EP0463157A1 - Lfa-3 utilise comme adjuvant de vaccins - Google Patents

Lfa-3 utilise comme adjuvant de vaccins

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Publication number
EP0463157A1
EP0463157A1 EP91904441A EP91904441A EP0463157A1 EP 0463157 A1 EP0463157 A1 EP 0463157A1 EP 91904441 A EP91904441 A EP 91904441A EP 91904441 A EP91904441 A EP 91904441A EP 0463157 A1 EP0463157 A1 EP 0463157A1
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EP
European Patent Office
Prior art keywords
lfa
tll
vaccine
adjuvant
hepatitis
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.)
Withdrawn
Application number
EP91904441A
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German (de)
English (en)
Other versions
EP0463157A4 (en
Inventor
Barbara P. Wallner
David W. Thomas
Mary A. V. Crimmins
Christopher D. Benjamin
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.)
Biogen Inc
Original Assignee
Biogen Inc
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Publication date
Application filed by Biogen Inc filed Critical Biogen Inc
Publication of EP0463157A1 publication Critical patent/EP0463157A1/fr
Publication of EP0463157A4 publication Critical patent/EP0463157A4/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70528—CD58
    • 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
    • A61K39/12—Viral antigens
    • 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
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • LFA-3 lymphocyte function associated antigen-3
  • This invention relates to the use of lymphocyte function associated antigen-3 (LFA-3) as an adjuvant in a vaccine mixture.
  • LFA-3 is capable of augmenting T-lymphocyte proliferation and thus enhancing the immune response to a vaccine's immunogenic component.
  • the immune system has evolved primarily to combat infection by pathogenic organisms. To accomplish this, the immune system has highly specialized effector components and complex regulatory mechanisms. The immune response is not confined to a single site in the body; effector cells move within and among lymphoid organs and various body compartments. On a cellular level, the immune, response to a pathogen depends on a complex system of communication between the various cell types of. the immune system. The mechanism of this communication among the various cells may be either by direct cell-to-cell contact or by soluble secreted mediators (cyto ines) which can act at a distance from the cell that secretes them.
  • cyto ines soluble secreted mediators
  • a cell mediated response primarily comprising the action of cytotoxic T cells which attack and kill foreign cells or virus-infected cells
  • a humoral response comprising the activation of B cells to plasma cells which secrete antibodies specific for foreign macromolecules.
  • T cells play a central role in both types of responses (1) by interacting directly with target Gells in the production of cytotoxic T cell-mediated responses and (2) by interacting with antigen- presenting cells (APCs) bearing foreign macromolecules in the initiation of antibody responses.
  • APCs antigen- presenting cells
  • the B cells are activated to plasma cells which secrete antibodies of pre-determined specificity.
  • helper T cells plays a unique role in increasing the specificity of the B cell antibody response and in the development of "memory" B cells, which are critical for anamnestic or secondary responses to a renewed challenge by a foreign antigen.
  • T cells with target and antigen-presenting cells is highly specific and depends on the recognition of a surface antigen on the target cell or APC (or B cell) by highly specific receptors on the T cells. This interaction may be facilitated by other antigens expressed on the surface of the interacting cells, e.g., the antigen receptor complex known as CD3 on T cells or other T cell accessory molecules such as CD4, LFA-1, CD8 and CD2, and accessory molecules such as LFA-3, ICAM-1 and MHC on antigen-presenting cells. Interaction between accessory molecules on T-lymphocytes and accessory molecules on target cells or APCs (or B cells) may be important in mediating intercellular adhesion and are thought to enhance the efficiency of lymphocyte/APC and lymphocyte/target cell interactions.
  • Classical vaccine compositions are designed to activate the immune system in order to confer immunity, prophylactically, to subsequent challenges from a living pathogen that might infect and debilitate an individual or animal.
