EP1311548A1 - Peptides ligands de fixation de lymphocytes t, constructions peptidiques contenant ces peptides et leur utilisation dans le traitement de troubles immunologiques - Google Patents

Peptides ligands de fixation de lymphocytes t, constructions peptidiques contenant ces peptides et leur utilisation dans le traitement de troubles immunologiques

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Publication number
EP1311548A1
EP1311548A1 EP01939376A EP01939376A EP1311548A1 EP 1311548 A1 EP1311548 A1 EP 1311548A1 EP 01939376 A EP01939376 A EP 01939376A EP 01939376 A EP01939376 A EP 01939376A EP 1311548 A1 EP1311548 A1 EP 1311548A1
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EP
European Patent Office
Prior art keywords
peptide
seq
hiv
construct
peptides
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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.)
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Application number
EP01939376A
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German (de)
English (en)
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EP1311548A4 (fr
Inventor
Daniel S. Zimmerman
Prem S. Sarin
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Cel Sci Corp
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Cel Sci Corp
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Publication of EP1311548A1 publication Critical patent/EP1311548A1/fr
Publication of EP1311548A4 publication Critical patent/EP1311548A4/fr
Withdrawn legal-status Critical Current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
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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

  • This invention relates to peptide constructs useful in modulating the immune system of humans and other mammals, and to a peptide useful as a T cell binding ligand (TCBL) for directing a CD4 related T helper cell response, when the TCBL is linked to a disease associated antigenic peptide to form the peptide conjugate. More particularly, this invention relates to a derivative of Peptide G (the fifteen-mer peptide sequence from MHC Il ⁇ chain (135-149), previously used as a T cell binding ligand for forming immunogenic peptide constructs, and which derivative enhances the immune response of immunogenic peptide constructs as compared to the same peptide conjugates but in which the non-derivatized Peptide G is used.
  • Peptide G the fifteen-mer peptide sequence from MHC Il ⁇ chain (135-149
  • this invention relates to an immunogenic composition useful to activate the immune system of a patient exposed to or at risk of infection by human immunodeficiency virus (HIV-1) which is the causative organism of the disease known as Acquired Immune Deficiency Syndrome (AIDS) .
  • HIV-1 human immunodeficiency virus
  • This invention also relates to other peptide constructs for treating other specific immunological disorders and diseases and to the methods for treating individual subjects to modify the subjects immune system response.
  • the antibodies derived from certain conjugated peptides were better able to recognize the native molecule than were the antibodies prepared using a conventional peptide-KLH conjugate. It was shown that antibodies induced by the conjugated peptide (also referred to as "peptide construct") had a broader specificity, so that they recognized the peptide epitope not only in the free linear peptide form, but also in the native molecule. In some cases, the use of the peptide conjugated to KLH was not able to recognize the epitope in the native molecule.
  • peptide G As exemplary of the T cell binding ligand portion of the above described peptide constructs, a portion of the MHC Class II ⁇ from residues 135-149 (Peptide G) was used and achieved good results.
  • the present invention is based, in part, on the discovery that a modified version of Peptide G (Asn Gly Gin Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu He - SEQ ID NO: 2) obtained by replacing Asn with Asp to form Peptide G' (Asp Gly Gin Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu He - SEQ ID NO:l) overcomes the long range stabilization problem of the peptide conjugates and, quite surprisingly, also enhances the immune response, particularly the CD4 related (cell mediated) response, of conjugated peptides (L.E.A.P.S. constructs) as previously described.
  • the present invention provides a novel peptide having SEQ ID NO:l, useful as a TCBL in forming conjugated peptides having immunological activity by directing a host to mount a Thl response to an antigenic peptide present in the peptide construct.
  • the present invention also provides conjugated peptide constructs obtained by covalently bonding the peptide having SEQ ID NO:l, directly, or preferably via a divalent linking group, to an antigenic peptide.
  • conjugated peptide constructs obtained by covalently bonding the peptide having SEQ ID NO:l, directly, or preferably via a divalent linking group, to an antigenic peptide.
  • a pharmacologically effective composition obtained from the above mentioned peptide construct and a pharmaceutically acceptable carrier.
  • new peptide constructs obtained by linking Peptide J (Asp Leu Leu Lys Asn Gly Glu Arg He Glu Lys Val Glu - SEQ ID NO: 34) to various antigenic Cancer Mucl peptides, CEA peptides, and others, and useful to modulate the immune response of individual subjects in need thereof.
