WO2009055936A1 - Anticorps humanisés de recombinaison contre le virus de l'encéphalite équine du vénézuéla - Google Patents

Anticorps humanisés de recombinaison contre le virus de l'encéphalite équine du vénézuéla Download PDF

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WO2009055936A1
WO2009055936A1 PCT/CA2008/001940 CA2008001940W WO2009055936A1 WO 2009055936 A1 WO2009055936 A1 WO 2009055936A1 CA 2008001940 W CA2008001940 W CA 2008001940W WO 2009055936 A1 WO2009055936 A1 WO 2009055936A1
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seq
antibody
veev
hu1a4a1
amino acid
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PCT/CA2008/001940
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Wei-Gang Hu
Leslie P. Nagata
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Her Majesty The Queen In Right Of Canada As Represented By The Minister Of National Defence
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Priority to GB201009145A priority Critical patent/GB2467491C/en
Priority to CA2704119A priority patent/CA2704119C/fr
Priority to US12/740,826 priority patent/US8501157B2/en
Publication of WO2009055936A1 publication Critical patent/WO2009055936A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/461—Igs containing Ig-regions, -domains or -residues form different species
    • C07K16/464—Igs containing CDR-residues from one specie grafted between FR-residues from another
    • 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
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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 humanized antibodies (Abs) and, more specifically, to humanized recombinant antibodies (rAbs) against infection by the Venezuelan equine encephalitis virus (VEEV).
  • VEEV Venezuelan equine encephalitis virus
  • the invention provides methods of prophylaxis and treatment against VEEV using such antibodies.
  • Venezuelan equine encephalitis virus a member of the alphavirus genus of the family Togaviridae, is an important mosquito-borne pathogen in humans and equides [1].
  • VEEV infections mainly target the central nervous system and lymphoid tissues causing severe encephalitis in equines and a spectrum of human diseases ranging from unapparent or sub- clinical infection to acute encephalitis. Neurological disease appears in 4-14% of cases. The incidence of human infection during equine epizootics could be up to 30%. Mortality associated with the encephalitis in children. is as high as 35%.
  • VEEV is highly infectious by aerosol inhalation in humans and other animals.
  • antiviral drugs available that are effective against VEEV although currently there are two forms of IND (investigational new drug) VEEV vaccines available for human and veterinary use: TC-83, a live-attenuated Trinidad donkey strain and C-84, a formalin-inactivated TC-83 [3,4].
  • these vaccines are far from satisfactory. For example, approximately 20% of recipients that receive the TC-83 vaccine fail to develop neutralizing Abs, while another 20% exhibit reactogenicity.
  • the TC-83 vaccine could revert to wild-type form.
  • the vaccine C-84 is well tolerated, but requires multiple immunizations, periodic boosts, and fails to provide protection against aerosol challenge in some rodent models.
  • VEEV is an enveloped virus, consisting of three structural proteins: a capsid encapsidating the viral RNA genome, and two envelope glycoproteins, E1 and E2. E1 and E2 form heterodimers, which project from the virus envelope as trimer spikes. Epitopes on the spikes are the targets of neutralizing Abs. Studies have shown that the viral neutralizing epitopes are mainly located on the E2 protein, and that the E2C epitope appears to be the hub of the neutralization epitopes [5,6]. The murine monoclonal Ab (mAb) 1A1A4 [14] is specific for E2C.
  • mAb monoclonal Ab
  • Murine mAbs have serious disadvantages as therapeutic agents in humans [8]. For example, one of the problems associated with using murine mAbs in humans is that they may induce an anti-mouse Ab response. Further, repeat administration of murine mAbs may result in rapid clearance of the murine mAbs and anaphylaxis, which can sometimes be fatal. To overcome this hurdle, the humanization of murine mAbs has been proposed, by which process murine Ab frameworks are replaced by human Ab ones in order to reduce immunogenicity of Abs in humans [9,10]. [0006] An effective means of immunization againt VEEV is needed. In particular, a means of prophylaxis against VEEV and/or a therapy for VEEV infection is desired.
  • the present invention provides prophylaxis and post-exposure therapy against VEEV infection.
  • the invention provides a humanized rAb comprising a human immunoglobulin (Ig) framework and having grafted thereon complementarity determining regions (CDRs) from the murine mAb 1 A4A1.