  • Vaccines generally contain two main components. The first is an immunogen which the host's immune system will recognize as foreign (not self) .
  • the immunogen is usually an attenuated viral or bacterial pathogen or proteins or polypeptides derived from the pathogen.
  • the second component is an adjuvant.
  • Classical adjuvants have ranged from simple metal salts, e.g., Al(OH) 3 (known as Alum), to complex emulsified mixtures such as Freund's adjuvant, a water- in-oil emulsion containing suspended tubercle bacilli.
  • Adjuvants act primarily by creating a depot from which immunogen is slowly released, thereby imitating the persistent challenge posed to the immune system during an infection by a functional replicating organism or virus.
  • Adjuvants may also contain a plethora of foreign biomolecules (e.g., tubercle bacilli) which strongly sensitize the cells of the immune system to both the adjuvant biomolecules themselves and the included immunogen. In this regard, adjuvants enhance the immunogenicity of a particular immunogen.
  • Previously used adjuvants have various shortcomings. Many adjuvants cause inflammation at the site of administration, resulting in discomfort. Adjuvants derived from inactivated viral or bacterial sources may also be toxic and, consequently, of limited usefulness despite their high potency.
  • One aspect of this invention is a composition comprising LFA-3 and an immunogen to form a vaccine mixture. Both the adjuvant and the immunogen are present in amounts sufficient to stimulate and augment the immune response in a host animal toward the immunogen.
  • Another aspect of this invention is a method of vaccination comprising administration of an LFA-3 adjuvant/immunogen mixture to a host animal.
  • a particularly preferred embodiment of the present invention is a vaccine comprising an immunogen and LFA-3 as an adjuvant, and also a co- - adjuvant comprising a ligand or an antibody recognizing other T-cell surface molecules than CD2 (e.g., CD3) , or the same or a different epitope of CD2 than that bound by LFA-3 (i.e.. Til.).
  • a co- - adjuvant comprising a ligand or an antibody recognizing other T-cell surface molecules than CD2 (e.g., CD3) , or the same or a different epitope of CD2 than that bound by LFA-3 (i.e... Til.).
  • Examples of antibodies recognizing one of the epitopes of CD2 include anti- Tll ⁇ anti-Tll 2 or anti-Tll 3 , 9.6, CD2.1, 9-1, 35.1, D66, and GT2.
  • the compositions and methods of . the present invention substantially avoid irritationi and inflammation at the site of administration as well as toxic side
  • Figure 1 is a graph showing levels of immunoglobulin secretion by monocyte-depleted human peripheral blood lymphocytes (PBLs) subjected to different stimuli.
  • the lymphocytes were cultured for 7 days in the presence of a fixed amount of anti-Til alone, different concentrations of PI-LFA-3 alone, or a combination of a fixed amount of anti-Tll 2 and different concentrations of PI-LFA-3.
  • Figure 2 is a graph showing level of immunoglobulin secretion by monocyte-depleted human peripheral blood lymphocytes subjected to different stimuli.
  • the lymphocytes were cultured for 7 days in the presence of PI-LFA-3 alone (10 ⁇ g/ l) , different concentrations of anti ⁇ Tll 2 alone, or a combination of a fixed amount of PI-LFA-3 (10 ⁇ g/ml) and different concentrations of anti-Til, or a murine myeloma immunoglobulin of the same isotype (I-J G 2a ) •
  • LFA-3 Any source and form of LFA-3 is suitable provided that the LFA-3 employed is compatible with the host animal.
  • human LFA-3 should be used as the adjuvant in vaccines according to this invention intended for use in humans.
  • this invention contemplates cross-species use of LFA-3 where cross- reactivity is observed without adverse side effects.
  • primate LFA-3 is determined to be active in humans and does not have any serious side- effects, this cross-species use is within the concept of the invention.
  • the form of LFA-3 employed should itself be of limited toxicity or immunogenicity to the host.
  • the LFA-3 is either the complete • LFA-3 molecule or a fragment capable of binding to CD2.
  • the LFA-3 will be the form having a carboxyterminal phophatidylinositol linkage (PI-LFA- 3), discussed completely in copending, commonly assigned U.S. application Ser. No. 237,309, filed August 26, 1988, incorporated herein by reference. Even more preferably, the LFA-3 will be in multimeric micellar form. Most preferably, the LFA-3 is PI-LFA-3 in an octameric, micellar form. The LFA-3 may be in any form that leads to a sufficiently high binding affinity which, in turn, enhances activated T-lymphocyte proliferation or leads to crosslinking of receptor sites on the T-lymphocyte cell surface.