  • This invention also provides a method for treating a patient suffering from an immunological disorder by administering to the patient a therapeutically effective amount of a peptide construct in which a peptide having SEQ ID NO:l or SEQ ID NO: 34 is covalently bonded to an antigenic peptide associated with the immunological disorder.
  • the present invention relates to a peptide construct comprising a first T cell specific binding peptide having SEQ ID NO:l and a second peptide covalently linked together, wherein the second peptide is an antigenic peptide of from about 25 to about 37 amino acids (which is referred to hereinafter as "modified HGP-30") and which is capable of eliciting preferentially TH1 associated antibodies when administered to a human in need thereof.
  • modified HGP-30 modified HGP-30
  • the antigenic or second peptide has sequence identity (including naturally occurring variants and alleles thereof) with the pl7 gag protein of HIV-1 wherein the peptide has a sequence originating with an amino acid residue chosen from residues 76 to 83 and ending with an amino acid residue chosen from residues 107 to 112 of pl7 gag protein of HIV-1.
  • the second or antigenic peptides disclosed in the aforementioned copending application SN 08/695,304, or copending application SN 08/695,301 the disclosures of which are incorporated herein in their entirety by reference thereto.
  • Peptide G' used as T cell specific binding molecule in the conjugated peptides of this invention will bind specifically or at least preferentially to specific class or subclass of T cells, such as helper T cells, T h , suppressor T cells, T s , cytotoxic T cells, CTL, and the like.
  • the second or antigenic peptide used to form the peptide construct of this invention may be any known or subsequently discovered antigenic peptide, including, for example, any of the antigenic peptides mentioned in any of the above patents or copending patent applications, including, without limitation, peptides associated with herpes simplex virus (HSV) , malaria, tuberculosis, cancers, AIDS, allergies, autoimmune diseases, such as, arthritis, Graves disease, multiple sclerosis (MS), myocarditis, diabetes, Lupus, and the like.
  • HSV herpes simplex virus
  • MS multiple sclerosis
  • the antigenic peptide is a portion of pl7 of human immunodeficiency virus (HIV-1) and, particularly, a peptide of from about 25 to 37 amino acids, extending in the range from residues 76 to 112, such as the amino acid sequences shown by the following representative cases: A T L Y S V H Q I D V K D T
  • a particularly preferred antigenic peptide for use in this invention has the following amino acid sequence A T L Y S V H Q R I D V K D T
  • SEQ ID NO: 6 (sometimes referred to, for convenience, as m-HGP- 30, or even more simply as "mH") , representing a modified version of HGP-30 (see, e.g., U.S. 4,983,387). While the following discussion will focus primarily on the conjugated peptides containing the sequence of Peptide G' and the sequence for a modified HGP-30, it is to be understood that other antigenic peptides, whether from HIV-1, HIV-2, or other disease causing organism, or antigenic peptide associated with a particular disease, disorder or condition, may be used in place of modified HGP-30 with expectation of similar results.
  • conjugated peptides formed from Peptide G' and a modified HGP-30 offer the advantages previously seen with other conjugated peptides, such as those more generally disclosed in the aforementioned U.S. 5,652,342, of inducing broad spectrum antibodies but, additionally providing a desired THl specificity believed to result from the second or antigenic peptide which incorporates a CTL epitope which may modify the response to the desired isotype.
  • the present invention also relates to pharmaceutically effective compositions containing such antigen-peptide G 1 constructs (for convenience, may sometimes be referred to as "heteroconjugate”) for eliciting immunization to infection against Human Immunodeficiency Virus, HIV-1, in a human subject.
  • Such compositions in addition to the heteroconjugate of this invention will, preferably, include suitable immunological adjuvant (s) .
  • the invention relates to the use of such heteroconjugate and the pharmaceutically effective composition containing same for treating or preventing HIV-1 infection and
  • AIDS Acquired Immunodeficiency Complex
  • the antigenic peptides useful in this invention will generally be between about 25 and 37 amino acids, as represented in the following exemplary cases including examples of a longer and shorter antigenic peptides :
  • any of the above amino acid sequences of antigenic peptides variations of specific amino acids which do not adversely effect the desired biological activity are contemplated and included within the scope of the invention.