  • the human Ig framework is obtained from IgGL
  • the invention provides a humanized rAb having specificity to the E2 envelope protein of VEEV. More specifically, the rAb has specificity to the E2 C epitope of the E2 protein.
  • the invention provides a humanized rAb wherein the complementarity determining regions CDR1 , CDR2 and CDR3 of the heavy chain variable region (VH) have the following amino acid sequences: CDR1: SEQ ID NO: 1 CDR2: SEQ ID NO: 2 CDR3: SEQ ID NO: 3.
  • the invention provides a humanized rAb wherein the complementarity determining regions CDR1 , CDR2 and CDR3 of the light chain variable region (VL) have the following amino acid sequences: CDR1: SEQ ID NO: 4 CDR2: SEQ ID NO: 5 CDR3: SEQ ID NO: 6. [0012] In a further aspect, the invention provides a humanized rAb having a VH comprising the amino acid sequence of SEQ ID NO: 7. [0013] In a further aspect, the invention provides a humanized rAb having a VL comprising the amino acid sequence of SEQ ID NO: 8.
  • the invention provides a DNA sequence which encodes a polypeptide corresponding to a CDR grafted VH having the amino acid sequence according to SEQ ID NO: 7. [0015] In another aspect, the invention provides a DNA sequence which encodes a polypeptide corresponding to a CDR grafted VL having the amino acid sequence according to SEQ ID NO: 8. [0016] In a further aspect, the invention provides a DNA construct having a nucleic acid sequence according to SEQ ID NO: 11 or SEQ ID NO:13. [0017] In another aspect, the invention provides an expressed protein comprising a humanized rAb having an amino acid sequence according to SEQ ID NO: 12 or SEQ ID NO: 14.
  • the invention provides vectors containing such DNA sequences and host cells transformed thereby. [0019] In other aspects, the invention provides methods and uses for treatment and/or prophylaxis against VEEV infection comprising the antibodies described herein. The invention also provides pharmaceutical preparations for such treatment or prophylaxis.
  • Figure 1 is a representation of the external structure of the VEEV.
  • Figures 2a to 2d schematically illustrate murine, human, chimeric and humanized Abs, respectively.
  • Figures 3a to 3c schematically illustrate the humanization of the murine Ab variable region.
  • Figure 4 schematically illustrates the cloning of the murine Ab VH and VL.
  • Figure 5 schematically illustrates the humanization of the Ab VH and shows its amino acid sequence.
  • Figure 6 schematically illustrates the humanization of the Ab VL and shows its amino acid sequence.
  • Figure 7 schematically illustrates the design of a full Hu1A4A1 IgGI rAb gene in a single open reading frame with two versions, Hu1A4A1 lgG1-furin and Hu1A4A1 lgG1-2A.
  • Figure 8 schematically illustrates the cloning of the Hu1A4A1 lgG1-furin and Hu1 A4A1 lgG1-2A genes into an adenoviral vector respectively.
  • Figure 9 schematically illustrates expression and purification of the Hu1A4A1 IgGI- furin and Hu1A4A1 IgGI -2A rAbs.
  • Figures 10 and 11 illustrate the results from the SDS-PAGE separation of the produced Hu1A4A1 IgGI -furin rAb.
  • Figure 12 illustrates the results from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of the produced Hu1A4A1 lgG1-2A rAb.
  • Figure 13 illustrates the results of the enzyme-linked immunosorbent assays (ELISA) for the reactivity of the Hu1A4A1 IgGI -furin and Hu1A4A1 lgG1-2A rAbs.
  • ELISA enzyme-linked immunosorbent assay
  • Figure 14 schematically illustrates Hu1A4A1 IgGI -2A was cleaved between the heavy and light chains as expected, whereas Hu1A4A1 IgGI -furin was not cleaved.
  • Figure 15 schematically illustrates the neutralization assay used in assessing the neutralizing activity of the Hu1A4A1 IgGI -furin and Hu1A4A1 lgG1-2A rAbs against VEEV.
  • monoclonal antibody or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • recombinant antibody refers to antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of immunoglobulin gene sequences to other DNA sequences.
