WO2020204923A2 - Vaccin à base de récepteur de thromboxane pour la gestion de la thrombogenèse - Google Patents

Vaccin à base de récepteur de thromboxane pour la gestion de la thrombogenèse Download PDF

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Publication number
WO2020204923A2
WO2020204923A2 PCT/US2019/025612 US2019025612W WO2020204923A2 WO 2020204923 A2 WO2020204923 A2 WO 2020204923A2 US 2019025612 W US2019025612 W US 2019025612W WO 2020204923 A2 WO2020204923 A2 WO 2020204923A2
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Prior art keywords
vaccine
peptide
tpr
amino acid
salts
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PCT/US2019/025612
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WO2020204923A3 (fr
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Fadi T. Khasawneh
Fatima Z. Alshbool
Zubair A. Karim
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Board Of Regents, The University Of Texas System
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Publication of WO2020204923A2 publication Critical patent/WO2020204923A2/fr
Publication of WO2020204923A3 publication Critical patent/WO2020204923A3/fr

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    • 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
    • A61K39/0005—Vertebrate antigens

Definitions

  • the invention generally concerns the field of medicine.
  • modulation of platelet aggregation In particular the modulation of platelet aggregation.
  • AA arachidonic acid
  • COX-1 cyclooxygenase enzyme
  • TXA2 thromboxane A2
  • TXA2 a well-studied platelet agonist
  • TPR thromboxane A2 receptor
  • TPRs including platelet aggregation.
  • TPRs' clear role in normal hemostasis is supported by the finding that "patients” have a bleeding disorder as a result of a point mutation in the receptor protein.
  • upregulated signaling through TPR has been implicated in the pathogenesis of multiple cardiovascular and thrombosis-based diseases.
  • thromboxane synthase (TS) inhibitors being the initial lead candidates.
  • the COX inhibitor aspirin remains the only clinically approved agent for therapeutic interventions that target the TXA 2 pathway.
  • the aspirin is in clinical use, it is associated with many inherent limitations, including sever adverse effects (e.g., bleeding), resistance, amongst others.
  • the aspirin was found to: (1) lack selectivity to TXA 2 as it also inhibits PGI 2 synthesis, (2) cause bleeding and gastric ulcers — adverse effects that in some instances mandate its discontinuation, (3) redirect AA metabolism to isoprostanes , which themselves modulate platelet function, and (4) be
  • the inventors describe a vaccine to induce an antagonistic TPR response that can be used to modulate thromboembolic
  • the inventors sought to further assess the contributions of the C-EL2 domain to in vivo TPR-dependent platelet activation (e.g., hemostasis/bleeding time) and the genesis of thrombosis, by employing a vaccine- based approach employing the cognate TPR C-EL2 peptide as an immunogen.
  • immunization of mice with the CEL2 peptide TPR vaccine did indeed lead to the production of a C-EL2 TPR antibody, and inhibit aggregation induced by the TPR agonist U46619, whereas it produced no effects on aggregation stimulated by separate agonists, namely ADP and TRAP4.
  • TPR C-EL2 vaccinated mice exhibited a prolonged time for occlusion, but their bleeding time was no different from the controls.
  • vaccinating mice with the random version of the C-EL2 peptide (i.e., C-EL2r Vac) or KLH exerted no effects on platelet
  • Embodiments generally provide compositions that induce antibodies that bind thromboxane (TX) A2 receptors (TPR) and inhibit thrombosis and other events within the cardiovascular, renal or pulmonary systems.
  • Compositions of the invention prevent TXA2 from binding to the TPR and stimulating platelet activation and aggregation, thereby decreasing the risk of a clinically significant thrombus or embolus.
  • the C-EL2 vaccine provides beneficial pharmaceutical properties for treating thrombosis and other events within the cardiovascular, renal or pulmonary systems .
  • compositions and methods of making and using the same of the present invention can "comprise,” “consist essentially of,” or “consist of” particular ingredients, components, blends, method steps, etc., disclosed throughout the specification.