  • the foregoing sequences are based upon a specific variant of HIV-1, namely, HIV-1 SF2 (actually, the Ser 86 analog of the natural Cys 86 sequence) and, although this region of interest of HIV-1 is generally fairly highly conserved, other naturally occurring and spontaneously occurring variants, including from one or several (e.g., up to about 10) variations of the amino acids are within the sequences of interest.
  • Such natural and spontaneously occurring amino acid variations are specifically contemplated and, in certain cases, it may be advantageous to use mixtures of peptides, the sequences of which, may, correspond to two or more natural and spontaneously occurring variants of HIV-1.
  • Examples of different consensus sequences of HIV-1 which are also specifically included within the scope of the modified HGP-30 antigenic peptides for use as the second peptide in the conjugated peptides of this invention include, for instance, the following, taken from "HIV-1 Sequence Database, Human Retroviruses and AIDS 1996: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences", edited by G. Myers, et al . , Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM, December 1996, and any of the subsequent yearly updates thereof.
  • CONSENSUS A kSL fNt vat Lye vHq rid vkD tKe Aid kiE eiq nKs k
  • CONSENSUS B rSL yNt vat Lye vHq rle vkD tKe Aid kiE eEq nKs k
  • CONSENSUS C rSL ?Nt vat LyC vH?
  • CONSENSUS D kSL ?Nt vat Lye vHe rle vkD tKe Ale kmE eEq nKs k
  • CONSENSUS F rSL yNt vav Lyf vHq rvE vkD tKe Aid KLE eEq nKs q SEQ ID NO: 11
  • CONSENSUS G kSL ?N? ?a? L?c ?Hq rle vkD tKe Ale EVE Kaq kns Q SEQ ID NO: 12
  • CONSENSUS H QSL fNL La? Lye vHq rid
  • Consensus A, Consensus B, Consensus C, Consensus D, as well as Consensus F, Consensus G, Consensus H, Consensus O can be used as the modified HGP-30 antigenic peptide in the peptide constructs of this invention.
  • these various consensus sequences are generally derived from, and are prevalent in different geographical regions of the world and are often referred to as "clades” (also known as "subtypes”) of the HIV-1 virus .
  • modified HGP-30 include the following consensus sequences (wherein the letter designations generally correspond to the consensus sequences as given above) and any allelic variations thereof: Thailand-B:
  • YCV HER IKV ADT KEA LDK IEE EQT KSK KKA SEQ ID NO: 25 As can be seen from the above aligned consensus sequences and species for the various consensus sequences, there is some variation amongst HIV-1 subtypes in the gag protein sequence. Moreover, there is considerable variation in the specific numbering of amino acids among different HIV-1 strains. In the present invention, the numbering of sequences is based on the sequence of clade B HIV-1 strain SF2 or MN (most recently revised to MNCG) ; however, it is the amino acid sequence itself, allowing for variations observed amongst HIV-1 subtypes, that is important.
  • HIV-1 SF2 (Clade B) has the following sequence in the region from residue 76 to residue 112:
  • HIV-1 MNCG (Clade B) has the following sequence in the region from residue 76 to residue 112: KSL YNT VAT LYC VHQ KIE IKD TKE ALE KIE EEQ NKS K
  • amino acids at the N-terminal and C-terminal may be present as the free acid (amino or carboxyl groups) or as the salts, esters, ethers, or amides thereof.
  • amide end groups at the C-terminal and acetylation, e.g., myristyl, etc. at the N- or C-terminal are often useful without effecting the immunological properties of the peptide.
  • the conjugated peptides and the constituent components thereof can be prepared by conventional processes for synthesizing proteins, such as, for example, solid phase peptide synthesis, as described by Merrifield, R. B., 1963, J. of Am. Chem. Soc, 85:2149-2154. It is also within the scope of the invention and within the skill in the art to produce the novel peptide constructs of this invention or the peptide components thereof by genetic engineering technology.
  • the above modified HGP-30 antigenic peptides are covalently linked to Peptide G* .
  • Peptide constructs prepared by linking the antigenic peptides based on the modified HGP-30 epitopes to Peptide G' have been shown by the inventors to elicit an immune response to HIV-1 that can be directed toward the desired THl response as evidenced by the numerous examples of the THl characteristic antibody IgG2a (mouse) or IgG3 (man) induction.