  • recombinant means such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from
  • the terms "expression vector” or "cloning vector” include vectors which are designed to provide transcription of the nucleic acid sequence. The transcribed nucleic acid may be translated into a polypeptide or protein product.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication). Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors” or "cloning vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • operatively-linked or “operably-linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are well known in the art such as, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).
  • Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences) or inducible promoters (e.g., induced in response to abiotic factors such as environmental conditions, heat, drought, nutrient status or physiological status of the cell or biotic such as pathogen responsive).
  • tissue-specific regulatory sequences e.g., tissue-specific regulatory sequences
  • inducible promoters e.g., induced in response to abiotic factors such as environmental conditions, heat, drought, nutrient status or physiological status of the cell or biotic such as pathogen responsive.
  • suitable promoters include for example constitutive promoters, ABA inducible promoters, tissue specific promoters and abiotic or biotic inducible promoters.
  • the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired as well as timing and location of expression, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.
  • the terms "host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell.
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a polypeptide of the invention encoded in an open reading frame of a polynucleotide of the invention. Accordingly, the invention further provides methods for producing a polypeptide using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a polypeptide of the invention has been introduced) in a suitable medium such that the polypeptide is produced. In another embodiment, the method further comprises isolating the polypeptide from the medium or the host cell.
  • Figure 1 illustrates the external structure of the VEEV.
  • the virus 10 includes a nucleocapsid 12 enveloping the viral RNA genome.
  • the envelope comprises glycoproteins E1 and E2, arranged in the form of heterodimers 14.
  • Protein E2 which is responsible for viral attachment to the host cell, contains neutralizing epitopes.
  • the murine mAb 1 A4A1 has been found to be specific to the VEEV E2 envelope protein and, further, has been found to have a strong neutralizing function against VEEV.
  • the murine mAb causes a sometimes fatal allergenic reaction in humans, resulting in the formation of human anti-mouse Abs (HAMA).
  • HAMA human anti-mouse Abs
  • the recombinant adenoviral vector can be delivered as a therapeutic agent for prophylaxis or treatment of VEEV infection in humans.
  • One advantage of this method is that the vector can express the humanized Ab in the human body for a long period of time.
  • the humanized Ab can also be produced in cell culture and delivered directly as a therapeutic.
  • the humanization of the present anti-VEEV mAb 1A4A1 has not been done previously and particularly not for the prophylaxis or treatment of VEEV infection.
  • the present invention provides in one embodiment a humanized Ab, referred to herein as Hu1 A4A1 IgGI , that retains the VEEV-binding specificity and neutralizing activity of murine 1A4A1 while not eliciting a HAMA response.
  • the humanized Ab comprises an Ig framework of human IgGI and CDRs obtained from murine mAb 1A4A1.
  • the rAb of the present invention is specific to an epitope of the E2 envelope glycoprotein of VEEV and, more specifically, to the E2 C epitope thereon.
  • Figure 2a illustrates schematically the structure of a murine Ab 16 containing murine CDRs 18 on the respective variable regions.
  • Figure 2b shows a human Ab 20 containing human CDRs 22.
  • a chimeric Ab 26 would comprise the murine variable regions 24, containing the murine CDRs 18, joined to the constant regions of the human Ab.
  • Figure 2d illustrates a humanized Ab 28 according to an embodiment of the invention, wherein only the murine CDRs 18 are grafted to the variable regions of the human Ab 20.
  • the substitution of the murine CDRs into the human Ig framework is illustrated also in Figures 3a to 3c.
  • the humanized Ab variable region comprises the grafted CDRs, 18, from the murine Ab.
  • the protein sequences of the rAbs of the invention include linker sequences.
  • the expressed rAbs of the invention have amino acid sequences as shown in SEQ ID NO:12 and SEQ ID NO: 14.
  • the nucleic acid constructs used in transforming cells to express the above rAbs are shown in SEQ ID NO:11 and SEQ ID NO:13.
  • the humanized recombinant antibodies of the present invention have been found to be effective as both a prophylaxis and a treatment against VEEV infection.
  • murine mAb 1A4A1 CDRs of VH, VL were grafted onto the frameworks of germline variable and joining (V, J) gene segments of human Ig heavy and light chains, respectively, which were chosen based on the CDR similarities between human lgs and murine mAb 1A4A1.
