the ratio of lactide to glycolide is from about 100:1 to 1:100 weight percent;
	the inherent viscosity of PLGA polymers used in the microspheres is about 0.1 to 1.2 dL/g;
	the median diameter of the microspheres is from about 20 to 100 μm; and
	the antigen is released from the microspheres in a triphasic pattern, wherein about 0.5 to 95% of the antigen is released in an initial burst, about 0 to 50% is released over a period of about 1 to 180 days, and the remaining antigen is released in a second burst after about 1 to 180 days.

Another aspect of the invention is a composition for use as a vaccine comprising antigen encapsulated in PLGA microspheres, and soluble antigen.

Another aspect of the invention is a composition for use as a vaccine comprising about one to 100 antigens encapsulated in a mixture of about two to 50 PLGA microsphere populations, wherein
 

	the ratio of lactide to glycolide is from about 100:1 to 1:100 weight percent;
	the inherent viscosity of PLGA polymers used in the microspheres is about 0.1 to 1.2 dL/g;
	the median diameter of the microspheres is from about 20 to 100 μm; and
	the antigen is released from the microspheres in a triphasic pattern, wherein about 0.5 to 95% of the antigen is released in an initial burst, about 0 to 50% is released over a period of about 1 to 180 days, and the remaining antigen is released in a second burst in one microsphere: population after about 1 to 30 days, in a second microsphere population after about 30 to 90 days, and in additional microsphere populations after about 90 to 180 days.

Another aspect of the invention is a method for encapsulating antigen in microspheres, comprising
 

	(a) dissolving PLGA polymer in an organic solvent to produce a solution;
	(b) adding antigen to the solution of (a) to produce a PLGA-antigen mixture comprising a first emulsion;
	(c) adding the mixture of step (b) to an emulsification bath to produce microspheres comprising a second emulsion; and
	(d) hardening the microspheres of step (b) to produce hardened microspheres comprising encapsulated antigen.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

General Methods

In general, microencapsulation of an antigen or adjuvant is performed according to the protocol briefly outlined in FIG. 3. In summary, PLGA of the desired ratio of lactide to glycolide (about 100:0 to 0:100, more preferably, about 65:35 to 35:65, most preferably about 50:50 weight percent) and inherent viscosity (generally about 0.1 to 1.2 dL/g, preferably about 0.2 to 0.8 dL/g) is first dissolved in an organic solvent such as methylene chloride, or ethyl acetate with or without benzyl alcohol or acetone to the desired concentration (generally about 0.05 to 1.0 g/mL, preferably about 0.3 to 0.6 g/mL). A concentrated antigen or adjuvant solution (for example, typically at least 0.1 mg/mL for polypeptides, preferably greater than about 100 mg/mL, depending, for example, on the type of polypeptide and the desired core loading) is then suitably injected (such as with a 25 gauge needle) into the polymer solution while homogenizing at about 15,000 to 25,000 rpm. Dry antigen or adjuvant can be used in place of aqueous antigen or adjuvant. After homogenization (generally about 0.5 to 5 minutes, more preferably for 1 minute), the emulsion is added to the reaction kettle (emulsification bath) or static mixer (not shown) to form a second emulsion. The emulsification bath is typically a polyvinyl alcohol solution, optionally including ethyl acetate. The reaction kettle is mixed at high speed (generally about 1700 to 2500 rpm) to generate small microspheres (about 20 to 100 μm median diameter). The second emulsion is transferred to a hardening bath after a sufficient period of time, generally about 0.5 to 10 minutes, preferably about 1 minute, and allowed to gently mix for a suitable time, generally about 1 to 24 hours, preferably about 1 hour. When hardening is complete, the microspheres are prefiltered (such as with a 150 μm mesh), concentrated and diafiltered. Diafiltering is suitably accomplished in an Amicon stirred cell (2500 mL), preferably with about a 16 or 20 μm filter. The microspheres are washed, typically with about 1 to 100 L, preferably about 15 L of prefiltered water and typically with about 1 to 100 L, more preferably 15 L of 0.1% Tween® 20. The final microspheres are removed from the filter and resuspended in water and filled in vials, preferably at about 500 μL/vial in 3 cc vials. The microspheres can then be dried. Drying includes such methods as lyophilization, vacuum drying, and fluidized bed drying.

