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WO1998014170A1 - Vaccin idiotypique associe a un transporteur lipidique et destine aux troubles des cellules b - Google Patents

Vaccin idiotypique associe a un transporteur lipidique et destine aux troubles des cellules b Download PDF

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Publication number
WO1998014170A1
WO1998014170A1 PCT/US1997/017513 US9717513W WO9814170A1 WO 1998014170 A1 WO1998014170 A1 WO 1998014170A1 US 9717513 W US9717513 W US 9717513W WO 9814170 A1 WO9814170 A1 WO 9814170A1
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WIPO (PCT)
Prior art keywords
idiotype
lipid
based carrier
binding moiety
tumor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1997/017513
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English (en)
Inventor
David B. Agus
Andrew S. Janoff
Andrew D. Zelenetz
Imran Ahmad
Eric Mayhew
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Elan Pharmaceuticals LLC
Memorial Sloan Kettering Cancer Center
Original Assignee
Liposome Co Inc
Memorial Sloan Kettering Cancer Center
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Liposome Co Inc, Memorial Sloan Kettering Cancer Center filed Critical Liposome Co Inc
Priority to AU45071/97A priority Critical patent/AU4507197A/en
Publication of WO1998014170A1 publication Critical patent/WO1998014170A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/804—Blood cells [leukemia, lymphoma]

Definitions

  • This application relates to a method and composition for treatment of B cell non-Hodgkin's Lymphoma and other B cell disorders such as chronic lymphocytic leukemia, multiple myeloma and Walderstrom's macroglobinemia.
  • B cell non-Hodgkin's Lymphoma is characterized by a clonal proliferation of malignant B cells.
  • One characteristic of most cases of B cell NHL is the expression of a tumor-specific antigen, immunoglobulin, on the cell surface.
  • This surface antigen termed the tumor idiotype, is composed of variable regions of an immunoglobulin molecule which contain unique determinants, called idiotypes, which themselves can be recognized as antigen.
  • a tumor idiotype derived from B cells from the patient bound to the surface of the lipid-based carrier (b) a tumor idiotype derived from B cells from the patient bound to the surface of the lipid-based carrier.
  • the idiotype may be absorbed directly to the surface of the lipid-based carrier or bound via an idiotype-binding moiety which is bound to the surface of the lipid-based carrier.
  • this moiety is preferably protein G or protein A or a combination thereof.
  • the idiotype can be captured directly from a tumor lysate or other patient- derived material, and does not have to be prepared from a hybridoma and the use of anti- idiotype murine-derived antibodies is not necessary.
  • the invention thus provides a rapid and patient-specific method for isolation of idiotype and preparation of compositions which can be used to promote an immune response against the patient's tumor.
  • the invention makes it possible to prepare such a composition in the accordance with the invention and to use it to initiate the development of an immune response within a period of hours or days, rather than a period of months.
  • the lipid-based carrier of this invention is selected from the group consisting of micelles, lipoproteins, non-liposomal lipidic complexes and liposomes, e.g., a unilamellar liposome such as a small or large unilamellar liposome or a multilamellar liposome.
  • the lipid- based carrier is preferably a liposome, and may be composed of such lipids as to be an effective adjuvant for the tumor vaccine.
  • the immunotherapeutic composition of the invention can also include additional adjuvants or immunotherapeutic agents incorporated within the liposome.
  • the lipid-based carrier may contain granulocyte- macrophage colony stimulating factor (gmCSF) or cytokines such as interleukin-12 (LL-12) which enhance vaccine-induced immune response.
  • gmCSF granulocyte- macrophage colony stimulating factor
  • LL-12 interleukin-12
  • Fig. 1 shows a first embodiment of a method for making compositions according to the invention
  • Fig. 2 shows a second embodiment of a method for making compositions according to the invention
  • Fig. 3 shows a third embodiment of a method for making compositions according to the invention.
  • tumor idiotype from tumor lysates or other patient-derived materials is bound to the surface of a lipid-based carrier molecule either directly or through an idiotype-binding moiety which is bound to the lipid-based carrier to form a vaccine composition.