  • the humanized VH and VL were, respectively, grafted onto human gamma 1 heavy chain constant regions (CHs) and kappa 1 light chain constant region (CL) to assemble the whole humanized Ab gene.
  • CHs gamma 1 heavy chain constant regions
  • CL light chain constant region
  • the resultant whole humanized mAb gene was synthesized and cloned to an adenoviral vector. After the humanized Ab was expressed in HEK 293 cells and purified with protein L column, the Ab was demonstrated to retain antigen-binding specificity and neutralizing activity.
  • Murine mAb 1A4A1 Humanization of murine mAb 1A4A1
  • Murine mAb 1A4A1 was provided by Dr. JT. Roehrig (Division of Vector-borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Colins, CO, USA).
  • the VH and VL of mAb 1A4A1 were cloned in a single chain variable fragment (ScFv) format, mA116 previously [7], which showed to retain the same binding specificity as mAb 1A4A1 [11].
  • the humanization of VH and VL of murine mAb 1 A4A1 was done by Absalus Inc. (Mountain View, CA, USA).
  • VH and VL frameworks 1-3 the VH and VL amino acid sequences of murine 1A4A1 were separately subjected to IgBlast and IMGT searches against the entire human Ig germline V gene segments and then human heavy and light chain germline V gene segments were selected based on their highest CDR 1 and 2 similarities with those of murine 1A4A1 VH and VL without consideration of framework similarity. Both human VH and VL framework 4 were selected, respectively, from human heavy and light chain J gene segments based on the highest similarities between human J gene segments and murine 1A4A1 VH and VL CDR3.
  • CDRs of murine 1A4A1 VH and VL were, respectively, grafted onto the frameworks of selected germline V and J gene segments of human Ab heavy and light chains, resulting in humanized 1A4A1 (Hu1A4A1).
  • the Hu1A4A1 VH and VL were, respectively, grafted onto human gamma 1 heavy chain CHs and kappa 1 light chain CL to assemble the whole humanized Ab gene, resulting in humanized 1 A4A1 IgGI (Hu1 A4A1 IgGI ). This process is illustrated in Figures 3 to 6.
  • Hu1A4A1 lqG1 Hu1A4A1 lqG1-furin and Hu1A4A1 lqG1-2A
  • the Hu1 A4A1 IgGI DNA sequence'( ⁇ 2 kb) is schematically illustrated in Figure 7.
  • the nucleic acid sequence of the Hu1A4A1 lgG1-furin rAb is provided in SEQ ID NO:11 and the nucleic acid sequence of the Hu1A4A1lgG1-2A rAb is provided in SEQ ID NO:13.
  • the Hu1A4A1 IgGI DNA sequences were synthesized as follows.
  • a light chain leader sequence was provided upstream from the light chain, followed by a furin or 2A linker (discussed further below) before the heavy chain.
  • the whole DNA sequence flanked by Kpn I and Hind III was synthesized by GenScript Corporation (Scotch Plaines, NJ, USA) and cloned into pUC57 vector, resulting in pUC57-Hu1A4A1 IgGI -furin or pUC57- Hu1A4A1 lgG1-2A.
  • Recombinant adenovirus vectors expressing either Hu1A4A1 IgGI -furin or Hu1A4A1lgG1-2A were constructed using AdEasyTM system (Qbiogene, Carlsbad, CA, USA) according to the manufacturer's protocol. Briefly, the Kpn I— Hind III fragment of Hu1A4A1 IgGI- furin or Hu1 A4A1 lgG1-2A was ligated to a Kpn I-Hind Ill-digested pShuttle-CMV vector.
  • the resulting pShuttle construct was co-transformed with the pAdEasy-1 vector into Escherichia coli BJ5183 cells to produce recombinant adenoviral genomic constructs for Hu1A4A1 IgGI -furin or Hu1A4A1lgG1-2A proteins.
  • adenoviral constructs pAd-Hu1A4A1lgG1-furin and pAd-Hu1A4A1 IgGI -2A were linearized with Pac I and transfected into HEK 293 cells (American Type Culture Collection, Manassas, VA, USA) cultured in Dulbecco's Modified Eagle's Medium supplemented with 5% fetal bovine serum (FBS) for amplification and then the amplified adenovirus was purified by a chromatographic method. This procedure is illustrated in Figure 8.