  • the order of Peptide G 1 and second modified HGP-30 peptide is not usually critical and may be reversed. For example, if modified HGP-30 (B) is linked to G 1 then the peptide construct may have the sequence G'-B or B-G' .
  • Peptide G' and modified HGP-30 may be directly coupled to each other, it is preferred that a small linker sequence or a larger heterolinker molecule may be used to couple the two peptides.
  • a small linker sequence or a larger heterolinker molecule may be used to couple the two peptides.
  • the spacer one or a few, up to about 5, preferably, up to about 3 or 4, neutral amino acids, such as glycine, may be used to link the peptides.
  • Preferred spacer peptides include, for example, GGG, and GGGGS, however, the spacer may be made larger or smaller and altered to include other molecules or amino acids, besides the amino acid glycine, such as in GGGGS .
  • Examples of other known spacers which may be used for covalently linking two or more peptides or proteins or equivalent DNA sequences include, for example, GGGSGGGS (SEQ ID NO:93); GGGGSGGGGSGGGGS (SEQ ID NO:94); GGGGSS (SEQ ID NO:28); GGGGSGGGGSGG (SEQ ID NO:29); GGGSGTGSGSGS (SEQ ID NO:30); GGGGSGGGSGGGS (SEQ ID NO: 31); KGKGKGL (SEQ ID NO: 32) and VAKLEAKVAKLEAKGKGKY (SEQ ID NO: 33) .
  • heterolinkers mention may be made of, for example, N-succinimidyl-3- (2-pyridylthio)propionate (SPDP) , m-maleimidobenzoyl-N-hydroxy-succimide (MBS) as well as any of the other reagents employed to link peptides, including without limitation those disclosed in the aforementioned US 5,652,342.
  • SPDP N-succinimidyl-3- (2-pyridylthio)propionate
  • MBS m-maleimidobenzoyl-N-hydroxy-succimide
  • the administration of the peptide constructs of this invention may be carried out alone or in conjunction with other therapy.
  • Examples of other therapies which may be used in conjunction with the peptide constructs of this invention include, in the case of treatments (prophylactic or therapeutic) for infection by HIV-1, for example, protease inhibitors, reverse transcriptase inhibitors, zinc binding inhibitors, and the like.
  • the peptide constructs of this invention may be represented by formula (I) : Peptide G'-x-P* (I) wherein Peptide G' is the peptide having SEQ ID NO:l x is a direct bond or divalent linking group; and P* is an antigenic peptide associated with AIDS, especially HIV-1.
  • the conjugated peptides of formula (I) may be used to direct the immune response prophylactically (e.g., to a CD4 directed immune response) to prevent infection or reduce the likelihood of infection by HIV-1, or to direct the immune response therapeutically (e.g., to a THl directed immune response) in HIV- 1 infected individuals, perhaps in conjunction with other therapies, to reduce viral load and to control or cure the infection by HIV-1.
  • the peptide constructs may also be used to direct the immune response prophylactically to induce a THl (cellular) , TH2 (antibody) or mixed TH1/TH2 directed immune response to prevent or reduce the infection by HIV-1, or to direct the immune response therapeutically to induce a THl, e.g., CD4 + , TH2 or mixed TH1/TH2 directed immune response against the AIDS virus, perhaps in conjunction with other therapies to reduce the viral load and to control or cure the infection by HIV-1 in HIV-1 infected subjects.
  • THl cellular
  • TH2 antibody
  • mixed TH1/TH2 directed immune response to prevent or reduce the infection by HIV-1
  • a THl e.g., CD4 + , TH2 or mixed TH1/TH2 directed immune response against the AIDS virus
  • the peptide constructs of this invention may be used as a component of an immunomodulatory composition, together with one or more pharmaceutically acceptable carriers or adjuvants, either prophylactically or therapeutically.
  • the immunomodulatory composition is provided in advance of any evidence of infection by HIV-1.
  • the prophylactic administration of the composition should serve to prevent or attenuate HIV-1 in mammals.
  • a human, at high risk for HIV-1 is prophylactically treated with the peptide conjugate of this invention, as such, or as a component of an immunomodulatory composition.
  • the peptide construct or composition containing same is provided to enhance the HIV-1 infected patient's own immune response to the HIV-1 antigen.