  • Figs. 1-3 show three different methods for making such a composition schematically.
  • a tumor lysate 10 or other patient-derived material containing tumor idiotype is purified to isolate the idiotype, I.
  • This can be accomplished rapidly using the methods described in Example 3 and 4 hereof. Basically, this method uses a protein G affinity column or other idiotype-binding solid support to capture idiotype from the tumor lysate and then recovers the separated idiotype by elution. Unlike other methods, this method does not require detergent and can be rapidly accomplished.
  • the purified tumor idiotype, I is then combined with a lipid-based carrier, LIP, and absorbed on the surface thereof to form an idiotype-lipid-based carrier complex, LIP-I.
  • Figs 2 and 3 show alternative approaches for making vaccine compositions in accordance with the invention using an intermediate idiotype-binding moiety to link the idiotype to the lipid-based carrier.
  • the idiotype- binding moiety is first associated with the lipid-based carrier, LIP, to form the intermediate composition, LIP-LB.
  • the LIP-LB bond may be covalent or non-covalent.
  • the LIP-LB is then combined with tumor lysate 10 or to other patient-derived material containing tumor idiotype, with the result that idiotype I is captured by the LIP-IB intermediate to produce the composition LLP-LB-I in accordance with the invention.
  • LIP-LB can also be combined with purified tumor idiotype to form the LLP-LB-I composition of the invention.
  • the idiotype-binding moiety IB is added to tumor lysate 10 or other patient-derived material containing tumor idiotype prior to association with the lipid-based carrier, thus forming the intermediate composition IB-I.
  • This composition is then captured by binding to a lipid-based carrier to form a LLP-LB-I composition according to the invention.
  • Idiotype-lipid-based carrier compositions made by any of these methods are then used as a tumor-specific therapeutic vaccine for the patient from which the tumor idiotype was derived.
  • the lipid-based carrier needs to absorb or bind to the idiotype with a sufficiently high affinity to provide for rapid binding of idiotype to the lipid-based carrier complex in the first instance, and low release of idiotype from the lipid-based carrier complex during vaccine use.
  • this can be accomplished in some cases using the inherent protein absorption capabilities of the lipid-based carrier.
  • an idiotype-binding moiety is used.
  • Isolation of Idiotype Isolation of idiotype for use in the embodiment of the invention depicts in Fig.
  • the method utilizes a solid support having an idiotype-binding moiety affixed to the surface thereof to capture idiotype from a tumor lysate.
  • the solid support is then washed, to separate unbound materials, and the idiotype is eluted.
  • solid supports which can be used include affinity columns or solid beads, such as for example magnetic beads, having idiotype-binding moieties attached thereto.
  • Suitable idiotype-binding moieties include protein G, protein A and anti-idiotype antibodies which will specifically bind idiotype to separate idiotype from a tumor lysate or other patient derived sample.
  • lipid-based carriers covalently or non-covalently coupled to molecules which bind to IgM or IgG are suitable for use as idiotype-binding moieties in the present invention.
  • molecules include bacterial proteins which bind to immunoglobulins with high affinity.
  • Specific examples of such molecules are Group C Streptococcal protein G ("protein G") or its isolated immunoglobulin binding domain which bind to all isotypes of IgG with high affinity, and
  • Staphylococcal protein A or its isolated immunoglobulin binding domain which bind to the VH3 domain of the IgM molecule.
  • the VH3 domain is utilized in approximately 45 percent of B cells and 40 percent of B cell follicular lymphomas.
  • lipid-based carriers refers to structures primarily composed of one or more lipids, such as the amphipathic phospholipids, which can, but are not required to contain lipid layers and enclosed aqueous volume. Suitable carriers include, without limitation nonliposomal lipid complexes, lipoproteins, micelles and liposomes.
  • the preferred lipid-based carriers for use in the present invention are liposomes.
  • “Liposomes” are self-assembling structures comprising one or more lipid bilayers, each of which surrounds an aqueous compartment and comprises two opposing monolayers of amphipathic lipid molecules.