  • Figure 1 is a graph of immune responses in mice elicited by vaccinations with one of C-EL2, C-EL2r or KLH;
  • Figures 2A—2C are graphs of aggregation responses of platelets from mice vaccinated with one of C-EL2, C-EL2r or KLH;
  • Figures 3A—3C are graphs of dense granule secretion
  • Figures 4A—4C are graphs of alpha (a) granule secretion responses of platelets from mice vaccinated with one of C-EL2, C-EL2r or KLH;
  • Figures 5A—5D are graphs of integrin activation responses of platelets from mice vaccinated with one of C-EL2, C-EL2r or KLH;
  • Figures 6A—6B are graphs of occlusion times of thrombosis in mice vaccinated with one of C-EL2, C-EL2r or KLH;
  • Figures 7A—7B are graphs of platelet-leukocyte aggregate formation responses in samples from mice vaccinated with one of C-EL2, C-EL2r or KLH;
  • Figure 8 is a graph of the expression levels of major platelet integrins of platelets from mice with one of vaccinated with one of C-EL2 , C-EL2r or KLH;
  • Figure 9 is a graph of occlusion times of thrombosis in mice vaccinated with one of C-EL2, C-EL2r or KLH, and
  • Figure 10 is a graph of aggregation responses of platelets from mice vaccinated with one of C-EL2, C-EL2r or KLH, and subsequently injected with the C-EL2 cognate peptide.
  • the illustrative embodiments recognize and take into account one or more different considerations.
  • the illustrative embodiments recognize and take into account that the thromboxane A2 /thromboxane A2 receptor (TXA2-TPR) signaling pathway is known to play an important role in platelet function in vivo, and has been implicated in the genesis of various forms of cardiovascular disorders.
  • TXA2-TPR thromboxane A2 /thromboxane A2 receptor
  • TPR antagonistic activity would be expected to exhibit a better/safer pharmacological profile, and perhaps be more effective in managing thrombosis-based diseases.
  • TS inhibitors exhibited minimal in vivo activity because the immediate precursor of TXA2, PGH2, binds to the same receptor, i.e., TPR and can therefore induce platelet aggregation.
  • TPR antagonists were designed throughout the years and tested for biological activity. While in vitro results were encouraging, the in vivo effectiveness of these molecules was limited by short biological half-life, toxicity or limited tissue distribution.
  • the illustrative embodiments recognize and take into account that, in spite of the clear involvement of TPR signaling in occlusive vascular disease, aspirin is still the only clinically effective drug for the prevention of TPR-mediated platelet activation. Thus, the availability of a
  • pharmacologically effective non-aspirin derivative or C-EL2- derived vaccine with anti-TPR activity could have widespread therapeutic applications, especially given the limitations of current thromboembolic therapy, e.g., resistance, and bleeding associated with COX inhibitor aspirin.
  • the illustrative embodiments described herein constitute the first investigation of vaccine-based antithrombotic agent, and of immunization-based inhibition of TPR function in vivo. Moreover, the illustrative embodiments also provide novel information concerning a potential target site, i.e., C-EL2, for therapeutic intervention protecting against thrombosis. Finally, the functionally active TPR sequence identified herein
  • the illustrative embodiments provide a method and vaccine for treating thrombotic conditions that selectively targets the TXA2/TPR pathway and blocking TXA2- mediated platelet aggregation.
  • C-EL2 segment of SEQ ID NO: 1 includes the amino acid sequence:
  • This extracellular segment contains ligand-amino acid coordination sites.
  • C-EL2Ab An antibody raised against this sequence (i.e., C-EL2Ab) inhibits TPR ligand binding, platelet aggregation in vitro, and protects from thrombogenesis in vivo without any apparent bleeding diathesis or interference with physiological
  • the illustrative embodiments are directed to a therapeutic C-EL2 TPR vaccine.
  • the effects of therapeutic C-EL2 TPR vaccine are predominantly limited to platelet TPRs, in part because the distribution of C-EL2 antibody to compartments other than the vascular system is, in general, restricted due to poor
  • C-EL2 TPR vaccine approaches address bleeding time and thrombosis under conditions of selective modulation of the platelet TPRs, without affecting TPRs of the smooth muscle - which are known to affect bleeding time.
