WO2008068048A2 - Nouveau vaccin passif pour infections de candida - Google Patents

Nouveau vaccin passif pour infections de candida Download PDF

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Publication number
WO2008068048A2
WO2008068048A2 PCT/EP2007/011075 EP2007011075W WO2008068048A2 WO 2008068048 A2 WO2008068048 A2 WO 2008068048A2 EP 2007011075 W EP2007011075 W EP 2007011075W WO 2008068048 A2 WO2008068048 A2 WO 2008068048A2
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WIPO (PCT)
Prior art keywords
candida
antibody
passive vaccine
seq
vaccine according
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PCT/EP2007/011075
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English (en)
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WO2008068048A3 (fr
Inventor
Antonio Cassone
Fabio Malavasi
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Istituto Superiore Di Sanita
Universita Di Torino
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Priority to EP07847070A priority Critical patent/EP2087004A2/fr
Publication of WO2008068048A2 publication Critical patent/WO2008068048A2/fr
Publication of WO2008068048A3 publication Critical patent/WO2008068048A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • A61P31/10—Antimycotics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to a passive vaccine for use in the prophylaxis and treatment of Candida sp. infection.
  • Passive vaccination is particularly attractive for the control of opportunistic infections which rely upon immune dysfunction to cause disease in a debilitated host. Absence or reduction of immune competence reduces the ability of the host to cooperate with antimicrobials and also makes it particularly hard to generate a protective vaccine.
  • Candida sp. including C. albicans, C. tropicalis and C. parapsilosis, are extracellular, opportunistic pathogens.
  • Candida albicans is a major mucosal and systemic pathogen, and is particularly opportunistic in immunocompromised hosts, especially in neutropenic patients or those with AIDS.
  • it is also a frank pathogen in the setting of vaginal candidiasis and other mucocutaneous pathologies, causing disease in both normal and immunocompromised patients. It has been estimated that three quarters of all normal women in fertile age experience at least one attack of acute vaginal candidiasis, and more importantly, about 5% of them suffer from chronic recurrences thereafter.
  • Candida albicans has defined virulence traits and, most importantly, studies with experimental animal models of infection have recently demonstrated that antibodies of the right specificity and isotypes can indeed protect against severe mucosal and systemic experimental infections. Some antibodies are in phase 1 clinical trials.
  • SAP enzymes are amply secreted by the fungus and degrade the keratinous layers of the skin.
  • vaginal infections where SAP enzymes are able to degrade both the keratinous layer overlying the epithelial cuboid tissue and protective soluble factors of innate or adaptive immunity such as the cytokines and antibodies
  • SAP family One of the ten members of SAP family, the S AP2 enzyme, is particularly relevant as a virulence factor, and so antibodies against SAP2 have been shown, in active vaccines, to be protective (Reference 5).
  • Table 1 shows a sequence alignment of the conserved catalytic sites of several aspartyl proteases of both viral and eukaryotic organisms, providing the rationale for some form of shared susceptibility to protease inhibitors 6 .
  • SAP2 of C. albicans is particularly similar at this site to HFV protease and the plasmepsins of P. falciparum.
  • the antibodies are not degraded by the enzymatic activity of the SAP proteases, which would normally be expected to cleave most proteins, including antibodies. These antibodies are, therefore, useful therapeutics against mucosal, cutaneous and systemic infections by Candida sp., especially in a passive vaccine.
  • the present invention provides a passive vaccine, for use in the treatment or prophylaxis of a Candida sp. infection, comprising an antibody, or a fragment thereof, capable of recognising at least one epitope from a Secreted Aspartyl Protease (SAP) enzyme from Candida sp., the antibody or fragment thereof being substantially resistant to degradation by the Secreted Aspartyl Proteases.
  • SAP Secreted Aspartyl Protease
  • Antibodies and polynucleotides encoding them are also provided.
  • the Antibody may be of any form, but monoclonal antibodies (mAb) are particularly preferred.