  • Hu1A4A1 IgGI -furin or Hu1A4A1 IgGI -2A was achieved by first infecting HEK 293 cells with the recombinant adenovirus pAd- Hu1A4A1 lgG1-furin or pAd-Hu1A4A1 lgG1-2A at a multiplicity of infection (MOI) of 1.
  • MOI multiplicity of infection
  • the infected cells were cultured for one week and the culture supernatant was harvested.
  • the expressed Hu1A4A1 lgG1-furin or Hu1A4A1 lgG1-2A was purified using protein L agarose gel from Pierce (Brockville, Ont., Canada).
  • culture supernatant was dialyzed against phosphate buffer saline (PBS) (Sigma-Aldrich, Oakville, Ont, Canada) for 12h and then concentrated using PEG (Sigma-Aldrich) to less than 50 ml.
  • PBS phosphate buffer saline
  • the concentrated sample was incubated with 2ml protein L agarose gel at 4°C for 1 h. The gel and supernatant mixture was then loaded to an empty column, which was subsequently washed with binding buffer.
  • Bound Hu1A4A1 lgG1-furin or Hu1 A4A1 lgG1-2A was eluted with elution buffer.
  • the eluted Ab was further desalted using an excellulose column (Pierce) and then concentrated by a CentraconTM YM-30 (Millipore Corp., Bedford, MA, USA).
  • the amino acid sequence of the expressed Hu1A4A1 lgG1-furin is shown in SEQ ID NO:12 and the amino acid sequence of the expressed Hu1A4A1lgG1-2A is shown in SEQ ID NO:14.
  • SDS-PAGE Abs were separated by 10% SDS-PAGE gels using a Mini-PROTEANTM Il apparatus (Bio-Rad Laboratories, Mississauga, Ont., Canada). The bands were visualized by SimplyBlueTM safestain staining (Invitrogen, Burlington, Ont., Canada). The molecular weights of the samples were estimated by comparison to the relative mobility values of standards of known molecular weights.
  • the SDS-PAGE analyses of the purified Hu1A4A1lgG1-furin are illustrated in Figures 10 and 11.
  • Figure 12 illustrates the SDS-PAGE analysis of the purified Hu1A4A1 lgG1-2A.
  • lanes 1 and 3 correspond to purified Hu1 A4A1 IgGI and control human IgGI in a non- reducing condition and lanes 2 and 4 correspond to purified Hu1A4A1 IgGI and control human IgGI in a reducing condition.
  • ELISA The reactivity of purified Hu1A4A1 lgG1-furin or Hu1A4A1 lgG1-2A to VEEV E2 antigen was determined by ELISA.
  • Nunc MaxisorpTM flat bottomed 96-well plates (Canadian Life Technologies, Burlington, Ont., Canada) were coated overnight at 4°C with recombinant VEEV E2 antigen at a concentration of 10 ⁇ g/ml in carbonate bicarbonate buffer, pH 9.6. The plates were washed five times with PBS containing 0.1% TweenTM-20 (PBST) and then blocked in 2% bovine serum albumin for 2h at room temperature.
  • PBST 0.1% TweenTM-20
  • Neutralization assay in vitro Neutralizing activity of each of Hu1A4A1 IgGI -furin and Hu1A4A1 IgGI -2A against VEEV (strain TC-83) was analyzed by a plaque reduction assay. Briefly, each Ab was serially two-fold diluted (1 :32, 1 :64, 1 :128, etc.) and mixed with an equal volume containing 50 plaque- forming units of virus per 100 ⁇ l. Afterwards, the mixtures were incubated for 1 h at room temperature, 200 ⁇ l of the mixture was inoculated in duplicate into wells of six-well plates containing confluent Vero cell monolayers and incubated at 37°C for 1 h.
  • the virus/Ab mixtures were removed from the wells before the wells were overlaid by tragacanth gum and then incubated for 2 days.
  • the wells were stained with 0.3% crystal violet and plaques were counted.
  • Neutralization titre was expressed as the highest Ab dilution that inhibited 50% of virus plaques. This procedure is illustrated in Figure 15.