  • a vaccine of the cognate TPR C-EL2 peptide is employed as an immunogen to in vivo TPR- dependent platelet activation, e.g., hemostasis/bleeding time, and the genesis of thrombosis.
  • the CEL2 peptide TPR vaccine induced production of a C-EL2 TPR antibody, and inhibited aggregation induced by TPR agonists. Additionally, the CEL2 peptide TPR vaccine produced no effects on aggregation stimulated by separate agonists, such as ADP and TRAP4.
  • the CEL2 peptide TPR vaccine inhibited TPR-dependent dense and alpha granule release, and GPIIb-IIIa.
  • the TPR C-EL2-based vaccine of the illustrative embodiments protects against thrombogenesis , without impairing hemostasis.
  • this active vaccination approach would not be expected to face some of the functional limitations an antagonist does, including frequent administration and high costs .
  • TPR A2 receptors
  • compositions of the invention prevent TXA2 from binding to the TPR and stimulating platelet activation and aggregation, thereby decreasing the risk of a clinically significant thrombus or embolus.
  • the C-EL2 vaccine provides beneficial pharmaceutical properties for treating thrombosis and other events within the cardiovascular, renal or pulmonary systems .
  • peptide-based vaccines e.g., C-EL2
  • C-EL2 peptide-based vaccines
  • Peptide vaccines are safer and more economic.
  • Peptide vaccine production is also relatively inexpensive due to the ease of production and simplistic composition. Additionally, peptide vaccines avoid the inclusion of unnecessary components possessing high
  • reactogenicity to the host, such as lipopolysaccharides , lipids, or toxins .
  • the present invention further provides compositions or vaccine compositions, comprising at least one active ingredient selected from (a) a peptide of the present invention; or (b) a polynucleotide encoding a peptide of the present invention.
  • compositions of the present invention can comprise as needed a carrier (s), an excipient (s) or such commonly used in pharmaceuticals without particular limitations, in addition to the active ingredient (s) described above.
  • a carrier that can be used in a pharmaceutical composition of the present invention includes sterilized water, physiological saline, phosphate buffer, culture fluid and such. Therefore, the present invention also provides compositions, comprising at least one active ingredient and a pharmaceutically acceptable carrier: (a) a peptide of the present invention; or (b) a polynucleotide encoding a peptide of the present invention in an expressible form.
  • vaccine compositions of the present invention can comprise, as needed, stabilizers, suspensions,
  • preservatives surfactants, solubilizing agents, pH adjusters, aggregation inhibitors and such.
  • compositions comprising an active agent as described herein can be used as a vaccine.
  • the term “vaccine” also called “immunogenic composition” refers to a composition that has a function of inducing an immune response that leads to TPR modulation when inoculated into an animal.
  • composition can be used to induce an immune response that leads to therapeutic action.
  • the immune response induced by a peptide or a polypeptide and a pharmaceutical composition of the present invention is not particularly limited as long as it is an immune response that leads to anti- TPR action.
  • peptides having the amino acid sequence of SEQ ID NO: 2 or a functional variant thereof are peptides that can induce a potent and specific immune response. Therefore, pharmaceutical compositions of the present invention comprising a peptide having the amino acid sequence of SEQ ID NO: 2 or a functional variant thereof is suitable for
  • compositions comprising a
  • compositions of the present invention may also optionally comprise the other therapeutic substances as an active ingredient, as long as they do not inhibit the anti- TPR effects of the above-described active ingredients such as peptides of the present invention.
  • the pharmaceutical composition of the present invention may include other components conventional in the art, in addition to the ingredients specifically mentioned herein.
  • Embodiments can include articles of manufacture or kits that comprise a vaccine composition or components described herein.
  • the articles of manufacture or kits of the present invention can include a container that houses a composition described herein.
  • An example of an appropriate container includes a bottle, a vial or a test tube, but is not limited thereto.
  • the container may be formed of various materials such as glass or plastic.
