RPMI media supplemented with Fetal Bovine Albumin (low endotoxin), 2-mercaptoethanol, Pen/Strep and 2001 U/ml GMCSF. Media was changed on days 3, 6 and 8 of culture and DCs were used on day 9. BMDC are then harvested and used as needed. To verify the differentiation of bone marrow cells into DCs, CD11c surface expression was confirmed by flow cytometry using anti-CD11c-FITC antibody with appropriate isotope controls.
Effect of OPLS on phenotypic maturation of BMDC: Next we investigated the effects of OPLS on phenotypic maturation of BMDC. BMDCs were exposed to 1 μg/ml FVIII in the presence and absences of OPLS for 24 hr. The cells were then harvested, washed twice with ice cold sterile PBS and suspended at approximately 1×106 cells/mL. Cells were blocked using BD Fc-block antibody before labeling with anti-CD40-PE or anti-MHCII-FITC antibodies. The cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, Calif.). This study was conducted in triplicates.
Effects of OPLS on cytokine profile of BMDCs in vitro: The effects of OPLS on cytokine secretion by dendritic cells were investigated. On day 9 BMDCs, generated as above, were treated with FVIII as an antigen in the presence of increasing dose of OPLS. Changes in production of pro-inflammatory cytokines TNF-α and IL12p70 as well as the regulatory cytokine TGF-β were monitored as a function of dose. BMDCs were exposed to 1 μg/ml FVIII with increasing dose of OPLS (0, 0.5, 1, 10, 25, 50 mM). 1 M stock of OPLS was prepared by adding the appropriate amount of OPLS directly to RPMI media, and pH was adjusted using sterile 3N and 1N NaOH. Endotoxin free stocks were used (Endosafe Endochrome-K® kit Charles River Laboratories, Charleston, S.C.). Cells were plated in quadruplicates (n=4) and cultured for 48 hours. Media was collected at the end of the incubation period for cytokine analysis. TGF-β, TNF-α and IL-12p70 concentrations were determined using standard ELISA (R&D System Inc. Minneapolis, Minn., USA or Invitrogen Amarillo, Calif.).
Effect of OPLS on migratory potential of BMDC: The effect of OPLS on in-vivo migratory potential of BMDCs treated ex-vivo with FVIII or FVIII/OPLS was investigated. On Day nine, 3×106 cells were incubated overnight with 5 μg FVIII or 5 μg/50 mM FVIII/OPLS. The next day, non-adherent cells were harvested and washed twice with ice-cold sterile PBS and were labeled with DilC(18)5 dye (Sigma, St. Louis, Mo.). Cells were incubated for 5 min at 37° C. with DilC(18)5 at 2 μg/ml (Sigma, St. Louis, Mo.). After 5 min, cells were washed twice with ice-cold sterile PBS. Incorporation of the dye was confirmed by imaging a sample of the cells with Ziess Axiovert 200M inverted microscope equipped with and AxiocamMRC colored camera and LEP motorized stage controller. Excitation was by Cy5 channel and emission filter was 695/655 nm. (Carl Zeiss Microscopy, LLC. Thornwood, N.Y.). Nine HA mice were divided into 3 groups: Naïve, FVIII and FVIII/OPLS. Total non-adherent cells recovered from above were divided equally between the three mice in each treatment group and were injected subcutaneously (sc) in the lower abdominal. 3 hr after injection the animals were sacrificed and the inguinal lymph nodes were removed and fixed in 10% paraformaldehyde for 24 hr. The lymph nodes were then imaged whole using a Bio-Rad ChemiDoc MP imaging system with an ImgeLab 4.0.1 software package (Bio-Rad Laboratories, Inc. Hercules, Calif.).
Immunogenic potential of BMDC exposed to FVIII and OPLS/FVIII: To ascertain the effects of OPLS on the ability of BMDC to initiate a robust immune response in vivo, we tested the immunogenic potential of BMDCs after ex vivo exposure to FVIII in the presence and absence of 50 mM OPLS. On day nine, BMDCs were treated with 5 μg FVIII or 5 μg/50 mM FVIII-OPLS for 24 hr. Then, media was removed and cells were washed twice with ice cold sterile PBS. About 5×105 cells/mouse were then injected sc into HA mice, in two groups, FVIII and FVIII-OPLS group. This was done once a week for 3 weeks followed by a 2-week washout period. At the end of the washout period animals were sacrificed and plasma was collected. Total anti-FVIII titer levels were measured by ELISA with the plate specific cutoff values calculated from plasma of naïve animals.