  • PI-LFA- 3 carboxyterminal phophatidylinositol linkage
  • LFA-3 Minor variations of the primary amino acid sequence of LFA-3 may occur and are within the scope of this invention, so long as the derivative LFA-3 binds to the CD2 receptor.
  • amino acid site- specific mutations, non-naturally-occurring amino acids, peptide structural analogs or phosphoinositol analogs may be used to augment the LFA-3 or to replace a portion of the LFA-3 molecule.
  • Soluble forms of LFA-3 may also be used. These modifications may be used to improve the biological stability of the LFA-3 adjuvant or to alter the structure of LFA-3 micelles formed from LFA-3 molecules. For example, a different hydrophobic moiety may be used to replace the carboxyr. terminal phosphoinositol appendage of PI-LFA-3.
  • an LFA-3 derivative having a hydrophobic moiety attached to the LFA-3, preferably at its carboxy-terminal end, may form a multivalent micelle.
  • the stoichiometry of such micelles will depend upon the hydrophobic moiety used as well as the particular LFA-3 derivative.
  • Suitable alternative hydrophobic moieties include, e.g., phosphatidylethanolamine.
  • the LFA-3 may be in the form of conjugates, such as fusion proteins, coupled proteins and immunoconjugates.
  • the LFA-3 may be expressed as a fusion protein linked to an immunogen, a co-adjuvant, or a protein sequence which causes aggregation of LFA- 3, e.g., domains of C4 binding protein or the amino acid sequence of gelsolin which binds to phosphoinositol.
  • LFA-3 may be chemically coupled in known ways directly to an immunogen, providing a single molecule with dual functionality with respect to the immune system.
  • LFA-3 linked to an immunogen by recombinant or chemical techniques would be expected to significantly enhance the antibody response to that immunogen by stimulating a productive cell-to-cell interaction between an immunogen-specific B cell and any T cell stimulated by the LFA-3 linked to that immunogen.
  • the LFA-3 may also be in the form of an immunoconjugate when coupled to antibodies, e.g., where anti-Til. or anti-CD3 antibodies are coupled with LFA- 3. Mixtures of LFA-3 conjugates may also be used. Regardless of the structure used, the LFA-3 should survive .in vivo for a sufficient time to augment lymphocyte activation.
  • LFA-3 may be obtained in substantially pure form from either native or recombinant sources.
  • the PI-LFA-3 may be isolated, purified, and converted to micellar form following methods described above (Springer, 1989) . A selection may be made among commonly used isolation and purification procedures without departing from the scope of this invention. Plasmids bearing genes coding for recombinant LFA-3 and recombinant Pi-linked LFA-3 have been deposited with In Vitro International under the terms of the Budapest Treaty, under accession nos. IVI-10133 and IVI-10180.
  • the immunogen may be any biological system capable of eliciting an immune response against the immunogen.
  • the immunogen may be derived from either natural, recombinant, or synthetic sources or mixtures thereof; furthermore, it may be used in substantially pure form or it may be a crude mixture of biomolecules.
  • Immunogens may be derived from viral or bacterial sources.
  • immunogens may be derived from agents responsible for acne vulgaris, caries, cholera, gonorrhea, haemophilus, klebsiella, lactobacillus, leprosy, measles, meningitis, otitis media, pertussis, rubella, syphilis, shigella, malaria, hoof and mouth disease, adenovirus, AIDS, HTLV, CMV, dengue, GBV, herpes simplex, hepatitis A, hepatitis B, hepatitis non A-non B, hepatitis C, influenza, lassa fever, parainfluenza, pneumonia, parvovirus, rotavirus, or RSV.
  • the amounts of the adjuvant and immunogen needed to evoke an immune response in the host are interrelated, but are within the ranges generally employed in conventional vaccines.
  • the preferred amount of adjuvant is between about 0.05 microgram and about 5.0 milligrams per dose. More preferably, between about 0.8 microgram and about 2.0 milligrams is used.
  • the preferred amount of immunogen is between about 0.05 microgram and about 5 milligrams per dose. More preferably, between about 0.8 micrograms and about 2 milligrams of the immunogen is used.