  • a label may be attached to the container, and the disease or disease state to which the pharmaceutical composition of the present invention should be used may be described in the label.
  • the label may also indicate directions for administration and such.
  • inventions may further comprise a second container that houses pharmaceutically acceptable diluents optionally, in addition to the container that houses the pharmaceutical composition of the present invention.
  • the articles of manufacture or kits of the present invention may further comprise the other materials desirable from a commercial standpoint and the user's
  • the vaccine composition or components thereof can be provided in a pack or dispenser device that can contain one or more units of dosage forms containing active ingredients.
  • the vaccine composition comprising a peptide described herein or functional variant thereof can be formulated by conventional formulation methods as needed.
  • the pharmaceutical compositions of the present invention may comprise as needed in addition to the active ingredient, carriers, excipients and such commonly used in pharmaceuticals without particular limitations. Examples of carriers that can be used in pharmaceutical
  • compositions of the present invention include sterilized water (for example, water for injection), physiological saline, phosphate buffer, phosphate buffered saline, Tris buffered saline, 0.3% glycine, and such. Further, the pharmaceutical compositions of the present invention may comprise as needed stabilizers, suspensions, preservatives, surfactants,
  • compositions of the present invention can induce specific immunity against TPR, and thus can be applied for the purpose of treatment or prevention
  • the vaccine compositions can be prepared by dissolving in pharmaceutically or physiologically acceptable water-soluble carriers such as sterilized water (for example, water for injection) , physiological saline, phosphate buffer, phosphate buffered saline, and Tris buffered saline and adding, as needed, stabilizers, suspensions, preservatives, surfactants, solubilizing agents, pH adjusters, aggregation inhibitors and such, and then sterilizing the peptide solution.
  • the method of sterilizing a peptide solution is not particularly limited, and is preferably carried out by filtration sterilization.
  • the filtration-sterilized peptide solution can be administered to a subject, for example, as an injection, without being limited thereto.
  • the vaccine compositions may be prepared as a freeze- dried formulation by freeze drying the above-described peptide solution.
  • the freeze-dried formulation can be prepared by filling the peptide solution into an appropriate container such as an ampule, a vial or a plastic container, followed by freeze drying and encapsulation into the container with a wash- sterilized rubber plug or such after pressure recovery.
  • the freeze-dried formulation can be administered to a subject after it is re-dissolved in pharmaceutically or physiologically acceptable water-soluble carriers such as sterilized water (for example, water for injection) , physiological saline, phosphate buffer, phosphate buffered saline, Tris buffered saline and such before administration.
  • compositions include injections of such a filtration-sterilized peptide solution, and freeze-dried formulations that result from freeze drying the peptide solution.
  • Certain embodiments encompass kits comprising such a freeze-dried formulation and redissolving solution.
  • the present invention also encompasses kits comprising a container that houses the freeze-dried formulation, which is a pharmaceutical composition of the present invention, and a container that houses a re-dissolving solution thereof.
  • the vaccine compositions can comprise a combination of two or more types of the peptides of the present invention.
  • the combination of peptides can take a cocktail form of mixed peptides, or can be conjugated with each other using standard techniques.
  • peptides can be chemically linked or expressed as single fusion polypeptide sequences.
  • a second peptide is an adjuvant for enhancing the immunogenicity of the first peptide.
  • Suitable adjuvants include peptides such as Keyhole Limpet hemocyanin protein (KLH) ; aluminum salts
  • aluminum phosphate aluminum hydroxide, aluminum oxyhydroxide and such
  • alum cholera toxin
  • Salmonella toxin Incomplete Freund's adjuvant (IFA); Complete Freund's adjuvant (CFA)
  • IFA Incomplete Freund's adjuvant
  • CFA Complete Freund's adjuvant
  • oligodeoxynucleotide containing the CpG motif (CpG7909 and such); oil-in-water emulsions; Saponin or its derivatives (QS21 and such) ; lipopolysaccharide such as Lipid A or its derivatives (MPL, RC529, GLA, E6020 and such); lipopeptides ; lactoferrin; flagellin; doublestranded RNA or its derivatives (poli IC and such); bacterial DNA; imidazoquinolines (Imiquimod, R848 and such); C-type lectin ligand (trehalose-6, 6 '-dibehenate (TDB) and such) ; CDld ligand ( alpha-galactosylceramide and such) ; squalene emulsions (MF59, AS03, AF03 and such); PLGA; virus-like
  • the adjuvant may be contained in the same or another container separate from the peptide composition in the kits comprising vaccine components.