  • the present invention also provides antibodies, or fragments thereof, capable of recognising an epitope from an SAP enzyme from Candida sp. the antibody or fragment thereof being substantially resistant to degradation by said enzyme.
  • a passive vaccine is provided, for use in the treatment or prophylaxis of a Candida sp. Infection.
  • the vaccine comprises an antibody, or a fragment thereof, capable of recognising an epitope from a Secreted Aspartyl Protease (SAP) enzyme from Candida sp.
  • SAP Secreted Aspartyl Protease
  • the antibody, or fragment thereof is substantially resistant to degradation by the Secreted Aspartyl Proteases.
  • references to one epitope or one SAPs may be understood to include one or more such epitopes or SAPs unless otherwise apparent.
  • the antibody discovered by the present inventor are substantially resistant to protease activity, in particular the aspartyl proteases, SAPl, SAP2 and SAP3, or other bacterial and host proteases possibly present in female vaginal fluids, such that they retain sufficient activity to allow immune clearance of the proteases. Immune clearance is thought to be through the stimulation of the complement pathways.
  • the antibodies are resistant to cleavage by the proteases, at least to extent that they retain at least 50% and more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 99% of their immune clearance ability in the presence of the protease, for instance under suitable conditions for the protease to cleave suitable target proteins.
  • Such ability is preferably the ability of the antibody to bind to a protease and thereafter stimulate the complement pathway and lead to the ultimate destruction of the protease. This may be measured by modifying the proteases themselves, for instance at the active site, so that their proteolytic function is reduced, thus allowing a comparison of the activity of the Antibodies in immune clearance of the albeit modified proteases.
  • the Ab according to the present invention binds to one or more SAPs as herein defined with a K 0S rate constant of between 5 xlO "1 and 1 xlO "7 s "1 .
  • the Ab according to the present invention binds to the SAP with a dissociation constant (Kd) of at least 100 microMolar to 1 picoMolar.
  • Candida infections of any species maybe treated using one or more Abs according to the present invention.
  • Suitable Candida spp infections for treatment using the Abs of the present include any of those in the group consisting of the following: Candida ciferrii, Candida famata, Candida lambica, Candida lipolytica, Candida norvegensis, Candida rugosa, Candida viswanathii, Candida zeylanoides, Candida albicans, Candida tropicalis, Candida glabrata, Candida par apsilosis, Candida krusei, Candida lusitaniae, Candida kejyr, Candida guilliermondii and Candida dubliniensis.
  • Candida spp. for treatment according to the invention include any of those in the group consisting of the following: Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida krusei and Candida lusitaniae.
  • Candida albicans is particularly preferred.
  • the present invention provides an Ab according to the present invention for use in the prophylaxis and/or treatment of Candida spp. infection.
  • the present invention provides an Ab according to the present invention for use in manufacture of a medicament for the prophylaxis and/or treatment of Candida spp. infection.
  • the present invention provides a method for the prophylaxis and/or treatment of azole resistant Candida spp. infection in a patient by administering to a patient in need of such treatment an Ab according to the present invention wherein the Candida is resistant to any one or more agent/s in the group consisting of the following: itraconazole, fluconazole and voriconazole, caspofungin, micafungin.
  • the present invention provides a method for the treatment of imidazole resistant Candida spp. infection in a patient by administering to a patient in need of such treatment an Ab according to the present invention wherein the Candida spp. infection is resistant to any one or more agents in the group consisting of the following: Clotrimazole, econazole, fenticonazole, sulconazole and tioconazole.
  • composition comprising an Ab according to the present invention and a pharmaceutically acceptable carrier, diluent and/or excipient.
  • the antibody has a Light Chain according to SEQ ID NO. 1, or a functional variant thereof.
  • the antibody has a Heavy Chain according to SEQ ID NO. 2, or a functional variant thereof.
  • the antibody has a Light Chain according to SEQ ID NO. 3, or a functional variant thereof.
  • CDRs Complementarity Determining Regions
  • the Abs are humanized Abs.
  • the Abs are human variable domains or comprise human framework regions (FWs) and preferably one or more heterologous CDRs which bind specifically to one or more SAP proteins described herein.