  • the peptide construct is, in the case of treatments for individuals exposed to the AIDS virus, preferably administered after disease symptoms and viral load have been reduced or stabilized by Highly Active AntiRetroviral therapy (HAART) . While it is possible for the immunogenic peptide construct to be administered in a pure or substantially pure form, it is preferable to present it as a pharmaceutical composition, formulation or preparation.
  • the formulations of the present invention both for clinical and for human use, comprise a conjugated peptide as described above, together with one or more pharmaceutically acceptable carriers and, optionally, other therapeutic ingredients, especially therapeutic immunological adjuvants.
  • the carrier (s) must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • formulations may conveniently be presented in unit dosage form and may be prepared by any method well-known in the pharmaceutical art. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, bringing the product into the desired formulation.
  • Formulations suitable for intravenous, intramuscular, subcutaneous, or intraperitoneal administration conveniently comprise sterile aqueous solutions of the active ingredient (s) with solutions which are preferably isotonic with the blood of the recipient.
  • Such formulations may be conveniently prepared by dissolving solid active ingredient in water containing physiologically compatible substances such as sodium chloride (e.g.
  • 0.1-2.0M glycine, and the like, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering the solution sterile.
  • glycine and the like, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering the solution sterile.
  • These may be present in unit or multi-dose containers, for example, sealed ampoules or vials.
  • the formulations of the present invention may incorporate a stabilizer or excipient.
  • Illustrative excipients include polyethylene glycol, glycerol, proteins, saccharides, amino acids, inorganic acids, and organic acids which may be used either on their own or as admixtures. These excipients, when used, are preferably incorporated in an amount of about 0.1 to about 10,000 parts by weight per part by weight of immunogen. If two or more stabilizers are to be used, their total amount is preferably within the range specified above. These excipients are used in aqueous solutions at the appropriate concentration and pH.
  • the specific osmotic pressure of such aqueous solutions is generally in the range of about 0.1 to about 3.0 osmoles, preferably in the range of about 0.8 to about 1.2.
  • the pH of the aqueous solution is adjusted to be within the range of about 5.0 to about 9.0, preferably within the range of 6-8.
  • anti-adsorption agent may be used. Additional pharmaceutical methods may be employed to control the duration of action. Controlled release preparations may be achieved through the use of polymer to complex or absorb the conjugated peptide.
  • the controlled delivery may be exercised by selecting appropriate macromolecules (for example polyester, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release.
  • appropriate macromolecules for example polyester, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate
  • concentration of macromolecules as well as the methods of incorporation in order to control release.
  • Another possible method to control the duration of action by controlled-release preparations is to incorporate the conjugated peptide into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylene vinylacetate copolymers .
  • microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxy- methylcellulose or gelatin-microcapsules and poly (methylmethacrylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions .
  • compositions may be combined with typical carriers, such as lactose, sucrose, starch, talc, magnesium stearate, crystalline cellulose, methyl cellulose, carboxymethyl cellulose, glycerin, sodium alginate or gum arabic among others.
  • typical carriers such as lactose, sucrose, starch, talc, magnesium stearate, crystalline cellulose, methyl cellulose, carboxymethyl cellulose, glycerin, sodium alginate or gum arabic among others.
  • the peptide constructs of the present invention may be supplied in the form of a kit, alone, or in the form of a pharmaceutical composition as described above.
  • the immunogenic peptide constructs and immunomodulatory compositions containing same can be conducted by conventional methods.
  • the immunogenic peptide construct can be used in a suitable diluent such as saline or water, or complete or incomplete adjuvants.
  • the immunogen can be administered by any route appropriate for immune system stimulation, such as intravenous, intraperitoneal, intramuscular, subcutaneous, nasal, oral, rectal, vaginal, and the like.
  • the immunogen may be administered once or at periodic intervals until, for example, a significant titer of CD4 + or CD8 + T cell and/or antibodies directed against the HIV-1 antigen is obtained.
  • the antigenic peptide constructs of the invention elicit THl associated antibodies and other aspects of a THl immune response.
  • the presence of such cells may be assessed by measuring cytokine secretion specific for TH-1 (e.g., IFN- ⁇ , IL-2) or TH-2 (e.g., IL-4, IL-10) in response to being pulsed with the immunogen.
  • TH-1 e.g., IFN- ⁇ , IL-2
  • TH-2 e.g., IL-4, IL-10
  • the antibody may be detected in the serum using conventional immunoassays.