  • the adjuvant and the peptide composition may be administered to a subject in succession, or mixed together immediately before administration to a subject.
  • kits comprising a vaccine composition comprising a peptide and an adjuvant are also provided.
  • the kit can further comprise a redissolving solution.
  • embodiments include kits comprising a container that houses a peptide composition and a container that stores an adjuvant. The kit can further comprise as needed a container that stores the re-dissolving solution.
  • composition may be prepared as an emulsion.
  • Emulsions can be prepared, for example, by mixing and stirring the peptide solution and an oil adjuvant.
  • the peptide solution may be one that has been re-dissolved after freeze drying.
  • the emulsion may be either of the W/O-type emulsion and O/W-type emulsion, and the W/O-type emulsion is preferred for obtaining a high immune response-enhancing effect.
  • IFA can be preferably used as an oil adjuvant, without being limited thereto.
  • Preparation of an emulsion can be carried out immediately before administration to a subject, and in this case, the vaccine composition may be provided as a kit comprising the peptide solution and an oil adjuvant. When the pharmaceutical composition is a freeze-dried formulation, the kit can further comprise a redissolving
  • the pharmaceutical composition of the present invention may be a liposome formulation within which a peptide is encapsulated, a granular formulation in which a peptide is bound to beads with several micrometers in diameter, or a formulation in which a lipid is bound to a peptide.
  • the peptide may also be administered in the form of a pharmaceutically acceptable salt.
  • salts include salts with alkali metals (lithium, potassium, sodium and such), salts with alkaline-earth metals (calcium, magnesium and such) , salts with other metals (copper, iron, zinc, manganese and such) , salts with organic bases, salts with amines, salts with organic acids (acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid and such) , and salts with inorganic acids (hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, nitric acid and such) .
  • pharmaceutically acceptable salt used herein refers to a salt that retains the biological, physiological,
  • compositions comprising a pharmaceutically acceptable salt of a peptide of the present invention are also encompassed by the present invention.
  • Suitable methods for administering the peptides or vaccine compositions include oral, epidermal, subcutaneous, intramuscular, intraosseous, peritoneal, intranasal, and
  • intravenous injections as well as systemic administration or local administration, but are not limited thereto.
  • systemic administration or local administration but are not limited thereto.
  • administration can be performed by single administration or boosted by multiple administrations.
  • the peptides can be
  • the dose of the peptides of the present invention can be appropriately adjusted according to the disease to be treated, the patient's age and weight, the method of administration and such.
  • the dose is usually 0.001 mg-1000 mg, for example, 0.01 mg-100 mg, for example, 0.1 mg-30 mg, for example, 0.1 mg-10 mg, for example, 0.5 mg-5 mg.
  • the dosing interval can be once every several days to several months, and for example, the dosing can be done in a once-per-week interval.
  • a skilled artisan can appropriately select a suitable dose (dosage) .
  • the vaccine compositions include a therapeutically
  • physiologically acceptable carrier and an adjuvant.
  • compositions of the present invention can be any pharmaceutical compositions of the present invention.
  • a vaccine composition when a vaccine composition is an injection, it can comprise a peptide at a concentration of 0.001 mg/ml-1000 mg/ml, preferably 0.01 mg/ml-100 mg/ml, more preferably 0.1 mg/ml-30 mg/ml, even more preferably 0.1 mg/ml-10 mg/ml, for example, 0.5 mg/ml-5 mg/ml.