Three other exemplary methods can be employed to produce microspheres. The first method utilizes a solvent evaporation technique. A solid or liquid active agent is added to an organic solvent containing the polymer. The active agent is then emulsified in the organic solvent. This emulsion is then sprayed onto a surface to create microspheres and the residual organic solvent is removed under vacuum. The second method involves a phase-separation process, often referred to as coacervation. A first emulsion of aqueous or solid active agent dispersed in organic solvent containing the polymer is added to a solution of non-solvent, usually silicone oil. By employing solvents that do not dissolve the polymer (non-solvents) but extract the organic solvent used to dissolve the polymer (e.g. methylene chloride or ethyl acetate), the polymer then precipitates out of solution and will form microspheres if the process occurs while mixing. The third method utilizes a coating technique. A first emulsion comprising the active agent dispersed in a organic solvent with the polymer is processed through an air-suspension coater apparatus resulting in the final microspheres.

When antigen and adjuvant are to be administered from within the same microspheres, a solution containing both antigen and adjuvant or solutions containing antigen and adjuvant separately can be added to the polymer solution. Similarly, soluble antigen and dry adjuvant, dry antigen and soluble adjuvant, or dry antigen and dry adjuvant, can be used. The microspheres of the instant invention are preferably formed by a water-in-oil-in-water emulsion process.

In general, both aqueous formulations and dry polypeptide antigens or adjuvants can be admixed with an excipient to provide a stabilizing effect before treatment with an organic solvent such as methylene chloride. An aqueous formulation of a polypeptide can be a polypeptide in suspension or in solution. Typically an aqueous formulation of the excipient will be added to an aqueous formulation of the polypeptide, although a dry excipient can be added, and vice-versa. An aqueous formulation of a polypeptide and an excipient can be also dried by lyophilization or other means. Such dried formulations can be reconstituted into aqueous formulations before treatment with an organic solvent.

The excipient used to stabilize a polypeptide antigen of interest will typically be a polyol of a molecular weight less than about 70,000 kD. Examples of polyols that can be used include trehalose (copending U.S. Ser. No. 08/021,421 filed Feb. 23, 1993), mannitol, and polyethylene glycol (PEG). Typically, the mass ratio of trehalose to polypeptide will be about 1000:1 to 1:1000, preferably about 100:1 to 1:100, more preferably about 1:1 to 1:10, most preferably about 1:3 to 1:4. Typical mass ratios of mannitol to polypeptide will be about 100:1 to 1:100, preferably about 1:1 to 1:10, more preferably about 1:1 to 1:2. Typically, the mass ratio of PEG to polypeptide will be about 100:1 to 1:100, preferably about 1:1 to 1:10. Preferred ratios are chosen on the basis of an excipient concentration which allows maximum solubility of polypeptide with minimum denaturation of the polypeptide.

The formulations of the instant invention can contain a preservative, a buffer or buffers, multiple excipients, such as polyethylene glycol (PEG) in addition to trehalose or mannitol, or a nonionic surfactant such as Tween® surfactant. Non-ionic surfactants include polysorbates, such as polysorbate 20 or 80, and the poloxamers, such as poloxamer 184 or 188, Pluronic® polyols, and other ethylene oxide/propylene oxide block copolymers, etc. Amounts effective to provide a stable, aqueous formulation will be used, usually in the range of from about 0.1% (w/v) to about 30%(w/v).

The pH of the formulations of this invention is generally about 5 to 8, preferably about 6.5 to 7.5. Suitable buffers to achieve this pH include, for example, phosphate, Tris, citrate, succinate, acetate, or histidine buffers, depending on the pH desired. Preferably, the buffer is in the range of about 2 mM to about 100 mM.

Examples of suitable preservatives for the formulation include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, benzalconium chloride, and benzethonium chloride. Preferred preservatives include about 0.2 to 0.4%(w/v) phenol and about 0.7 to 1%(w/v) benzyl alcohol, although the type of preservative and the concentration range are not critical.

In general, the formulations of the subject invention can contain other components in amounts not detracting from the preparation of stable forms and in amounts suitable for effective, safe pharmaceutical administration. For example, other pharmaceutically acceptable excipients well known to those skilled in the art can form a part of the subject compositions. These include, for example, salts, various bulking agents, additional buffering agents, chelating A agents, antioxidants, cosolvents and the like; specific examples of these include tris-(hydroxymethyl)aminomethane salts ("Tris buffer"), and disodium edetate.