  • Amphipathic lipids comprise a polar (hydrophilic) headgroup region covalently linked to one or two non-polar (hydrophobic) acyl chains. Energetically unfavorable contacts between the hydrophobic acyl chains and the aqueous medium are generally believed to induce lipid molecules to rearrange such that the polar headgroups are oriented towards the aqueous medium while the acyl chains reorient towards the interior of the bilayer.
  • Liposomes useful in this invention can have a single lipid bilayer (unilamellar liposomes, "ULVs"), or multiple lipid bilayers (multilamellar liposomes, "MLVs").
  • UUVs unilamellar liposomes
  • MLVs multilamellar liposomes
  • Liposomes can be made by a variety of methods (for a review, see, for example, Deamer and Uster, "Liposome Preparation: Methods and Mechanisms", in Liposomes, ed. M. Ostro, Marcel Dekker, Inc. NY, pp. 27-51 (1983)). These methods include without limitation: Bangham's methods for making multilamellar liposomes (MLVs), J.
  • 5,008,050 can be used to size reduce liposomes, that is to produce liposomes having a predetermined mean size by forcing the liposomes, under pressure, through filter pores of a defined, selected size.
  • Tangential flow filtration can also be used to regularize the size of liposomes, that is, to produce liposomes having a population of liposomes having less size heterogeneity, and a more homogeneous, defined size distribution.
  • Liposome sizes can also be determined by a number of techniques, such as quasi-elastic light scattering, and with equipment, e.g., NICOMP® particle sizers, well within the possession of ordinarily skilled artisans.
  • the lipid-based carriers used in the present invention can be made using a variety of amphipathic lipids. These include, without limitation: phospholipids, such as phosphatidylcholines (PCS"), phosphatidylethanolamines (“PEs”), phosphatidylserines ("PS's”) and phosphatidylglycerols (“PGs”); sterols, such as cholesterol and alpha-tocopherol; and, glycolipids, e.g., galactolipids and glycosphingolipids. Ordinarily skilled artisans are well aware of the varied properties of each of these types of lipids.
  • PCS phosphatidylcholines
  • PEs phosphatidylethanolamines
  • PS's phosphatidylserines
  • PGs phosphatidylglycerols
  • glycolipids e.g., galactolipids and glycosphingolipids.
  • glycolipids e.g., galact
  • PCS have a neutral charge; as such, when incorporated into liposomes, PCS do not change the overall charge of the vesicles, and hence, their attraction/repulsion to cell surfaces.
  • PEs because of the nature of their headgroup, tend to adopt hexagonal-phase conformations instead of lamellar strictures, and hence, tend to destabilize bilayers into which they are incorporated.
  • the lipid-based carrier may include one or more materials selected to act as adjuvants to increase the immune response generated by the vaccine composition.
  • lipid-based carriers including liposomes, can be formulated organic acid-derivatized sterols such as cholesterol hemisuccinate. Such lipids, which are further described in US Patents Nos.
  • lipid-based carriers of the invention may be used as a part of, and even as the principal lipid component of the lipid-based carriers of the invention.
  • Another lipid adjuvant which can be used as an immuno-potentiator is Lipid A, the lipid fraction of endotoxin derived from Gram negative bacteria. See, US Patent No. 5,026,557, which is incorporated herein by reference.
  • the liposomes employed may be prepared to include one or more "bioactive agents" within the encapsulated volume or coupled to or incorporated in the liposome membrane.
  • Bioactive agents which may be associated with this invention's lipid-based carrier include, but are not limited to: antiviral agents such as acyclovir, zidovudine and the interferons; antibacterial agents such as aminoglycosides, cephalosporins and tetracyclines; antifungal agents such as polyene antibiotics, imidazoles and triazoles; antimetabolic agents such as folic acid, and purine and pyrimidine analogs; antineoplastic agents such as the anthracycline antibiotics and plant alkaloids; sterols such as cholesterol; carbohydrates, e.g., sugars and starches; amino acids, peptides, proteins such as cell receptor proteins, immunoglobulins, enzymes, hormones, neurotransmitters and glycoproteins; dyes; radiolabels such as radioisotopes and radioisotope-labeled compounds; radiopaque compounds; fluorescent compounds; mydriatic compounds; bronchodilators; local anesthetics
  • coupling of idiotype-binding moiety to lipid- based carriers can be performed before or after complexation of the idiotype-binding moiety with patient-derived idiotype.