  • a vaccine composition when a vaccine composition is an injection, it can comprise a peptide at a concentration of 0.001 mg/ml-1000 mg/ml, preferably 0.01 mg/ml-100 mg/ml, more preferably 0.1 mg/ml-30 mg/ml, even more preferably 0.1 mg/ml-10 mg/ml, for example, 0.5 mg/ml-5 mg/ml.
  • a vaccine composition when a vaccine composition is an injection, it can comprise a peptide at a concentration of 0.001 mg/ml-1000 mg/ml, preferably 0.01 mg/ml
  • 0.1 to 5 ml preferably 0.5 ml to 2 ml of the
  • composition of the present invention can be administered to a subject by injection.
  • polynucleotide or peptide refers to a polynucleotide or peptide differing from a specifically recited polynucleotide or peptide of the invention by insertions, deletions, and substitutions, created using, e.g., recombinant DNA techniques.
  • recombinant variants encoding these same or similar peptides may be synthesized or selected by making use of the "redundancy" in the genetic code.
  • Various codon substitutions such as the silent changes that produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system.
  • Mutations in the polynucleotide sequence may be reflected in the peptide or domains of other peptides added to the peptide to modify the properties of any part of the peptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.
  • variant C-EL2 peptide refers to a molecule that differs in amino acid sequence from a "parent" C-EL2 peptide amino acid sequence (e.g., SEQ ID N0:2) by virtue of addition, deletion and/or substitution of one or more amino acid residue (s) in the parent antibody sequence.
  • the variant may comprise at least one to about three
  • a variant C-EL2 peptide may also comprise one or more additions, deletions and/or substitutions in one or more amino acids.
  • the variant peptide will retain the ability to induce an immune response to TPR C-EL2.
  • substitutions can result in replacing one amino acid with another amino acid having similar structural and/or chemical properties, e.g., conservative amino acid replacements.
  • Constant amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • “Insertions” or “deletions” are generally in the range of about 1 to about 20 amino acids, more specifically about 1 to about 10 amino acids, and even more specifically, about 2 to about 5 amino acids. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. For example, amino acid substitutions can also result in
  • Certain embodiments are directed to an expression vector and/or a host cell that comprises one or more polynucleotide sequence encoding a peptide described herein.
  • the host cell or expression vector comprises any one or more of the polynucleotides or polynucleotides encoding the peptides and/or functional variants thereof.
  • the host cell or expression vector comprises one or more polynucleotides encoding peptide (s) provided herein.
  • the illustrative examples use a mouse keyhole limpet hemocyanin/peptide-based vaccination approach rationalized over the TPR ligand-binding domain, i.e., the C-terminus of the second extracellular loop.
  • the mouse and the human platelet TPRs are identical in 17 out of the 21 amino acid that are located in the second extracellular loop region of the receptor protein. The biological activity of this vaccine was assessed in the context of platelets and thrombotic diseases, and using a host of in vitro and in vivo platelet function experiments .
  • mice were immunized with keyhole limpet hemocyanin- (KLH) coupled peptide corresponding to SEQ ID NO. 2.
  • Control animals are immunized with a randomized peptide having the amino acid sequence
  • SEQ ID NO. 3 STLACGFDGEL
  • cysteine synthesized at the end of the peptides allow coupling with KLH (CSTLACGFDGEL) , or with KLH alone.
  • Mice received intraperitoneal injections of peptides (35 pg) or KLH dissolved in Freund's complete adjuvant. The animals were boosted 3 times with peptides (and Freund's incomplete adjuvant) .
  • Mouse blood was collected from a ventricle and the citrated (0.38%) blood was mixed with phosphate-buffered saline, pH 7.4, and was incubated with PGI2 (10 ng/mL; 5 minutes), followed by centrifugation at 237 c g for 10 minutes at room temperature (RT) .