Antigens of interest useful in the instant invention include, for example, HIV antigens such as gp120, gp160, gag, pol, Nef, Tat, and Rev; malaria antigens such as CS proteins and sporozoite 2; hepatitis B antigens, including Pre-S1, Pre-S2, HBcAg, HBsAg, and HBeAg; influenza antigens such as HA, NP, and NA; hepatitis A surface antigens; Herpes virus antigens such as EBV gp340, EBV gp85, HSV gB, HSV gD, HSV gH, and HSV early protein product; cytomegalovirus antigens such as gB, gH, and IE protein gP72; respiratory syncytial virus antigens such as F protein, G protein, and N protein. Polypeptides or protein fragments defining immune epitopes, and amino acid variants of proteins, polypeptides, or peptides, can be used in place of full length proteins. Polypeptides and peptides can also be conjugated to haptens.

Multivalent vaccines can be formulated with mixtures of antigens, either first mixed together and then encapsulated, or first encapsulated and then mixed together in a formulation for administration to a patient. Such mixtures can consist of two to upwards of about 100 antigens. The antigens can represent antigenic determinants from the same organism, such as gp120 polypeptides isolated from geographically different strains of HIV, or from different organisms, such as diphtheria-pertussis-tetanus vaccine.

Exemplary adjuvants of interest include saponins such as QS21, muramyl dipeptide, muramyl tripeptide, and compounds having a muramyl peptide core, mycobacterial extracts, aluminum hydroxide, proteins such as gamma interferon and tumor necrosis factor, phosphatidyl choline, squalene, Pluronic® polyols, and Freund's adjuvant (a mineral oil emulsion) (see the Background of this application for specific references). Although antigen is desirably administered with an adjuvant, in situations where the initial inoculation is delivered with an adjuvant, boosts with antigen may not require adjuvant. PLGA or other polymers can also serve as adjuvants.

Typically, an antigen of interest will be formulated in PLGA microspheres to provide a desired period of time between the first and second bursts of antigen and to provide a desired amount of antigen in each burst. The amount of antigen in the initial burst can be augmented by soluble antigen in the formulation. Preferably, an adjuvant is microencapsulated, although soluble adjuvant can also be administered to the patient.

The microspheres, soluble antigen, and/or adjuvant are placed into pharmaceutically acceptable, sterile, isotonic formulations together with any required cofactors, and optionally are administered by standard means well known in the field. Microsphere formulations are typically stored as a dry powder.

The amount of antigen delivered to the patient to be used in therapy will be formulated and dosages established in a fashion consistent with good medical practice taking into account the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Similarly, the dose of the vaccine administered will be dependent upon the properties of the antigen employed, e.g. its binding activity and in vivo plasma half-life, the concentration of the antigen in the formulation, the administration route, the site and rate of dosage, the clinical tolerance of the patient involved, the pathological condition afflicting the patient and the like, as is well within the skill of the physician. Generally, doses of from about 0.1 to 1000 μg per patient per administration are preferred. Different dosages can be utilized during a series of sequential inoculations; the practitioner can administer an initial inoculation and then boost with relatively smaller doses of vaccine.

It is envisioned that injections (intramuscular or subcutaneous) will be the primary route for therapeutic administration of the vaccines of this invention, although intravenous delivery, or delivery through catheter or other surgical tubing is also used. Alternative routes include suspensions, tablets, capsules and the like for oral administration, commercially available nebulizers for liquid formulations, and inhalation of lyophilized or aerosolized microcapsules, and suppositories for rectal or vaginal administration. Liquid formulations can be utilized after reconstitution from powder formulations.

The adequacy of the vaccination parameters chosen, e.g. dose, schedule, adjuvant choice and the like, can be determined by taking aliquots of serum from the patient and assaying antibody titers during the course of the immunization program. Alternatively, the presence of T cells or other cells of the immune system can be monitored by conventional methods. In addition, the clinical condition of the patient can be monitored for the desired effect, e.g. anti-infective effect. If inadequate vaccination is achieved then the patient can be boosted with further vaccinations and the vaccination parameters can be modified in a fashion expected to potentiate the immune response, e.g. increase the amount of antigen and/or adjuvant, complex the antigen with a carrier or conjugate it to an immunogenic protein, or vary the route of administration.