  • the administration of the peptide constructs of the present invention and the immunomodulatory compositions containing same may be for either a prophylactic or therapeutic purpose.
  • the immunogen is provided in advance of any evidence or in advance of any symptom due to HIV-1, or other disease causing organism, especially in patients at significant risk for occurrence.
  • the prophylactic administration of the immunogen serves to prevent or attenuate HIV-1, or other disease or condition associated with the antigenic peptide component of the conjugated peptide in a human or other animal.
  • the immunogen is provided at (or after) the onset of the disease or at the onset of any symptom of the disease.
  • the therapeutic administration of the immunogen serves to attenuate the disease.
  • the invention also concerns a method for treating or preventing human acquired immunodeficiency syndrome (AIDS) caused by infection with HIV-1, by administering to a human patient in need thereof a therapeutically effective amount of the peptide construct of this invention, such as the peptide construct of formula (I) , wherein the antigenic peptide P* is an antigen associated with AIDS, especially, one of the antigenic peptides from pl7 , as previously described.
  • the antigenic peptide P* will be chosen from the antigenic peptides associated with or causing the particular disease, disorder or condition, such as previously described, for example, in U.S. 5,652,342, or any of the other copending applications described above, or any other of the myriad known antigenic peptides associated with disease or causing disease.
  • peptide constructs of the following formula (II) are also provided by the present invention: P ⁇ x-P 2 (II) where P 1 represents the peptide having SEQ ID NO:l or SEQ ID NO: 34; x represents a direct bond or divalent linking group; and P 2 represents a peptide associated with disease, including immunological disorders, autoimmune diseases, and conditions;
  • P 1 represents the peptide having SEQ ID NO:l or SEQ ID NO: 34
  • x represents a direct bond or divalent linking group
  • P 2 represents a peptide associated with disease, including immunological disorders, autoimmune diseases, and conditions
  • antigenic peptides represented by P 2 mention may be made of, for example, antigenic peptides associated with autoimmune myocarditis, such as Peptide My: DSA FDV LSF TAE EKA GVY K (SEQ ID NO: 42); antigenic peptides associated with cancer, especially Mucl, such as Mia, having the sequence APD TRP AP (SEQ ID NO
  • SLK MAD PNR FRG KDL P (SEQ ID NO: 51) gDl ⁇ : KYA LAD ASL KMA DPN RFR GKD LP (SEQ ID NO: 52) ; extgBl: ERI KTT SSI EFA RLQ FTT DHI Q (SEQ ID NO:53); antigenic peptides associated with HIV-1, including not only those previously described, but also those associated with p24, GP41, GP120, or GP160.
  • the divalent linking group x may be a direct bond or any of the divalent linking groups or spacers as previously described.
  • DGQ EEK AGV VST GLI GGG APD TRP AP SEQ ID NO: 54
  • DGQ EEK AGV VST GLI GGG STA PPA HGV SEQ ID NO: 55
  • APG STA PPA H (SEQ ID NO: 64)
  • FRG KDL P (SEQ ID NO: 72) DLL KNG ERI EKV EGG GSL KMA DPN RFR GKD LP (SEQ ID NO-.73)
  • the immune response induced by the conjugated peptide is at least predominantly directed toward at least the desired THl response as evidenced by the THl characteristic antibody IgG2a (mouse) and presumably thereby IgG3 (man) .
  • THl characteristic antibody IgG2a mouse
  • IgG3 man
  • These peptide conjugates may, however, in addition to a THl elicited immune response, elicit a TH2 immune response, and in particular, a mixed TH1/TH2 immune response.
  • the antigenic peptide constructs of this invention provide potentially powerful vaccines for preventing infection by, or treating cells infected by, HSV and many other infectious and viral diseases or other immunogenic disorders and conditions. Therefore, the present invention provides such vaccine compositions which can be used to immunize patients at risk for, or exposed to the causative organism associated with a particular disease or disorder, such as, HSV, and various forms of cancer. The present invention, therefore, provides antigenic peptide constructs, which provide powerful vaccines for neutralizing and/or killing infected cells.
  • the vaccines of this invention can be used to immunize patients at risk for or exposed to various diseases (e.g., bacterial or viral caused diseases, e.g., herpes simplex virus, tuberculosis, diabetes, and the like) as well as other immunological disorders caused by exposure to an antigen, including, for example, allergies, asthma, autoimmune diseases, such as myocarditis, and the like.