  • PGI2 10 ng/mL; 5 minutes
  • RT room temperature
  • PRP Platelet-rich plasma
  • HEPES/Tyrode buffer (HT; 20 mM HEPES/KOH, pH 6.5, 128 mM NaCl , 2.8 mM KC1, 1 mM MgCl 2 , 0.4 mM NaH 2 P0 4 , 12 mM NaHC0 , 5 mM d-glucose) supplemented with 1 mM EGTA, 0.37 U/mL apyrase, and 10 ng/mL PGI2. Platelets were washed and resuspended in HT (pH 7.4) without EGTA, apyrase, or PGI2. Platelets were counted with an automated hematology analyzer (Drew Scientific Dallas, TX) and adjusted to the indicated concentrations.
  • HEPES/Tyrode buffer (HT; 20 mM HEPES/KOH, pH 6.5, 128 mM NaCl , 2.8 mM KC1, 1 mM MgCl 2 ,
  • the TPR C-terminus of the second extracellular loop vaccine (1) triggered an immune response, which resulted in the development of a C-terminus of the second extracellular loop antibody; (2) did not affect expression of major platelet integrins (e.g., glycoprotein Ilb-IIIa) ; (3) selectively inhibited TPR-mediated platelet aggregation, platelet-leukocyte aggregation, integrin glycoprotein Ilb-IIIa activation, as well as dense and a granule release; (4)
  • major platelet integrins e.g., glycoprotein Ilb-IIIa
  • Enzyme-linked immunosorbent assays were performed to determine antibody development in the immunized mice (C-EL2 Vac, CEL2r Vac, and KLH) .
  • Nunc-ImmunoTM MicroWellTM 96-Well plates were coated with 12.5 pg/well C-EL2 peptide for 18-24 h at room temperature.
  • the plates were washed three times with 200 pL/well modified Tyrode ' s buffer (0.1% bovine serum albumin, 20 mM HEPES/KOH, pH 7.4, 128 mM NaCl, 2.8 mM KC1, 1 mM MgCl2, 0.4 mM NaH2PC>4, 12 mM NaHCCg, 5 mM d-glucose) , and then nonspecific sites were blocked by incubation for 1 h with 5% bovine serum albumin (200 pL/well) in the same buffer.
  • 200 pL/well modified Tyrode ' s buffer 0.1% bovine serum albumin, 20 mM HEPES/KOH, pH 7.4, 128 mM NaCl, 2.8 mM KC1, 1 mM MgCl2, 0.4 mM NaH2PC>4, 12 mM NaHCCg, 5 mM d-glucose
  • C-EL2 As indicated in Figure 1, significant levels of the C-EL2 antibody was observed when C-EL2 was used as an immunogen, unlike C-EL2r and KLH .
  • the relative antibody concentrations illustrated in indicates that C-EL2 vaccination successfully elicits an immune response targeting the C-terminus of the second extracellular loop of the thromboxane A2 receptor.
  • Vaccinations with the C-EL2 antigen show a normal platelet count, and other blood parameters, relative to the control. As indicated, the C-EL2 TPR vaccine elicits an immune response, without altering peripheral blood count.
  • Platelet count were measured 1, 2, and 3 months after the final boost of C-EL2 vaccine. Time Course of Platelet Count and Antibody Titer in C-EL2 Vaccinated Mice are indicated in Table 2.
  • Aggregation response was measured to determine whether in vivo immunizations with the C-EL2 peptide treatment would also translate into inhibition of platelet aggregation by the C-EL2 vaccine (i.e., antibody generated from this vaccine) .
  • Platelet secretion is an important and early event in platelet activation, and is known to be triggered by TXA2. As illustrated in Figures 3A-3C, C-EL2 TPR vaccine inhibits platelet dense granule and alpha granule secretion.
  • Platelets from vaccinated mice were prepared as described above (250 pL; 2.5 c 108/mL) before being placed into siliconized cuvettes and stirred for 5 min at 37°C at 1200 rpm.
  • the luciferases substrate/luciferase mixture (12.5 pL, Chrono-Log) was then added, followed by the addition of the agonists U46619 (1 mM) , ADP (5 mM) or TRAP4 (80 mM) .
  • the C-EL2 (35 pg) vaccine inhibited platelet dense granule secretion (ATP release) , and alpha granule secretion (P-selectin expression), in response to the TPR agonist U46619 (ImM; Fig. 3A and 4A) , when compared with C-EL2r vaccine or KLH .