  • CDR-grafting involves the grafting of all murine CDRs onto a human Ab frameworks.
  • the human Ab frameworks are chosen based on their similarities to the frameworks of the murine Ab to be humanized.
  • the CDR-grafting approach has been proven successful in some cases. However, in many more instances, this humanization process could result in CDR conformation changes, which affect the antigen-binding affinity. To restore the affinity, additional work for back-mutation of several murine framework amino acids, which are deemed to be critical for CDR loop conformation, have to be done.
  • [12] employed an approach which consisted of grafting CDRs onto human germline Ab frameworks based on the CDR sequence similarities between the murine and human Abs while basically ignoring the frameworks. Because the selection of the human frameworks is driven by the sequence of the CDRs, this strategy minimizes the differences between the murine and human CDRs. This approach has the potential to generate humanized Abs that retain their binding affinity to their cognate antigen. Further, since all residues in frameworks are from human Ab germline sequences, the potential immunogenicity of non-human Abs is highly reduced. [0074] Using the above approach, and as disclosed herein, the present inventors humanized an anti-VEEV murine mAb 1 A4A1.
  • the amino acid sequences of VH and VL from murine 1 A4A1 were first aligned with human Ig germline V and J genes. As shown in Figure 5, the human heavy chain V gene segment H5-51 and J gene segment JH4 were selected to provide the frameworks for the murine 1A4A1 VH. Similarly, as shown in Figure 6, for the murine 1A4A1 VL, the human light chain V gene segment L15 and J gene segment Jk3 were selected.
  • the CDRs of murine 1A4A1 VH were then grafted onto the frameworks of selected human Ig germline H5-51 and JH4 gene segments, while the CDRs of murine 1A4A1 VL were grafted onto human L15 and Jk3 gene segments.
  • the hu1A4A1 VH was further grafted onto the human gamma 1 heavy chain CHs to form a complete heavy chain, while the VL was grafted onto the human kappa 1 light chain CL to form a whole humanized light chain. This procedure is schematically illustrated in Figures 5 and 6 with the end structure being illustrated in Figure 7.
  • the murine 1A4A1 VH CDRs grafted onto the human framework comprised the following amino acid sequences: VH CDR1 : DYHVH (SEQ ID NO: 1) VH CDR2: MTYPGFDNTNYSETFKG (SEQ ID NO: 2) VH CDR3: GVGLDY (SEQ ID NO: 3) [0077] As shown in Figure 6, the murine 1A4A1 VL CDRs grafted onto the human framework comprised the following amino acid sequences: VL CDR1 : KASQDVDTAVG (SEQ ID NO: 4) VL CDR2: WSSTRHT (SEQ ID NO: 5) VL CDR3: HQYSSYPFT (SEQ ID NO: 6)
  • the VH of the humanized Ab according to the present invention comprises the following amino acid sequence: Hu-VH: EVQLVQSGAEVKKPGESLKISCKGSGYSFTDYHVHWVRQMPGKGLEWMGMTYPGFDNTNYS ETFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGVGLDYWGQGTLVTVSS (SEQ ID NO: 7).
  • the VL of the humanized Ab according to the present invention comprises the following amino acid sequence:
  • Hu-VL DIQMTQSPSSLSASVGDRVTITCKASQDVDTAVGWYQQKPEKAPKSLIYWSSTRHT GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYSSYPFTFGPGTKVDIKR (SEQ ID NO: 8).
  • Furin is a ubiquitous subtilisin-like proprotein convertase, which is the major processing enzyme of the secretory pathway [13].
  • the furin minimal cleavage site is R-X-X-R; however, the enzyme prefers the site R-X-(K/R)-R.
  • An additional R at the P6 position appears to enhance cleavage.
  • the FMDV-derived 2A linker is able to cleave at its own C terminus between the last two residues through an enzyme-independent but undefined mechanism, probably by ribosomal skip, during protein translation.
  • a leader sequence was added upstream to the Ab gene.
  • Figure 7 illustrates the synthesized DNA sequence, of approximately 2 kb, including the human Ab kappa light chain L15 leader sequence, the humanized light chain (VL + CL), the furin or 2A linker, and the humanized heavy chain (VH + CH 1 + CH2 + CH3). This sequence was then cloned into an adenoviral vector.