  • This coupling may be either covalent or non-covalent.
  • Techniques for affixing proteins to the exterior of liposomes are known in the art. For example, Heath et.al., Biochim. Biophys. Acta, 640:66-81 (1981), which is incorporated herein by reference, describe the covalent attachment of immunoglobulins to liposomes containing glycosphingolipid. Leserman et. al. Liposome Technology, III, CRC Press, Inc., California, p.
  • Streptavidin derivatives of idiotype-binding moieties may be made by various methods known in the art, including expression of fusion proteins from recombinant DNA constructs encoding both the idiotype-binding moiety.
  • a sandwich can also be formed using biotin-containing lipid-based carriers (formed, e.g, using biotinylated phosphatidylethanolamine) and biotinylated idiotype-binding moiety linked by a bifunctional avidin molecule. See, Ahmad et al, Cancer Res. 52: 4817-4820 (1992); Ahmad et al., Cancer
  • the liposomes or other lipid-based carrier may be coupled with a molecule which binds to the idiotype with high affinity.
  • a molecule which binds to the idiotype with high affinity are protein A, protein G, or derivatives thereof such as truncated forms of the protein G molecule, hyperiodinated protein G truncated protein A, or hyperiodinated protein
  • liposomes or other lipid-based carrier coupled to protein A and protein G with each other or with other idiotype-binding proteins can also be used.
  • Covalent coupling of proteins such as protein A to liposomes or other lipid- based carrier can be accomplished using a using a heterobifunctional reagent such as N- hydroxy-succinimidyl 3-(2-pyridyldithio)propionate (SPDP, Pharmacia).
  • SPDP N- hydroxy-succinimidyl 3-(2-pyridyldithio)propionate
  • SPDP reacts with primary amino groups on proteins to produce a protein-dithiopyridine which in turn is activated by reduction to the thiol, for example with dithiothreitol.
  • Liposomes or other lipid- based carrier incorporating a dithiopyridine-modified lipid, such as dithiopyridine-phosphatidyl ethanolamine can be covalently coupled to the protein-dithiopyridine.
  • thiol groups of a protein modified with SPDP succinimidylacetylthioacetate (SAT A) or succinimidylacetylthio- proprionate (SATP) and similar bifunctional reagents can also be coupled directly to the maleimido groups of a maleimide-modified lipid incorporated in a liposome. See, U.S. Patent No. 5,399,331 which is incorporated herein by reference.
  • Non-covalent coupling of the proteins to liposomes or other lipid-based carrier can also be accomplished by coupling anti-idiotype-binding protein antibodies to the liposome surface. This can be accomplished, for example through a biotin/streptavidin interaction or other binding of an affinity binding pair.
  • an antibody against protein A or protein G could be incorporated in the lipid-based carrier for non-covalent attachment of the idiotype-binding moiety, provided that such non-covalent attachment does not block the site of interaction between the idiotype-binding moiety and the idiotype.
  • This indirect approach is particularly suitable if the idiotype is complexed to the idiotype-binding protein prior to coupling with the liposome or other lipid-based carrier.
  • composition of the invention is prepared by absorbing substantially purified tumor idiotype to the lipid-based carrier, or by combining an idiotype-binding moiety, either before or after coupling of the idiotype-binding moiety to a liposome or other lipid-based carrier, with a patient-derived sample in which B cell idiotype is accessible for binding.
  • a patient-derived sample in which B cell idiotype is accessible for binding.
  • NHL this may be a tumor lysate, while in the case of myeloma, idiotype may be found in patient serum. Rescued idiotype may also be used.
  • Tumor lysates when needed, can be prepared from one to several grams of tumor tissue which is disrupted to make the normally membrane bound idiotype accessible for binding.