  • CDRs and framework regions are those regions of an immunoglobulin variable domain as defined in the Kabat database of Sequences of Proteins of Immunological Interest.
  • the CDRs are retained verbatim, and preferably within a similar framework, it is necessary for any variant to retain the ability to substantially resist degradation by the proteases. This will be readily apparent to the skilled person as any variant Abs can be readily assayed for their resistance to the proteases.
  • An example is provided in the Examples section.
  • the CDRs of the Light Chain from NL2/2A8 are provided as SEQ ID NOS. 11, 12 and 13. Thus, it is particularly preferred that these are retained in any variant.
  • the CDRs of the Heavy Chain from NL2/2A8 are provided as SEQ ID NOS. 14, 15 and 16. Thus, it is particularly preferred that these are retained in any variant.
  • the CDRs of the Heavy Chain from NL2/9B9 are provided as SEQ ID NOS. 17, 18 and 19. Thus, it is also particularly preferred that these are retained in any variant.
  • the antibody or functional variant thereof has 85% or more sequence identity to the respective SEQ NO., provided that at least two CDRs are retained.
  • Suitable frameworks also referred to as scaffolds, are known in the art and include human domain Abs (from Domantis), Camel/Llama V H H (from Ablynx), Human TCRs (from Avidex) and so forth.
  • the vaccine comprises a mixture of antibodies NL2/ 2A8 and NL2/ 9B9, including fragments or functional variants thereof.
  • the functional variants have preferably 70% or greater sequence identity to one of said sequence ID numbers, preferably 80%, preferably 85%, more preferably 90%, more preferably 95%, more preferably 99% and most preferably 99.9% sequence identity to any one of sequence ID numbers 1 to 3.
  • the antibody is a monoclonal antibody and, preferably, may also be a hybrid or chimeric antibody.
  • the antibody is Immunoglobulin type IgM or IgG.
  • the antibodies are those referred to herein as NL2/2A8, NL2/9B9, according to SEQ ID NOS. 1-2 and 3, respectively.
  • the present invention also provides polynucleotide sequences, preferably DNA or mRNA, encoding the amino acid sequences of SEQ NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3.
  • the antibodies are NL2/9B9 (SEQ ID NO. 3) or a functional variant thereof, or more preferably, NL2/2A8 (SEQ ID NOS. 1 and 2) or a functional variant thereof. These two antibodies show the greatest inhibition of SAP2 activity, as shown in Table 2, thereby providing the most therapeutically effective options for inclusion in a passive vaccine.
  • the passive vaccine may comprise only one type of the antibodies of the present invention, or may comprise a mixture of 2 or more antibodies. Particularly preferred is a vaccine comprising a mixture of the antibodies according to SEQ ID NOS. 1 - 2, and 3, including fragments or functional variants thereof.
  • the A8 and B9 antibodies recognise different epitopes on each of the SAP sub-family of enzymes, comprising SAP 1, 2 and 3.
  • these antibodies are shown in the example to be at least as effective as Pepstatin A, a known aspartyl protease inhibitor, and the known anti-Candidiasis drug, fluconazole.
  • the passive vaccine has equivalent, i.e. statistically significant, or greater, efficacy compared to known treatments for Candidiasis, particularly Pepstatin A or fluconazole.
  • Passive vaccines are well known in the art, but the skilled person will understand that therapeutically effective and pharmaceutically acceptable titres of antibodies according to the present invention will be required.
  • An active vaccine stimulates the host's immune system to produce specific antibodies or cellular immune responses or both, which would protect against or eliminate a disease.
  • a passive vaccine is in general a preparation of antibodies that neutralizes a pathogen and may be administered before or around the time of known or potential exposure.
  • Most references to the term "vaccine" per se are to active vaccines, which are the object of the vast majority of research and development activities in the field.
  • the passive vaccine may be administered intranasally or intravaginally. However, it is also envisaged that other modes of administration, such as oral, rectal, intravenous or parental administration could also be effective.