  • Protein G and A columns are widely used for a quick purification for Abs because of protein G and A binding to the Fc portion of Ig.
  • protein G and A cannot only bind to human Ig, but also bind to bovine Ig, therefore they cannot be used for purification of Hu1A4A1 lgG1-furin or Hu1A4A1 lgG1-2A in our study since pAd-Hu1A4A1 IgGI -furin or pAd- Hu1A4A1 lgG1-2A-infected HEK 293 cells were cultured in the medium with 5% FBS containing a high percentage of bovine Ig.
  • protein L binds Ig through interactions with the light chains. Protein L only binds to Ig containing light chains of type kappa 1, 3 and 4 in human and kappa 1 in mouse.
  • Hu1A4A1 IgGI- 2A showed the same patterns as a control human IgGI , one band of -15OkDa in non-reducing condition (intact disulfide bridges) and two bands, 5OkDa for heavy chains and 25kDa for light chains (broken disulfide bridges) in reducing condition, indicating that the 2A linker underwent self-processing perfectly.
  • Hu1A4A1 IgGI -furin showed only one clear band of ⁇ 75kDa in reducing condition observed as illustrated in Figures 10 and 11 , indicating that the furin linker was not cleaved.
  • both versions of the Hu1A4A1 lgG1 were found to bind to VEEV E2 in a dose-dependent manner, similar to the binding to VEEV E2 of its parental murine 1 A4A1 , indicating this non-cleaved Ab was still reactive to VEEV E2 antigen in ELISA. Furthermore, both versions were evaluated for their ability to block VEEV infection in Vera cells using a standard plaque-reduction assay.
  • the Hu1A4A1lgG1-fruin showed a neutralizing activity with 50% plaque reduction neutralization titre at 0.78 ⁇ g/ml, whereas Hu1A4A1 lgG1-2A showed a much higher neutralization titre at 0.1 ⁇ g/ml.
  • Example 2 In vivo study - Protection or pre-exposure prophylaxis of mice from VEEV challenge by passive immunization with Hu1A4A1lgG1-furin or Hu1A4A1lgG1-2A
  • mice aged 6-8 weeks were injected intraperitoneal ⁇ (i.p) with 50 ⁇ g of Hu1A4A1 lgG1-furin or Hu1A4A1 lgG1-2A in 100 ⁇ l PBS, human IgG in 100 ⁇ l PBS (positive control) or 100 ⁇ l PBS alone (negative control) 24 h prior to VEEV challenge.
  • VEEV challenge [0090] Each mouse was challenged subcutaneously (s.c.) with 30-50 plaque forming units (pfu) of virulent VEEV (Trinidad donkey, TRD) in 50 ⁇ l of Leibovitz L15 maintenance medium (L15MM) 24 h after passive immunization. The challenge dose approximated to 100 x 50% lethal dose (LD50). Mice were examined frequently for signs of illness for 14 days, and humane endpoints were used.
  • pfu plaque forming units
  • L15MM Leibovitz L15 maintenance medium
  • mice Hu1A4A1 loG1-furin or Hu1A4A1 loG1-2A clearance in mice
  • groups of 4 mice were injected i.p. with 50 ⁇ g, each mouse, of either Hu1A4A1 lgG1-furin or Hu1A4A1 lgG1-2A, or human anti-VEEV IgG and bled from the vein at increasing time intervals after injection.
  • the quantity of Ab present in serum samples was estimated by immunoassay.
  • Hu1A4A1lgG1-furin or Hu1A4A1lgG1-2A had a similar half-life as human anti- VEEV IgG, around 10 days.
  • Protection of mice from VEEV challenge by passive immunization with Hu1 A4A1 IgGI -furin or Hu1A4A1 IqGI -2A [0095] Groups of 8 mice were injected i.p. with the Hu1A4A1 IgGI -furin, Hu1A4A1 IgGI -2A, human IgG (positive control) or PBS alone (negative control) and 24h later challenged s.c. with 100 x LD50 of VEEV.
  • mice The half-lives of the humanized anti-VEEV rAbs in mice was around 10 days suggesting that the humanized anti- VEEV rAbs of the invention would be an effective prophylactic against VEEV for at least several weeks.