  • diseases e.g., bacterial or viral caused diseases, e.g., herpes simplex virus, tuberculosis, diabetes, and the like
  • other immunological disorders caused by exposure to an antigen including, for example, allergies, asthma, autoimmune diseases, such as myocarditis, and the like.
  • the vaccine can be introduced into the host most conveniently by injection, intramuscularly, intradermally, parenterally, orally or subcutaneously .
  • Any of the common liquid or solid vehicles may be employed, which are acceptable to the host and which do not have any adverse side effects on the host or any detrimental effects on the
  • Phosphate buffered saline at physiological pH, e.g. pH 6.8 to 7.2, preferably pH 7, may be used as a carrier, alone or with a suitable adjuvant.
  • concentration of immunogenic peptide construct may vary from about 0.5 to 200 ⁇ g/kg, such as about 25 ⁇ g/kg per injection, in a volume of clinical medium (e.g., solvent) generally from about 0.1 to 1 ml, such as about 0.2 ml, preclinical studies in animals, and from about 0.5 ml to about 2 ml, such as about 1 ml in humans.
  • Multiple injections may be required after the initial injections and may be given at intervals of from about 2 to 8 weeks, or other suitable time interval, for example, about 2 weeks in animals and about 8 weeks in humans, when multiple injections are given.
  • a preferred concentration of immunogenic peptide construct in the vaccines of the present invention may be in the range of from 10 to 25 ⁇ g/kg, however, a higher or lower dose may be administered as needed.
  • Example 1 This example demonstrates the improved biological activity of peptide constructs according to the present invention in comparison to similar peptide constructs and conventional peptide-immunogenic carrier constructs.
  • Peptides Peptide constructs were prepared using as T cell binding ligand, either Peptide G' (SEQ ID NO:l), Peptide G (SEQ ID NO:2),
  • Peptide J from a region of ⁇ -2 microglobulin (38-50) DLL KNG ERI EKV E SEQ ID NO: 34 or Peptide F, from IL-l ⁇ (163-171) VQG EES NDK SEQ ID NO: 35. or using a conventional immunogenic carrier protein, Keyhole
  • the peptide constructs are prepared using Peptide J or
  • YSVHQRIDVKDTKEALEKIEEEQNKSKKKA (SEQ ID NO: 36) and a spacer GGG (however, similar results would be obtained using a different spacer, e.g., GGGS, GGGSGGGS (SEQ ID NO:93), GGGGSGGGGSGGGGS (SEQ ID NO: 94); GGGSGGSS (SEQ ID NO:95), GGGGSGGGGS (SEQ ID NO:96), etc. ) .
  • the peptide constructs used in this example have the following formulas: DGQ EEK AGV VST GLI GGG ATL YSV HQR IDV KDT KEA LEK IEE EQN KS
  • SEQ ID NO: 41 where the double underlined portion represents HGP-30 (SEQ ID NO: 36) .
  • the peptides may be synthesized using, for example, the F- moc or t-Boc procedure and a double coupling protocol for the first 8 residues. Usually the peptide is prepared with the carboxyl terminus as an amide form. All of the peptides are purified using preparative HPLC, and analyzed by an analytical HPLC, amino acid analysis and mass spectrophotometer . The peptides are greater than 95%, usually greater than 98%, pure by HPLC criteria. The dry peptides are stored in vials with desiccant at -8°C. II. Preparation of KLH Conjugates
  • KLH Keyhole Limpet Haemocyanin
  • cGMP grade, Biosyn is conjugated to m-HGP-30 (mH) peptide by a glutaraldehyde conjugation method using a 1:1 mg weight ratio of peptide to KLH.
  • the peptide constructs may be synthesized as a single peptide without any conjugation step or by conjugation of the
  • T cell binding ligand peptide G 1 , G, F or J
  • antigenic peptide e.g., mH or HGP-30
  • the final products are analyzed for protein or peptide using the BCA protein, or other suitable, assay, and adjusted to contain between 200-400 ⁇ g/ml of total protein or peptide, and stored frozen (-20°C) in suitable (e.g., 1.5 ml) aliquots ready for thawing and administered in combination with an adjuvant ISA-51 (Seppic) or microfluidized MPL S/E (Corixa) .