  • One gram of tumor should yield approximately 10' 4 molecules of immunoglobulin (approximately 25 ug).
  • Disruption may be accomplished by sonication, mechanical homogenization, or chemical lysing agents such as detergents.
  • the vaccine can be produced from viably frozen single cell suspensions which are lysed to render the idiotype accessible.
  • idiotype-binding moiety When the idiotype-binding moiety has been previously coupled to a liposome or other lipid-based carrier, lysed tumor cells, serum or other source of patient-derived idiotype protein are combined with the idiotype-binding moiety-liposome and incubated to bind to the B cell idiotype creating idiotype-lipid-based carrier complexes. After incubation, the idiotype-lipid-based carrier complex is then purified from the patient-derived sample. This can, for example, be accomplished by precipitation in high calcium solutions.
  • idiotype-binding moiety When the idiotype-binding moiety has not been previously coupled to a liposome or other lipid-based carrier, lysed tumor cells, serum or other source of patient- derived idiotype protein are combined with the idiotype-binding moiety and incubated to bind to the B cell idiotype creating idiotype-idiotype-binding moiety complexes.
  • One way in which this could be carried out is by passing a patient-derived sample through an idiotype-binding moiety column, such as commercially available protein A columns, to capture idiotype from the sample, and then eluting the idiotype off with a concentrated protein A eluent.
  • the idiotype-binding moiety complex can be exchanged off of the column, resulting in the elution of a purified idiotype-idiotype-binding moiety complex.
  • This complex is then coupled to a liposome which is adapted to permit such coupling, for example through the incorporation of thiolated or maleimido-lipids, or by coupling of an anti-idiotype-binding moiety antibody.
  • the idiotype-idiotype-binding moiety complex is not prepurified, or if additional purification is needed after incubation, the idiotype-liposomal complex can be purified, for example by precipitation in high calcium solutions.
  • lipid-based carrier or idiotype-binding moiety Prior to combining the tumor idiotype (either in purified form or as part of a lysate) with the lipid-based carrier or idiotype-binding moiety, flow cytometry can be used to determine heavy chain isotypes and thus the appropriate type of idiotype-binding moiety to incorporate in the vaccine. Hyperiodination assays can also be employed to determine the isotype of the idiotype. Alternatively, liposomes or other lipid-based carrier including a mixture of idiotype-binding moieties may be employed in which case this step may be omitted.
  • composition of the invention can administered to the patient from whom the tumor idiotype is derived immediately upon preparation or it may be stored for future use.
  • the composition is preferably administered by intramuscular injection in an amount effective to stimulate a therapeutic immune response. It will be appreciated that this amount may vary with the specific formulation of the composition and in some cases with the patient, and that the determination of the appropriate amount to be administered is a routine matter well within the skill in the art.
  • the invention provides a timely tumor-specific vaccine in which the unique epitopes of a patient's own tumor cells are presented as antigen to elicit an anti-idiotypic immune response. Timeliness is important therapeutically, and the present invention permits treatment of NHL without relying upon a method which takes 4 to 6 months to complete production of a therapeutic product.
  • utilization of the liposome or the lipid-based carrier makes it possible to deliver cytokines locally to the vaccine microenvironment which can help to potentiate immunologic responses and facilitate the patient's response to the tumor vaccine.
  • liposomes or other lipid-based carrier themselves have adjuvant properties which can further potentiate the vaccine-induced immune response.
  • EXAMPLE 1 PREPARATION OF CHS LIPOSOME Multilamellar liposomes (MLVs) were prepared with the Tris salt of cholesterol hemisuccinate (CHS), as set forth in US Patent No. 4,721,612 at col. 1 1, line 45 - col. 12, line 3 (Example 6.1 - 6.1.1). These liposomes were then extruded, in succession, through filters of pore sizes: 1000 nm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, and 100 nm, so as to obtain liposomes of between 100 nm and 200 nm in size
  • EXAMPLE 2 PREPARATION OF TUMOR LYSATE Murine B cell lymphoma A20 cells (American Type Culture Collection) were maintained in RPMI-1640 medium supplemented with 10% FBS. Cells were prepared for injection by rinsing cells in T flasks with PBS, at room temperature, transferring to a centrifuge tube and centrifuging for 300 g for 10 minutes. The cell pellets were washed twice in PBS, then the cells were gently resuspended in PBS for injection into mice. The cells were counted and viability of cells (> 90%) was checked by trypan blue exclusion.