  • the rAbs of the invention have been demonstrated to have functionality as an immunization agent against VEEV infection.
  • Example 3 In vivo study - Treatment or post-exposure therapy of mice after VEEV challenge by passive immunization with Hu1A4A1lgG1-furin or Hu1A4A1lgG1-2A [0099] Materials and methods [00100] Post-exposure Therapy [00101] Balb/c mice aged 6-8 weeks were challenged s. c. with 100 x LD50 of virulent VEEV in 50 ⁇ l of L15MM per mouse. At 24 h post-challenge, mice were injected i.p with 50 ⁇ g of Hu1A4A1 lgG1-furin or Hu1A4A1 lgG1-2A in 100 ⁇ l PBS, or 100 ⁇ l PBS alone. Mice were examined frequently for signs of illness for 20 days, and humane endpoints were used. [00102] Results
  • mice were challenged s.c. with 100 x LD50 of VEEV. Twenty-four hours later, the infected mice were administered i.p. with Hu1A4A1 lgG1-furin, Hu1A4A1 lgG1-2A or PBS alone (50 ug/mouse). All the Hu1A4A1 lgG1-2A-treated mice survived throughout the observation period (20 days post-challenge) with minor clinical signs. All Hu1A4A1 lgG1-furin or PBS-treated mice died. [00106] Discussion
  • the neutralization site on the E2 glycoprotein of Venezuelan equine encephalomyelitis (TC-83) virus is composed of multiple conformationally stable epitopes.

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Abstract

L'invention porte sur un anticorps humanisés de recombinaison greffé par CDR contre le virus de l'encéphalite équine du Vénézuéla (VEEV) comprenant une structure d'ig humaine présentant les CDRs murins mAb 1A4A1 VH et VL. L'invention porte également sur des séquences d'ADN, des vecteurs d'expression incorporant lesdites séquences et des cellules hôtes transformées. L'invention porte en outre sur des préparations pharmaceutiques, des méthodes prophylactiques et des traitements, contre les infections par le VEEV, utilisant les anticorps humanisés de recombinaison de l'invention.
PCT/CA2008/001940 2007-11-01 2008-11-03 Anticorps humanisés de recombinaison contre le virus de l'encéphalite équine du vénézuéla WO2009055936A1 (fr)

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WO2011036435A1 (fr) * 2009-09-22 2011-03-31 The Secretary Of State For Defence Anticorps anti-veev humanisé
GB2473934B (en) * 2009-09-22 2012-11-28 Secr Defence Anti-VEEV antibody
GB2473934A (en) * 2009-09-22 2011-03-30 Secr Defence Humanised anti-VEEV antibody
US8778346B2 (en) 2010-11-04 2014-07-15 Boehringer Ingelheim International Gmbh Anti-IL-23 antibodies
US9441036B2 (en) 2010-11-04 2016-09-13 Boehringer Ingelheim International Gmbh Anti-IL-23 antibodies
US10202448B2 (en) 2010-11-04 2019-02-12 Boehringer Ingelheim International Gmbh Anti-IL-23 antibodies
US11078265B2 (en) 2012-05-03 2021-08-03 Boehringer Ingelheim International Gmbh Anti-IL-23 antibodies
US10507241B2 (en) 2014-07-24 2019-12-17 Boehringer Ingelheim International Gmbh Biomarkers useful in the treatment of IL-23A related diseases
US10059763B2 (en) 2014-09-03 2018-08-28 Boehringer Ingelheim International Gmbh Compound targeting IL-23A and TNF-alpha and uses thereof
US10793629B2 (en) 2014-09-03 2020-10-06 Boehringer Ingelheim International Gmbh Compound targeting IL-23A and TNF-alpha and uses thereof
US11680096B2 (en) 2014-09-03 2023-06-20 Boehringer Ingelheim International Gmbh Compound targeting IL-23A and TNF-alpha and uses thereof
CN107586335B (zh) * 2016-07-06 2020-12-29 中国科学院微生物研究所 一种人源化单克隆抗体及应用
CN107586335A (zh) * 2016-07-06 2018-01-16 中国科学院微生物研究所 一种人源化单克隆抗体及应用

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