Effect of OPLS on in-vivo immunogenicity of FVIII: 21 HA mice between 6 and 8 weeks of age were divided into 3 groups. Group one received 1 μg FVIII SC in the abdominal region. Groups two and three received 1 μg FVIII with 0.4 mg OPLS/injection (group 2) or 5 mg OPLS/injection (group 3) sc in the abdominal region. This treatment was given on day 1, 8, 10 and 12. Three days after the last injection the animals were sacrificed. Blood collected and centrifuged at 7500 rpm for 5 min at 4° C. Plasma was then removed and stored at −80° C. until analysis.
Statistics: Non-parametric one-way ANOVA with Tukey's post hoc analysis was used for the analysis of titer values where appropriate. Student t-test was used for comparing mean intensity of fluorescence (MIF) for flow cytomerty data. Non-parametric one way ANOVA with Kruskal-Wallis post hoc analysis was used for the analysis of titers values where appropriate.
Results: OPLS induces hypo-responsiveness to FVIII re-challenge in HA mice: In order to investigate whether OPLS is the critical structural component responsible for induction of tolerance, a re-challenge study was conducted. During the pre-treatment stage both OPLS and Dexamethasone reduced anti-FVIII antibody response as expected. Upon re-challenge with FVIII, animals pre-treated with FVIII, and FVIII-Dexamethasone showed a robust anti-FVIII antibody response (25438±5275 and 33694±7598 inverse dilution factor respectively, mean±SEM) as compared to animal pre-treated with FVIII-OPLS (14712±1853 inverse dilution factor mean±SEM,
OPLS mediated changes to BMDC maturation in vitro: The mechanism of antigen processing and presentation involves the following sequential steps; antigen uptake by dendritic cells is the first step in mounting an effective immune response. This is followed by phenotypic maturation, cytokine production and migration to afferent lymphatic for T-cell presentation. To determine whether OPLS interferes with antigen processing and presentation by DC, we monitored its effects on BMDC phenotypic maturation, cytokine secretion and migratory potential.
OPLS reduced the expression of co-stimulatory marker CD40 in BMDCs in vitro: Exposure of BMDC to FVIII resulted in an increase in CD40 expression. In the presence of 50 mM OPLS, the number of CD40 expressing BMDCs dropped from an average of 47.8% in FVIII treated cells to 26.5% in cells treated with FVIII-OPLS (n=3, p<0.05
MHCII expression levels were not affected by exposure of BMDC to OPLS as seen in
OPLS induces a dose dependent decrease in pro-inflammatory cytokines and an increase in regulatory cytokine production by BMDCs: DC exposed to FVIII in the presence of OPLS showed a dose dependent decrease in the pro-inflammatory cytokines TNF-α and IL-12p70 (n=4). At 50 mM a total shut down of IL-12p70 cytokine production and a substantial reduction in TNF-α (
OPLS effects on migration potential of BMDCs: Next step in antigen uptake and processing involved migration of DC from tissue to afferent lymphatics. Tolerogenic DC are expected to have enhanced migration to the afferent lymphatic. We followed the migration of BMDC exposed ex vivo to FVIII or FVIII/OPLS in host HA mice by incorporating the fluorescent membrane label DilC18(5). This dye only fluoresces in the hydrophobic environment of the cell membrane, any detected fluorescence will be attributed to DilC(18)5/BMDC complex and not the free dye.
OPLS curtails the immunological potential of BMDC exposed ex vivo to FVIII in HA mice: In this study we investigated the effect of OPLS on BMDC's ability to initiate an immune response by monitoring antibody production in host HA mice after reintroduction of ex vivo treated BMDC. Our study shows statistically higher anti-FVIII titers in mice injected with BMDC treated ex-vivo with FVIII compared to those injected with FVIII/OPLS treated BMDC (1894±667 vs. 81±15.6 arbitrary titer units respectively) (mean±SEM p=0.03
Effect of OPLS on in-vivo immunogenicity of FVIII
This data clearly shows an in vivo dose dependent response to OPLS. In the present study OPLS was not complexed to FVIII. This allows for sequential administration by which OPLS is administered first to prime the immune system then the biologic is administered second.