  • the antibodies may be expressed in the patient by means of gene therapy.
  • the vaccine will, therefore, comprise a suitable means for in situ expression of the antibodies, such as a suitable viral vector comprising polynucleotides encoding the antibody, preferably under the control of a suitable promoter, for instance.
  • the passive vaccine may be administered in the form of a cream or ointment, the cream or ointment comprising a therapeutically effective and pharmaceutically acceptable titre of the above-mentioned antibodies according to the present invention.
  • the antibody can be part of a therapeutic cocktail which may include other drugs or other anti-Candida antibodies Where reference to antibodies is made, it will be understand that this also includes functional variants thereof, as defined above.
  • the present invention also provides method of treatment or prophylaxis of a Candida sp. infection, comprising selecting a patient and administering an appropriate quantity of antibody according to the present invention, in the form of a passive vaccine, where the passive vaccine is as described above.
  • the invention also provides the use of an antibody or a functional variant thereof as described above, in the manufacture of a medicament for the treatment or prophylaxis of a Candida sp. infection.
  • the present invention is suitable for the treatment or prophylaxis of a range of Candidiasis infections, preferably mucosal or systemic Candidiasis. Vaginal Candidiasis is most particularly preferred.
  • the Candida species is Candida albicans, but is also effective against other Candida species, particularly C. tropicalis and/or C. parapsilosis.
  • the antibodies target and recognise epitopes on the SAP family of Candida proteases enzymes, most preferably the SAP 1-3 sub-family, including SAPl, SAP2 and SAP3. SAP2 is particularly preferred.
  • the epitopes recognised by the different antibodies of the invention may be the same, but are preferably different, such that each antibody preferably recognises a different epitope on SAP2, for instance. Due to the conserved nature of the protease family, it is preferred that each antibody recognises the same broadly conserved epitope on the different family members.
  • the fragment or functional variant of the antibody is capable of specifically binding to and recognising the epitope of the target enzyme.
  • the fragment may be a particular portion of the Heavy or Light chains of the invention, and not necessarily just the variable domain, although this is preferred.
  • restriction of the sequence may alter the folding or reveal new protease sites, so the ability to resist degradation must still be assayed, although this I relatively simple, as discussed above.
  • Single domain antibodies are preferred, for instance using the methods those taught in WO 2006/097689 (Domantis Ltd), although resistance to degradation will need to be assessed.
  • the Ab may consist of only a single chain according to the present invention, or may simply comprise a single chain of the invention. Alternatively, the Ab may consist of more two, or possibly more, chains according to the present invention.
  • the advantage of being able to deliver the antibodies in a passive vaccine is that active vaccines cannot be used in some circumstances, particularly in immunocompromised hosts in whom vaccines are unable to mount an effective immune response, even though it is in these hosts that opportunistic infections like Candida sp. axe most likely to occur. Furthermore, as mentioned above, one cannot be sure, when generating a protective immune response using an active vaccine, for instance, that the antibodies generated will not be degraded by their protease targets.
  • mice Two female Balb/c mice (Harlan) were immunized intrasplenically with 15 mg each of highly purified, recombinant, 6-his tailed SAP2 protein (Sandini et ah, submitted), suspended in 200 microliters of saline, followed by chronic boosting via subcutaneous injections at two weeks intervals of the same amount of protein suspended in Freund's adjuvant for three months. Four days after the last booster, the spleen was removed and fused with the X63.Ag8.653 cell line according to standard hybridoma protocols.
  • Clones were screened through ELISA against the recombinant antigens, excluding all those reactive with another recombinant antigen (enolase), thus bona fide recognizing the histidine tail.
  • Genuine SAP-binders were further cloned and selected for their capacity to inhibit SAP2 enzymatic activity using pepstatin A as positive control 6 .
  • pepstatin A as positive control 6 .
  • two mAbs named NL/2.A8 and NL/9.B9, were propagated as ascites and used for biochemical and functional testing.