  • Balb/C mice (5 weeks of age) were inoculated with A20 cells s.c, and then sacrificed after approximately 30 days using CO 2 . Skin was removed from s.c. tumor and a scalpel was used to slice into the solid mass. Tumor material was recovered by scraping with a scalpel and place into pre-weighed glass scintillation vials. 5mL of cold PBS was added to each vial, and the samples were placed on ice, and then homogenized with a tissue homogenizer (high speed and about 10 strokes). The supernatant was removed and saved. An additional 5mL cold PBS to each vial with the remaining sample and the sample was homogenized again at high speed for 10 strokes.
  • the supernatant was then returned to the vial and the contents homogenized again at high speed for 5 strokes.
  • the entire sample is then transferred into a 15mL conical tube and sonicated in cold water for 1 Omin. Centrifugation at 3500rpm for 30min at 4°C was then performed to separate the supernatant from a pellet. The pellet was discarded. The supernatant was then centrifuged at 10,000rpm for 1 Omin, separated from the resulting pellet and diluted 1 : 1 with basic buffer (pH ⁇ 8-9)
  • Binding Buffer supplied with the column, prior to running the sample through the column.
  • the column was then washed column with lOmL of Binding Buffer, after which the idiotype is eluted with 6 X lmL portions of Elution Buffer (acidic buffer. ⁇ pH 3 provided with the column).
  • Elution Buffer acidic buffer. ⁇ pH 3 provided with the column.
  • Other elution buffers which separate idiotpye from the column may also be employed. Fractions are monitored at 280nm and those fractions with elevated readings
  • the combined fractions are passed through a desalting column (Pierce, 5 ml), that has been previously equilibrated with lOmL of distilled water.
  • the column is eluted with 10 X 1 mL portions of distilled water to recover the idiotype. Again, the eluted fractions are monitored at 280nm and those fractions with elevated readings are combined. The amount of idiotype recovered can be quantitated based on the absorption or using ELISA techniques.
  • EXAMPLE 4 PURIFICATION OF TUMOR IDIOTYPE Idiotype is purified from a tumor lysate prepared as in Example 2 using Goat anti Mouse Immunoglobulin Dyna-beads.
  • the Goat anti Mouse Dyna-beads are washed twice with 0. IN NaOH, and finally washed with neutral buffer such as PBS.
  • Homogenized tumor sample is then added to the beads, and incubated with mixing for 2hrs. at 4°C.
  • the beads are separated from the supernatant by centrifugation or other appropriate means (for example using a magnet if magnetic beads are employed), and washed twice with a neutral buffer such as PBS.
  • idiotype is eluted from the separated beads by adding 5mL 0.5M glacial acetic acid. The supernatant containing the idiotype is then separated form the beads, and the pH of the sample is adjusted with 0. IN NaOH to about 6.5 to 7. The amount of idiotype recovered can be quantitated based on UV absorption or using ELISA techniques.
  • mice (20 g) were injected IM with CHS alone, CHS mixed with 25 meg of purified A20 murine monoclonal IgG, 25 meg purified A20 murine monoclonal IgG alone or PBS control.
  • EXAMPLE 6 Experiments are ongoing to evaluate the ability of the vaccine to prevent tumor engraftment in the murine host. These experiments are utilizing the murine A20 lymphoma cell line which forms subcutaneous tumors in syngeneic Balb/c mice. Idiotype was purified from A20 tumors harvested from Balb/c mice by the procedures described in Example 4 above. Balb/c female mice (20 g) were administered IP CHS alone, CHS mixed with 25 meg of purified A20-derived tumor idiotype, 25 meg purified A20-derived tumor idiotype alone or PBS control. The animals were injected IP with the vaccine on day 0 and day 21 and tumor is introduced ip on day 28. Vaccine-treated mice are expected to exhibit increased survival time relative controls or tumor rejection with proper dosage scheduling.