The microspheres of the instant invention are designed to release their contents in a triphasic manner consisting of an initial burst, a slow release, and a second burst. The degradation rate for the microspheres of the invention is determined in part by the ratio of lactide to glycolide in the polymer and the molecular weight of the polymer. Polymers of different molecular weights (or inherent viscosities) can be mixed to yield a desired degradation profile. Furthermore, populations of microspheres designed to have the second burst occur at different times can be mixed together to provide multiple challenges with the antigen and/or adjuvant at desired intervals. Similarly, mixtures of antigens and/or adjuvants can be provided either together in the same microspheres or as mixtures of microspheres to provide multivalent or combination vaccines. Thus, for example, rather than receive three immunizations with traditional DTP (diphtheria, tetanus, and pertussis) vaccine at 2, 4, and 6 months, a single microencapsulated vaccine can be provided with microspheres that provide second bursts at 2, 4, and 6 months.

The microspheres of the instant invention can be prepared in any desired size, ranging from about 0.1 to upwards of about 100 μm in diameter, by varying process parameters such as stir speed, volume of solvent used in the second emulsion step, temperature, concentration of PLGA, and inherent viscosity of the PLGA polymers. The relationship of these parameters is discussed in detail below. The microspheres used for the gp120 vaccine of the instant invention are of a median diameter of generally about 20 to 100 μm, preferably about 20 to 50 μm, more preferably about 30 μm.

The HIV vaccine of the instant invention will typically comprise three populations of PLGA microspheres: microspheres containing 1-5% w/w gp120, generated with a 50:50 mass ratio of PLGA polymers having inherent viscosities of 0.2 and 0.75 dL/g, wherein the ratio of lactide to glycolide is 50:50 (preparation 1); microspheres containing 1-8% w/w QS21, generated with a 50:50 mass ratio of PLGA polymers having inherent viscosities of 0.2 and 0.75 dL/g, wherein the ratio of lactide to glycolide is 50:50 (preparation 2); and microspheres containing 1-5% gp120, generated with PLGA polymers having inherent viscosities of 0.7 to 1.2 dL/g, wherein the ratio of lactide to glycolide is 50:50 (preparation 3). Soluble gp120 will also be provided in the vaccine at a concentration of about 300 to 1000 μg/dose, more preferably, 300 to 600 μg/dose. Soluble QS21 will also be provided in the vaccine at a concentration of about 50 to 200 μg/dose, more preferably, 50 to 100 μg/dose. This vaccine formulation will result in an initial exposure by the patient to about 300 to 600 μg gp120 and 50 to 100 μg QS21 at the time of parenteral inoculation, a slow release of less than 50 μg gp120 and less than 10 μg QS21 over about 120 to 180 days, a challenge ("autoboost") with about 300 to 600 μg gp120 and 50 to 100 μg QS21 at about 30 to 60 days resulting from the second burst from microsphere preparations 1 and 2; and another autoboost with about 300 to 600 μg gp120 at about 30 to 60 days resulting from the second burst of microsphere preparation 3.

Claim 1 of 13 Claims

1. A composition comprising a homogeneous population of polylactide or poly (lactide-co-glycolide) (PLGA) polymer microspheres encapsulating an antigen, wherein said homogeneous population is produced from an emulsion comprising aqueous antigen and a polylactide or PLGA polymer, and

(a) the polymer has a ratio of lactide to glycolide of about 100:0 to 50:50 weight percent;

(b) the polymer has an inherent viscosity of about 0.1 to 1.2 dL/g;

(c) the microspheres in said homogeneous population have a median diameter of about 20 to 100 μm; and

(d) the microspheres in said homogeneous population have an in vitro antigen release profile characterized by three phases: a first antigen burst phase, wherein about 0.5 to 30 percent of the antigen is released from the microspheres over a period of about three days after-suspension of the microspheres in a release medium; a second slow release phase after the first phase, extending from about the fourth to at least about the thirtieth day after suspension, wherein the daily release of antigen from the microspheres is less than in the first antigen burst phase or a third antigen burst phase; and the third antigen burst phase after the second phase, wherein antigen is released from the microspheres at a rate of greater than 10 percent per week, during a period of from about seven to about 30 days, starting from about 30 to about 180 days after suspension.
 

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