  • Both mAbs were determined to belong to IgM class by their SDS-PAGE profile and their reactivity in ELISA and Western blot assays with affinity isolated, alkaline phosphatase-conjugated goat anti- mouse M or G chain antibodies (Sigma).
  • Table 2 shows the ability of the NL/2.A8 and NL/9.B9 mAbs to neutralise the activity of SAP2.
  • the data refers to a typical determination out of three performed with similar results.
  • Ref.5 incorporated herein by reference.
  • Tables 3 and 4 show the antibody titres required for the NL/2.A8 and NL/9.B9 antibodies to recognise the SAPl, SAP3, SAP2 (full) and the two SAP2 fragment antigens.
  • Table 3 - Antibody reactivity with SAP 1-3 protein family and SAP-2 fragments*,** Reactivity with mAb NL2/2A8
  • Antigen used at a concentration of 200ng/ml Antibodies were ascitic fluids. All ELISA positive reactivities were confirmed in Western Blot using polyvalent anti- mouse IgG. There were no ELISA reactivities with any SAP or fragment using the irrelevant anti- enolase mAb.
  • both mAbs recognised SAPl and SAP3, in addition to SAP2, as expected, but differed from each other in their binding strength to the three SAP antigen proteins.
  • both mAbs target an epitope common to the three SAP members.
  • the epitope is not shared by the two mAbs as demonstrated by the observation that mAbNL2/2.A8 does not recognize either SAP2 fragment whereas mAb NL2/9.B9 strongly reacts with the IB 1 N-terminus fragment of SAP2.
  • each mAb binds to a different epitope, each epitope being common to all three SAP members. No reaction occurs of any SAP with the anti-enolase mAb.
  • the anti-enolase antibody (see above) was used a as negative control.
  • the model efficiency, significance and relevance for human disease has been discussed elsewhere 4
  • the treatment with either anti-SAP mAb was as efficacious as the treatment with pepstatin A and a therapeutic fluconazole dosage (Figure 2).
  • Both anti-SAP mAbs neutralize SAP2 enzymatic activity, though recognizing distinct epitopes of this enzyme, shared by at least other two members of the ten-member SAP family, i.e. SAPl and SAP3. It has to be stressed here that, firstly, SAP 1-3 sub-family expression is critically required for mucosal infection by Candida albicans, as demonstrated by gene knock-out experiments 9 and, secondly, that SAP virulence is explained by the capacity of these enzymes to hydrolyse a wide number of protein substrates, including immunoglobulins. Therefore, these mAbs are the first to neutralize SAP activity, rather than be degraded by the enzyme.
  • both anti-SAP mAbs exert a therapeutic effect in an estrogen-dependent rat vaginitis model, an effect which compares with the activity exerted by a prototypal inhibitor of aspartyl proteases (pepstatin A) and, more importantly, by fluconazole, a widely used and efficacious anticandidal drug.
  • pepstatin A prototypal inhibitor of aspartyl proteases
  • SEQ ID NO. 1 (SEQ ID NOS 11 , 12 and 13 are underlined in order) Sequence of Light Chain for Ab from clone NL2/ 2A8:

Abstract

L'invention concerne un vaccin passif, destiné à une utilisation pour le traitement ou la prophylaxie d'une infection de Candida sp. Le vaccin comprend un anticorps, ou un fragment d'anticorps, capable de reconnaître un épitope d'une enzyme protéase aspartyl sécrétée (SAP) de Candida sp. L'anticorps, ou le fragment d'anticorps, est sensiblement résistant à la dégradation par les protéases aspartyl sécrétées. L'invention concerne également des anticorps et des polynucléotides codant pour ces anticorps.
PCT/EP2007/011075 2006-12-07 2007-12-07 Nouveau vaccin passif pour infections de candida WO2008068048A2 (fr)

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EP07847070A EP2087004A2 (fr) 2006-12-07 2007-12-07 Nouveau vaccin passif pour infections de candida

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GBGB0624500.5A GB0624500D0 (en) 2006-12-07 2006-12-07 A novel passive vaccine for candida infections
GB0624500.5 2006-12-07

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