  • the C-EL2 vaccine did not inhibit ATP release and P-selectin
  • TPR C-EL2 vaccine has the capacity to inhibit platelet aggregation, it was determined whether the TPR C-EL2 vaccine would be associated with a commensurate inhibition of integrin allbb3 activation.
  • Flow cytometric analysis was carried out on platelets from vaccinated mice (C-EL2, C-EL2r and KLH) as discussed before. Briefly, platelets (2x108) were stimulated 1 mM U46619, 5 mM ADP, or 80 mM TRAP4 for 3 minutes. The reactions were stopped by fixing the platelets with 2% formaldehyde for 30 min at room temperature. Finally, platelets were incubated with FITC- conjugated JON/A or anti- P-selectin antibodies at room
  • C- EL2 Vac The C-terminus of the second extracellular loop vaccine (C- EL2 Vac) , but not the random C-EL2 (C-EL2r) or keyhole limpet hemocyanin (KLH) , inhibits integrin activation.
  • Immunization with C-EL2 antigen (35 pg) results in significant inhibition of U46619-triggered JON/A binding (1 mM; Figure 5A) , but not that induced by ADP (5 mM; Figure 5B) or TRAP4 (80 mM; 5C) , which indicates abrogation of allbb3
  • TPR antagonist SQ29,548 inhibited U46619 (1 mM) -mediated glycoprotein Ilb-IIIa activation in the
  • C- EL2 Vac The C-terminus of the second extracellular loop vaccine (C- EL2 Vac) prolongs the time for occlusion, but not the tail bleeding time, whereas the random C-EL2 (C-EL2r) or keyhole limpet hemocyanin (KLH) has no effect.
  • mice C-EL2, C-EL2r and KLH were anesthetized with isoflurane. Then, the left carotid artery was exposed and cleaned, and baseline carotid artery blood flow was measured with Transonic micro-flowprobe (0.5 mm, Transonic
  • mice Hemostasis in the vaccinated mice (C-EL2, C-EL2r and KLH) was examined using the tail transection technique. Briefly, mice were anesthetized with isoflurane and place on a 37°C
  • C-EL2 vaccinated mice had tail bleeding time that was no different from those that were vaccinated with C-EL2r or KLH (Fig. 6B) . These results provide evidence that a C-EL2 TPR vaccine exerts anti-thrombotic activity, without increasing the risk of bleeding.
  • Platelet-leukocyte aggregates are known to contribute to the pathogenesis of thrombotic disorders, and TPR antagonists were found to reduce their formation.
  • the C-terminus of the second extracellular loop vaccine (C-EL2 Vac) inhibits
  • C-EL2 Vac second extracellular loop vaccine
  • mice were injected with 8 mg/kg of the C-EL2 cognate peptide, and one-hour post injection were
  • the C-EL2 vaccine prolongation of the time for occlusion is reversed by administering the C-EL2 cognate peptide ( Figure 9) .
  • the C-EL2 vaccinated mice were first injected with 8 mg/kg of the C-EL2 cognate peptide, and one-hour post injection their platelets, along with those from non-cognate peptide injected C- EL2 as well as KLH vaccinated mice were collected. Platelets were then stimulated with 1 mM U46619 and their aggregation response was monitored using an aggregometer . Each experiment was repeated 3 times, with blood pooled from at least 6-8 mice each time.
  • the C-EL2 vaccine-mediated inhibition of aggregation is reversed by administering the C-EL2 cognate peptide ( Figure 10) .

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Abstract

L'invention concerne un vaccin contre le domaine de liaison de ligand de domaine tétratricopeptide (TPR), à savoir l'extrémité C de la seconde boucle extracellulaire (C-EL2), inhibant l'activation plaquettaire et la formation de thrombus, sans exercer d'effets sur l'hémostase.
PCT/US2019/025612 2019-04-03 2019-04-03 Vaccin à base de récepteur de thromboxane pour la gestion de la thrombogenèse WO2020204923A2 (fr)

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