  • the dosage will depend upon the host receiving the vaccine as well as its size, weight, metabolism, etc. Higher doses of the immunogen may produce side effects such as chills, fever, and the like. Higher doses of the immunogen are within the scope of this invention should the clinical advantage outweigh these side effects.
  • compositions of this invention may be formulated using methods and compositions similar to those used for other pharmaceutically important polypeptides.
  • the adjuvant and immunogen may be stored in lyophilized form and reconstituted in a physiologically acceptable vehicle prior to administration.
  • the adjuvant and immunogen may be stored in the vehicle.
  • Preferred vehicles are sterile solutions.
  • Preferred vehicles include sterile buffer solutions, such as phosphate buffered saline. Any method of combining the adjuvant and the immunogen in the vehicle that retains the immunoreactivity of the mixture is appropriate.
  • the vehicle may contain preservatives or other known additives which are used to improve the. shelf stability or the efficacy of the mixture. Suitable preservatives include e.g., thimerosal.
  • the vaccine mixture also may contain additional materials that supplement the ability of LFA-3 to stimulate lymphocyte activation. For example, the combination of LFA-3 and a co-adjuvant reactive with surface molecules of T-lymphocytes (or other cells) may be used. The immune system of a host is stimulated and reacts against the vaccine of this invention very rapidly. Thus, a vaccine mixture containing LFA-3 and optionally a co-adjuvant may be used in a therapeutic manner.
  • Suitable co-adjuvants may be other surface molecules of cells involved in the immune response or fragments thereof that recognize such molecules, antibodies to such cell surface molecules (including r V and F v antibody fragments that bind to surface antigens) or mixtures thereof.
  • these additional materials are antibodies which recognize T-cell surface molecules, most preferably antibodies which recognize the same or a different epitope of CD2 than recognized by LFA-3.
  • the co-adjuvant will be an antibody recognizing the Tllj epitope, the same CD2 epitope recognized by LFA-3 (e.g., anti-Til !
  • the volume of a single dose of the vaccine of this invention may vary but will be generally within the ranges commonly employed in conventional vaccines.
  • the volume of a single dose is preferably between about 0.1 ml and about 1.0 ml, more preferably between about 0.2 ml and about 0.5 ml.
  • the adjuvan /immunogen mixture may be administered by any convenient means.
  • Preferred methods of administration include subcutaneous, intraperitoneal, intramuscular, or intravenous injection.
  • the mixture may be released from a biodiffusible implant.
  • a single administration may be used, or preferably a series of administrations may be made over the course of several days or weeks.
  • the following examples are intended to illustrate the invention but are not to be construed as limiting the same.
  • Multimeric PI-LFA-3 was isolated in the following manner. CHO cells, transfected with LFA-3 cDNA coding for the phosphoinositol linked form of LFA-3 (PI-LFA-3) were grown on collagen beads in roller bottles to 1.2 x 10 7 cells/ml. 150 ml of cell coated beads in DMEM medium were treated with 50 g collagenase for 30 min at 37 ⁇ C. Cells were pelleted, washed 2 times in 100 ml conditioned medium, then in 50 ml lx PBS, followed by 50 ml Dulbecco's medium.
  • Lymphocyte Separating Medium ORGANON TEKNIKA
  • PBLs peripheral blood lymphocytes
  • the PBLs were taken up in- RPMI-1640 (GIBCO) .
  • the growth medium was made to be 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units penicillin G/ml growth medium, 100 micrograms streptomycin G/ml growth medium, and 5.5 x 10 ⁇ 5 M 2-mercaptoethanol.
  • the cells suspended in growth medium were placed on tissue culture treated plastic dishes (COSTAR®; Cambridge, MA) ; the cells were incubated for two cycles of 1 hr per cycle at 37°C to adhere the onocytes to the dishes. T-lymphocytes and B- lymphocytes, still suspended in solution, were removed by pipetting and transferred to the flat bottom wells of a 96-well tissue culture plate. 1 x 10 5 cells in 50 microliters growth medium were added to each well.
  • the 96 wells (all containing suspended cells) were grouped to receive phorbol myristate acetate (PMA), anti-Tll 2 (a gift of E. Reinherz, Dana-Farber Cancer Institute, Boston, MA), PI-LFA-3, various combinations of these three additives, or none of these additives (control).