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Abstract

Conformément à la présente invention, on prépare un vaccin spécifique à un patient et destiné à traiter des lymphomes qui ne sont pas du type de Hodgkin et autres troubles des cellules B, à partir d'un transporteur lipidique à la surface duquel on lie un idiotype tumoral dérivé de cellules B prélevées chez ledit patient. L'iodiotype peut être absorbé directement à la surface du transporteur lipidique ou lié par l'intermédiaire d'une fraction de liaison à un idiotype qui est liée à la surface du transporteur lipidique. Lorsqu'on utilise une fraction de liaison à un idiotype, ladite fraction est de préférence une protéine G ou une protéine A ou une combinaison de ces protéines. Il n'est pas nécessaire de préparer l'idiotype à partir d'un hybridome et il n'est pas nécessaire d'utiliser des anticorps anti-idiotype dérivés de la souris. Au contraire, il est possible d'obtenir l'idiotype directement à partir d'un lysat de tumeur ou d'un autre échantillon prélevé sur le patient. Il est ainsi possible de préparer une composition conforme à l'invention et de l'utiliser pour faire naître une réaction immunitaire en quelques heures ou quelques jours, plutôt que sur une période de plusieurs mois. Le transporteur lipidique est de préférence un liposome et il peut être composé de lipides propres à en faire un adjuvant efficace du vaccin anti-tumeur. La composition immunothérapeutique de la présente invention peut également comporter des d'autres adjuvants ou des agents immunothérapeutiques incorporés au liposome. Le transporteur lipidique peut, par exemple, contenir un facteur stimulant les colonies de granulocytes-macrophages (gmCSF) ou des cytokines telles que l'interleukine-12 (IL-12) qui renforcent la réaction immunitaire induite par le vaccin.
PCT/US1997/017513 1996-09-30 1997-09-30 Vaccin idiotypique associe a un transporteur lipidique et destine aux troubles des cellules b Ceased WO1998014170A1 (fr)

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AU45071/97A AU4507197A (en) 1996-09-30 1997-09-30 Idiotypic vaccine in lipid based carrier for b cell disorders

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Cited By (4)

* Cited by examiner, † Cited by third party
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WO2000076319A1 (fr) * 1999-06-16 2000-12-21 Biocrystal Ltd. Formulations de vaccins et procedes d'immunisation d'un sujet contre les lymphocytes b specifiques d'un antigene excrete
WO2010042644A3 (fr) * 2008-10-07 2010-07-15 Biovest International, Inc. Procédés d'induction d'une réponse immunitaire prolongée contre un idiotype de cellules b, au moyen de vaccins autologues anti-idiotypiques
WO2014036488A1 (fr) * 2012-08-31 2014-03-06 Biovest International, Inc. Procédés de production de vaccins idiotypiques autologues haute fidélité
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WO2000076319A1 (fr) * 1999-06-16 2000-12-21 Biocrystal Ltd. Formulations de vaccins et procedes d'immunisation d'un sujet contre les lymphocytes b specifiques d'un antigene excrete
US8722091B2 (en) 2001-09-26 2014-05-13 Baxter International Inc. Preparation of submicron sized nanoparticles via dispersion lyophilization
WO2010042644A3 (fr) * 2008-10-07 2010-07-15 Biovest International, Inc. Procédés d'induction d'une réponse immunitaire prolongée contre un idiotype de cellules b, au moyen de vaccins autologues anti-idiotypiques
WO2014036488A1 (fr) * 2012-08-31 2014-03-06 Biovest International, Inc. Procédés de production de vaccins idiotypiques autologues haute fidélité
US9725768B2 (en) 2012-08-31 2017-08-08 Biovest International, Inc. Methods for producing high-fidelity autologous idiotype vaccines

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