Tolerance is induced by tolerogenic DCs. Tolerogenic dendritic cells are thought to be generated in vivo in response to an apoptotic event. They are phenotypically distinct from tissue resident immature DCs. They are characterized by increase in regulatory cytokine production and lower pro-inflammatory cytokine secretion. They show some degree of maturation as well as loss of adhesion molecules. The latter is required for migration out of the tissue into the afferent lymphatics. In the afferent lymphatics, these DCs can present antigens to T-cells in the absence of co-stimulatory markers resulting in T-cell non-responsiveness. Our data (
Both immunogenic and tolerogenic DCs can migrate to the afferent lymphatics. In
Data presented in this disclosure show that the observed reduction in anti-FVIII titers in HA mice treated with OPLS/FVIII is the result of immunoregulation by OPLS and not immunosuppression. Furthermore, our data showed that, unlike Dexamethasone pretreated animals, OPLS pretreated animals were hyporesponsive to re-challenge with free FVIII. This not only indicates that OPLS is an immunemodulator but also indicates that the hyporesponsiveness to FVIII was antigen specific. This antigen specificity necessitates processing and presentation of FVIII by DCs in the context of lower CD40 expression accompanied by increased TGF-β production and reduced TNF-α and IL-12p70 production by DC in response to OPLS. This is further supported by our flow cytometry data showing no change in MHCII expression in response to OPLS. This mimics the properties of tolerogenic DCs. This is accompanied by an increase in the migratory potential of DC in response to OPLS and a curtailed immunogenic potential of DCs. Taken together our data indicates that OPLS has the critical structural properties required to alter the maturation of DC, leading to generation of DCs with tolerogenic properties.
The tolerogenic properties of OPLS can be utilized in a reverse vaccination strategy in which administration of low dose therapeutic protein with OPLS will desensitize the immune system of the patient to subsequent administration of the protein effectively reducing the immunogenicity of the therapeutic protein. We observed that this approach can be used to induce tolerance and reduced immunogenicity towards Acid Alpha-Glucosidase (GAA) in murine pompe disease models suggesting that this approach is generalizable. This reverse vaccination strategy with OPLS offers advantages over concomitant immune suppressive therapy such as dexamethasone, by offering antigen specific tolerance as opposed to general immune suppression.
This example describes an OPLS-FVIII sequential administration study.
To determine if OPLS can exert its immunomodulatory effects on the immune response to FVIII when the two are administered in separate injections temporally spaced apart, at the same injection site the following experiments were carried out. Hemophilia A mice (n=6 animals/group) were immunized with 4 weekly sc injections as follows. The control group of animals received one injection of sterile TRIS buffer (100 μL volume) then, after a 5 minute wait, they received a second injection of 1 μg FVIII in sterile TRIS (100 μL volume) at the same injection site. The second group of mice received one injection of 5 mg OPLS (100 μL volume) then, after a 5 minute wait, they received a second injection of 1 μg FVIII in sterile TRIS (100 μL volume) at the same injection site. After a two-week rest period (week 6) all of the mice were then rechallenged with 4 weekly intravenous injections of 1 μg of FVIII in 100 μL sterile TRIS (one single injection). Mice were sacrificed after two weeks and a terminal blood sample was collected using cardiac puncture. Analysis was done by ELISA for total titers. The results are shown in
While the invention has been described through specific embodiments, routine modifications will be apparent to those skilled in the art. Such modifications are intended to be within the scope of this disclosure.
This application is a continuation-in-part of U.S. application Ser. No. 15/024,071, filed on Mar. 23, 2016, which is a national stage application of international application PCT/US2014/057234, filed on Sep. 24, 2014, which claims priority to U.S. Provisional Application No. 61/881,857, filed on Sep. 24, 2013, the disclosures of which are incorporated herein by reference.
This invention was made with government support under contract HL-70227 awarded by the National Institutes of Health. The government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 61881857 | Sep 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15024071 | Mar 2016 | US |
| Child | 17443562 | US |