  • PMA phorbol myristate acetate
  • anti-Tll 2 a gift of E. Reinherz, Dana-Farber Cancer Institute, Boston, MA
  • PI-LFA-3 various combinations of these three additives, or none of these additives (control).
  • an equal number of wells comprised, in addition to the cells, no additive (control), PMA, anti-Tll 2 , PI-LFA-3, PMA and anti-Tll 2 , PMA and PI-LFA-3, PI-LFA-3 and anti-Tll 2 , and PMA, anti- Tll 2 and PI-LFA-3.
  • a stock solution of phorbol myristate acetate (PMA) in growth medium was added so that the final concentration of PMA in the well was 0.6 ng/ml.
  • PMA phorbol myristate acetate
  • anti-Tll 2 50 microliters of a stock solution containing anti-Tll 2 in growth medium was used to obtain a final dilution in anti- Tll 2 -containing wells of 1:1000.
  • the antibody stock solution was prepared by diluting 1:250 ascites drawn from a mouse injected intraperitoneally with a hybridoma that produces anti-Til- The ascites drawn directly contained approximately 1 mg/ml of anti-Tll 2 .
  • micellar PI-LFA-3 For wells receiving PI-LFA-3, 50 microliters of micellar PI-LFA-3 in growth medium was then added to the well. Varying concentrations of PI-LFA-3 were tested, i.e., 3 ng/ml, 30 ng/ml and 300 ng/ml.
  • the total volume of each well was 200 microliters. If PMA, anti-Tll 2 or PI-LFA-3 was omitted from a particular well, then sufficient growth medium was added to make the total volume 200 microliters.
  • the number of counts obtained from a control experiment identical to that described except that PI-LFA-3 was omitted was 2000 counts per minute.
  • PBLs were isolated as in Example 1, except that the PBLs were not incubated in tissue culture plastic treated dishes. The cells were taken up in growth medium, and 1 x 10 5 cells in 50 microliters growth medium added to each well of a 96-well U bottom tissue culture plate. Test solutions were added to specific wells in 50 microliter aliquots to give the indicated final concentration. Control wells contained medium and PBLs only. Test wells contained a combination of anti-CD2 monoclonal antibodies anti- Tll 2 and anti-Tll 3 at a final dilution of 1:900 of - ascites; or anti-Tll 2 with PI-LFA-3; or anti-Tll 3 with PI-LFA-3; or each compound individually.
  • PI-LFA-3 (prepared as in Example 1) was added at increasing concentrations ranging from 0.015 to 0.5 microgram/ml. All wells were brought to a final volume of 150 microliters with growth medium. Cells were incubated at 37 ⁇ C for 3 days, after which 50 microliters of 20 microCurie/ml [ 3 H]-thymidine was added and the cells incubated for 12 hrs at 37 ⁇ C. At this point, the cells were lysed and harvested in a manner similar to Example 1. Proliferation of T-lymphocytes was directly measured by detecting the incorporation of [ ]- thymidine. Results are presented in the following table:
  • PBLs were isolated from buffy coat cells purchased from the American Red Cross (Northeast Regional Blood Services) by the method described in Example 1. After isolation from a lymphocyte separating medium (Ficoll-Paque; Pharmacia, Piscataway, NJ) , removal of the monocytes and washing, the cells were taken up in growth medium, and 2 x 10 5 cells in 50 microliters growth medium were added to each well of a 96-well U-bottom tissue culture plate. Positive control wells received no additions (other than growth medium to compensate for volume on a like basis for additions to the test wells) .
  • a lymphocyte separating medium Ficoll-Paque; Pharmacia, Piscataway, NJ
  • Test wells in one experiment contained a constant amount of anti-Tll 2 antibody (final dilution 1:900 of ascites) alone or in combination with varying amounts of PI- LFA-3.
  • Test wells in another experiment contained a constant amount of PI-LFA-3 (10 ⁇ g/ml) alone or in combination with varying amounts of anti- Tll 2 antibody.
  • Negative control wells contained a murine myeloma immunoglobulin of an isotype identical to that of the anti-Tll 2 antibody (IgG 2a ) , either alone or in combination with PI-LFA-3 (10 ⁇ g/ml) . All additions were dissolved and/or diluted in growth medium.
  • each culture well contained 200 ⁇ l of growth medium.
  • Cells were incubated 37°C, in a 5% C0 2 atmosphere for seven days. After seven days, 100 microliters of conditioned culture medium supernatant (i.e., devoid of cells) was removed; triplicates were pooled and held at -20 ⁇ C until assayed.