  • mice in groups (5-10 animals per group as designated) are immunized with the LEAPS constructs (25 ⁇ g/dose) of SEQ ID NO's:37, 38 and 39, in 0.2 mL emulsified 1:1 with adjuvant
  • mice are evaluated by Delayed Type of Hypersensitivity (DTH) by inoculation on day 26 with 20 ⁇ l of a solution of saline or mH (SEQ ID NO: 6) (25 ⁇ g in saline) in the left or right ear respectively.
  • the DTH response is determined by measurement of the ear thickness 48 hours later and expressed as increase in ear thickness (mm) of experimental ear compared to that of the control ear.
  • Individual animal sera are collected for measurement of antibody on day 28, 42, 63 and 77.
  • Sera from individual animals and group pools are evaluated for specific anti-mH antibody, for total antibody, IgGl, IgG2a and IgG3 antibody isotype and cross clade titer at selected time points as indicated.
  • Table 2 shows the results of the ELISA antibody screening of the sera from the above animals, but with test bleeding collected at day 63 and for cross clade recognition.
  • the mouse sera for the ELISA was used at a dilution of 1:200. Again, clearly, the construct provides higher signals indicative of higher levels of the antibodies that recognized other clades besides the parental one (Clade B) of mH.
  • This example is designed to demonstrate "mechanism of action” as further “proof of principle” of the LEAPS technology.
  • the analysis is based on use of peptide J with mH (SEQ ID NO:39) and Peptide G 1 with mH (SEQ ID NO:37), for mechanism and types of cells involved.
  • mice are pretreated with the antisera as indicated for 5 days before the initial immunization with peptide-construct having SEQ ID NO:37 or SEQ ID NO:39 on day 1. No pretreatment is done before the booster immunization on day 14 or DTH on day 26-28.
  • the mice are then inoculated with the test antigen or saline (control) on day 26 in the left and right ears, respectively, and the DTH response is measured on day 28.
  • the difference between the values for the test and saline control ears is calculated for each group of five animals along with the standard error of the mean (sem) and the results are plotted (see Fig. 1) . Also shown in Fig. 1 are values for the untreated animals and animals immunized on day 14 only.
  • This example is similar to Example 2 but using a more sensitive ELISA test for antibody type.
  • No antibody data is shown for SEQ ID NO: 39, as for this type of MHC-I TCBL construct day 28 is too early to detect any antibodies to the immunogen.
  • mice None Average of group (5 mice) 0.224 0.274 22.10% 0.222 0.288 29.30%

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CN108342405A (zh) * 2017-01-23 2018-07-31 复旦大学 Il21融合蛋白及其制备方法和在制备靶向治疗肿瘤药物中的用途

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SG11202010454PA (en) * 2018-03-23 2020-11-27 Gavish Galilee Bio Appl Ltd Genetically reprogrammed tregs expressing membrane-bound il-10

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PISAREV V M ET AL: "Enhanced immunogenicity of an HIV gag peptide by conjugation to MHCIIbeta2 or IL-1beta epitopes is augmented, with type 1 T-cell responses, by Flt3 ligand" FASEB JOURNAL, vol. 14, no. 6, 20 April 2000 (2000-04-20), page A943, XP009040567 & JOINT ANNUAL MEETING OF THE AMERICAN ASSOCIATION OF IMMUNOLOGISTS AND THE CLINICAL IMMUNOLOGY SOCIET; SEATTLE, WASHINGTON, USA; MAY 12-16, 2000 ISSN: 0892-6638 *
WANG R ET AL: "Protective immunity against malaria by immunization with PYSSP2 CD4+ T helper epitope conjugated to MHC class II binding ligand MHC-II beta-chain peptide 135-149" AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, vol. 62, no. 3 Supplement, March 2000 (2000-03), pages 331-332, XP009040577 & 49TH ANNUAL MEETING OF THE AMERICAN SOCIETY OF TROPICAL MEDICINE AND HYGIENE; HOUSTON, TEXAS, USA; OCTOBER 29-NOVEMBER 02, 2000 ISSN: 0002-9637 *
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Publication number Priority date Publication date Assignee Title
CN108342405A (zh) * 2017-01-23 2018-07-31 复旦大学 Il21融合蛋白及其制备方法和在制备靶向治疗肿瘤药物中的用途

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