  • the conditioned medium was assayed for human immunoglobulin of the G and M classes by incubating said medium on microtiter ELISA plates coated with a purified goat antibody raised to human IgG and IgM (purchased from Jackson Immunoresearch,- Malvern, PA) .
  • Human immunoglobulin present in the conditioned culture medium that bound to the goat anti- human IgG and IgM coated ELISA plates was detected with an alkaline phosphatase conjugate of goat anti-human IgG and IgM.
  • the bound enzyme was illuminated by conversion of an uncolored substrate to a colored product. Color development, which directly correlates to the amount of human Ig in the culture supernatant, was quantified on a Molecular Devices Thermomax ELISA reader at a wavelength of 405 nm.
  • Vaccine mixtures containing human LFA-3 and hepatitis B core antigen are tested to investigate the effect of LFA-3 as an adjuvant in an jLn vivo model.
  • Vaccine mixtures containing 0.01-1.0 mg micellar PI- LFA-3 and 0 * 01-1.0 mg hepatitis B core antigen (Biogen, Inc., Cambridge, MA) are injected intramuscularly into rhesus monkeys three times at monthly intervals. Control groups of rhesus monkeys are injected with PI- LFA-3 alone, hepatitis B core antigen alone and buffer alone.
  • PI-LFA-3 Adjuvant activity of PI-LFA-3 is demonstrated by either greater anti-hepatitis B core antigen antibody titers or by a more prolonged presence of such antibodies in serum from monkeys receiving both PI- LFA-3 and hepatitis B core antigen when compared to serum titers obtained from monkeys of the control groups.
  • PBLs are isolated from heparinized whole blood in a manner similar to Example 1 and proliferation of activated T-lymphocytes is assayed by culturing PBLs with 0.001-0.1 mg hepatitis B core antigen with and without PI-LFA-3 under standard tissue culture conditions in microliter tissue culture plates. Proliferation is measured by incorporation of [ 3 H]- thymidine after 3-5 days of culture as in Example 1. Evidence of adjuvant efficacy is demonstrated by augmented proliferation of T-lymphocytes from rhesus monkeys receiving core antigen and PI-LFA-3, compared against T-lymphbcyte proliferation in monkeys of the control groups.
  • Human CD2 (h-CD2) transgenic mice (a gift from Dimitri Kioussis, Mill Hill, UK) are used as a model system to determine the ability of PI-LFA-3 to augment T-lymphocyte activation in vivo. Human CD2 has been shown to transduce T-cell activation signals to murine T-cells in the same manner as to human T-cells.
  • h-CD2-transgenic mice are injected with increasing amounts of PI-LFA-3 and ovalbumin; h- CD2-transgenic mice in one control group are injected with ovalbumin alone.
  • a second group of control h- CD2-transgenic mice are injected with PI-LFA-3 alone.
  • 0.1-8 micrograms PI-LFA-3 and 0.1-100 micrograms ovalbumin in 0.1 ml sterile PBS to form increasing doses are injected subcutaneously into h-CD2- transgenic mice.
  • Control mice are injected with 0.1- 100 micrograms ovalbumin in 0.1 ml sterile PBS or with 0.1-8 micrograms PI-LFA-3 in 0.1 ml sterile PBS.
  • groups of non-transgenic (normal) mice are immunized with the same moieties listed above. After 8-14 days, to assess cellular immunity, the draining lymph nodes are removed and a single cell suspension of lymphocytes is isolated.
  • the lymphocytes are cultured in a growth medium of RPMI-1640 made to be 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units penicillin G/ml growth medium, 100 micrograms stretomycin G/ml growth medium, and 5.5 x 10 ⁇ 5 M
  • the cells are transferred to flat bottom wells of a 96-well tissue culture plate. 2 x 10 5 cells are added to each well in 50 microliters of growth medium. The wells are grouped to receive increasing amounts of ovalbumin ranging from 0.1 microgram to 100 micrograms per milliliter, and the final volume of the wells is made to be 200 microliters.
  • the lymphocytes are incubated for 2-5 days, at which time 50 microliters of growth medium containing 1 microCurie/ml of [ 3 H]-thymidine are added to each well. The cultured lymphocytes are incubated for 6 hours more. The cells are harvested and [ 3 H]- thymidine incorporation measured as described in Example 1.
  • anti-ovalbumin antibody responses are determined using conventional ELISA techniques. Mice are bled prior to immunization and two weeks following immunization; the blood is allowed to clot and serial dilutions of the aspirated serum are assayed for anti-ovalbuim activity.

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Abstract

Vaccins comprenant un immunogène et, à titre d'adjuvant, un LFA-3 (antigène-3 associé aux fonctions lymphocytaires) ou un fragment de LFA-3 capable de se lier à la CD2, un récepteur superficiel sur des lymphocytes T. On a découvert que le LFA-3 augmente d'une façon marquée la prolifération de lymphocytes T activés, et est ainsi capable d'augmenter la réponse immunitaire. L'invention concerne également des procédés de vaccination dans lesquels on utilise le LFA-3 conjointement avec un inoculum immunogène.
EP19910904441 1990-01-24 1991-01-24 Lfa-3 as a vaccine adjuvant Withdrawn EP0463157A4 (en)

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US46914090A 1990-01-24 1990-01-24
US469140 1999-12-21

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EP0463157A4 EP0463157A4 (en) 1992-06-03

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EP (1) EP0463157A4 (fr)
JP (1) JPH04506666A (fr)
AU (1) AU7300091A (fr)
CA (1) CA2049931A1 (fr)
WO (1) WO1991011194A1 (fr)

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Publication number Priority date Publication date Assignee Title
US5223394A (en) * 1989-04-10 1993-06-29 Biogen, Inc. Recombinant dna molecule comprising lymphocyte function-associated antigen 3 phosphatidylinositol linkage signal sequence
ATE210454T1 (de) * 1991-10-07 2001-12-15 Biogen Inc Verfahren zur verbesserung der toleranz für allotransplantaten und xenotransplantaten durch verabreichung eines lfa-3- oder cd2- bindungsproteins
US6764681B2 (en) 1991-10-07 2004-07-20 Biogen, Inc. Method of prophylaxis or treatment of antigen presenting cell driven skin conditions using inhibitors of the CD2/LFA-3 interaction
US5795572A (en) * 1993-05-25 1998-08-18 Bristol-Myers Squibb Company Monoclonal antibodies and FV specific for CD2 antigen
DK0749323T3 (da) * 1994-03-08 2001-02-05 Dana Farber Cancer Inst Inc Fremgangsmåder til modulering af T-celle-anergi
DK1282702T3 (da) 2000-05-10 2007-04-02 Sanofi Pasteur Ltd Immunogene polypeptider, som er kodet af KAGE-minigener, og anvendelser deraf
CA2454618C (fr) 2001-07-24 2012-04-03 Biogen Idec Ma, Inc. Methodes de traitement ou de prevention de troubles sclereux par l'usage d'agents se liant a cd2
US7786278B2 (en) 2002-04-09 2010-08-31 Sanofi Pasteur Limited Modified CEA nucleic acid and expression vectors
CN101124327A (zh) 2003-10-08 2008-02-13 圣诺菲·帕斯图尔公司 经修饰的cea/b7载体
WO2005115436A1 (fr) * 2004-05-07 2005-12-08 Astellas Us Llc Polypeptide lfa-3 soluble destine a traiter de troubles viraux
CN101835488B (zh) 2007-09-04 2018-10-26 美国政府(由卫生和人类服务部、疾病控制和预防中心的部长所代表) 轮状病毒的热灭活
WO2014025198A2 (fr) * 2012-08-09 2014-02-13 주식회사 한독 Mutant de lfa3, protéine de fusion dans laquelle des polypeptides spécifiques d'une cible sont connectés au mutant ou à la région de liaison à cd2 de lfa3, et leur utilisation

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WO1989010938A1 (fr) * 1988-05-04 1989-11-16 Dana-Farber Cancer Institute Micelles de proteine
WO1989012458A1 (fr) * 1988-06-14 1989-12-28 Cell Med, Inc. Reactifs immunologiques cellulaires heterofonctionnels, vaccins les contenant et leurs modes d'utilisation

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US4956281A (en) * 1987-06-03 1990-09-11 Biogen, Inc. DNA sequences, recombinant DNA molecules and processes for producing lymphocyte function associated antigen-3

Patent Citations (2)

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WO1989010938A1 (fr) * 1988-05-04 1989-11-16 Dana-Farber Cancer Institute Micelles de proteine
WO1989012458A1 (fr) * 1988-06-14 1989-12-28 Cell Med, Inc. Reactifs immunologiques cellulaires heterofonctionnels, vaccins les contenant et leurs modes d'utilisation

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Title
CHEMICAL ABSTRACTS, vol. 108, no. 11, 14th March 1988, page 502, abstract no. 92752w, Columbus, Ohio, US; G. TIEFENTHALER et al.: "Purified lymphocyte function-associated antigen-3 and T11 target structure are active in CD2-mediated T cell stimulation" & EUR. J. IMMUNOl. 1987, 17(12), 1847-50 *
See also references of WO9111194A1 *

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JPH04506666A (ja) 1992-11-19
WO1991011194A1 (fr) 1991-08-08
AU7300091A (en) 1991-08-21
EP0463157A4 (en) 1992-06-03
CA2049931A1 (fr) 1991-07-25

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