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WO2013186752A1 - Anticorps monoclonaux aptes à se lier à la protéine e2 virale du vhc, leur préparation et leur utilisation - Google Patents

Anticorps monoclonaux aptes à se lier à la protéine e2 virale du vhc, leur préparation et leur utilisation Download PDF

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WO2013186752A1
WO2013186752A1 PCT/IB2013/054884 IB2013054884W WO2013186752A1 WO 2013186752 A1 WO2013186752 A1 WO 2013186752A1 IB 2013054884 W IB2013054884 W IB 2013054884W WO 2013186752 A1 WO2013186752 A1 WO 2013186752A1
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seq
hcv
antibodies
antibodies according
sequences
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PCT/IB2013/054884
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Antonio LEONARDI
Luca SANGUIGNO
Ruvo Menotti
Luigi Vitagliano
Anna Maria SANDOMENICO
Claudio Farina
Ester Ascione
Livio MUSCARIELLO
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Universita' Degli Studi Di Napoli "Federico Ii"
Kedrion S.P.A.
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Publication of WO2013186752A1 publication Critical patent/WO2013186752A1/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
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • Hepatitis C infections are major public health emergencies of the XXI century. In fact, approximately 170 million people worldwide are estimated to be HCV infected; in addition, chronic patients have a high likelihood of developing cirrhosis and hepatocellular cancer; HCV infections are also the leading cause of liver transplantation in USA.
  • HCV comprises multiple genotypes, moreover some heterogeneity exists between different subgroups of genotypes. This results in high drug resistance, and obviously there is an intensive search for combined therapies.
  • HCV is an enveloped virus with a positive strand RNA genome of 9.5 kb.
  • the 5' end of the viral genome is characterized by a highly conserved non-coding region of 341 base pairs, while downstream of this region there is an Open Reading Frame (ORF) encoding a polypeptide of about 3010 amino acids.
  • ORF Open Reading Frame
  • this polypeptide is processed by a pool of viral and endogenous proteases, generating 10 mature proteins. These include 2 envelope glycoproteins anchored to the viral membrane, designated E1 (or gp35) and E2 (or gp72), containing 5 or 6 and 11 N-glycosylation sites, respectively.
  • HCV envelope proteins E1 and E2 are responsible for binding and entry of the virus into host cells, therefore they represent selective targets for drugs that block the virus starting from the early stages of infection.
  • HCV due to its high in vivo replication rate and error-prone RNA polymerases, HCV exhibits high genetic variability which is particularly evident in the coding regions of envelope proteins; more specifically two hypervariable regions found at the N-terminus of the E2 protein, known as HVR1 and HVR2, are mostly involved in the binding to host cell receptors.
  • a region immediately downstream of HVR1 was reported to contain epitopes for antibodies capable of very effective inhibition of virus binding to CD81 receptor [18].
  • a linear epitope corresponding to residues 412-423 and defined by the AP33 monoclonal antibody is involved in inhibition of the interaction between CD81 and a variety of E2 genotypes, E1-E2 dimers and virus-like particles.
  • Patents WO 2006/100449 and, more recently, 20110002926 published on Jan. 6, 2011 report that the AP33 monoclonal antibody and its humanized variant bind and can neutralize the six HCV genotypes; thus the linear epitope 412-423 is clearly a target for anti-HCV ligands and is an immunogen for generating anti-HCV polyclonal and monoclonal antibodies [Owsianka A, Tarr AW, Juttla VS, Lavillette D, Bartosch B, Cosset FL, Ball JK, Patel AH. Monoclonal Antibody AP33 Defines a Broadly Neutralizing Epitope on the Hepatitis C Virus E2 Envelope Glycoprotein. 2005, J. Virol. , 79( 7): 11095- 1 04].
  • the invention relates to monoclonal antibodies of murine or human origin capable of binding and neutralizing to a varying extent the entry of the HCV virus into cells, and to the antigens used to generate aforesaid antibodies.
  • Figure 1 shows ELISA assays relative to the binding between synthetic peptides corresponding to the Cyclic epitope (Peptide I) and the corresponding Linear epitope (linear Peptide II) to Mabs I - VII, respectively.
  • Figure 2 shows Sensorgrams relative to the binding of Peptide I to Mabs I - VII.
  • Figure 3 shows Biacore Sensorgrams relative to the binding of linear Peptide II to monoclonal antibodies I - VII, immobilized on a biochip.
  • Figure 4 shows Biacore Sensorgrams relative to the binding of Cyclic peptides: III Mut1 , IV Mut2, V Mut3, III Mut1 , IV Mut2, V Mut3 e VI Mut4 respectively with: monoclonal antibody II (Mab II), monoclonal antibody III (Mab III) and monoclonal antibody VI (Mab VI).
  • Figure 5 shows the results of western blot analysis of the recognition of E2 of genotypes 1a and 3 by a mixture of MAb MAbV l-ll monoclonal antibodies.
  • Figure 6 shows the neutralizing activity of the monoclonal antibodies that were developed, and of the AP33 control.
  • Figure 7 shows the neutralizing activity of the monoclonal antibodies that were developed and of the AP33 control.
  • Figure 8 shows the neutralizing activity of MAb II, MAb VI e MAb VII assayed individually, in binary combination with each other, and all together.
  • the present invention makes possible to meet the above mentioned demands, making available monoclonal antibodies against epitopes derived from the E2 protein of hepatitis C virus (HCV) and their use for therapeutic and diagnostic applications. Therefore, the monoclonal antibodies according to the invention are able to bind the viral E2 protein and neutralize HCV infections in mammals.
  • HCV hepatitis C virus
  • the antibodies according to the invention are of human or murine origin and are characterized in that at least one of the chains (heavy or light) of the complementarity determining region (CDR) has the sequence (1 ) (heavy chain): AELVRPGGSVKLSCKASGYSFTTYWMNWVKQRPGQGLEWIGMIQPSDSETRLN QKFKDKATLTLDRSSSTVYMQLSSPTSDDSAVYYCARTGRGDAWLTYWGQG
  • sequence (2) (light chain): [SEQ. ID. No 1] PASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESG VPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRS [SEQ. ID. No 2] and at the same time, the other chain has a percent identity comprised between 50% and 100% to the respective sequences 1 or 2.
  • amino acid sequence is shown according to the single letter code, well known to the experts in the field, and refers to amino acids in L configuration.
  • the monoclonal antibody bearing CDR sequence 1 as heavy chain and sequence 2 as light chain is hereinafter referred to as Mab II.
  • human or murine monoclonal antibodies comprised in the present invention in that they are characterized by having the following heavy and light chains as defined above, are:
  • Tables I and II show the percent identity between light chains (Table I) and heavy chains (Table II) of monoclonal antibodies Mab II, Mab V and Mab VI.
  • Such antibodies are characterized primarily by their ability to bind the E2 protein of HCV virus in a specific manner.
  • Nucleotide sequence 2p - heavy chain of Mab II [SEQ. ID. No. 64]
  • Nucleotide sequence 21 - light chain of Mab II [SEQ. ID No. 65]
  • Nucleotide sequence 5a - heavy chain of Mab V [SEQ. ID. No. 66]
  • Nucleotide sequence 6a - heavy chain of Mab VI [SEQ. ID. No. 68]
  • Nucleotide sequence 6b - light chain of Mab VI [SEQ. ID. No. 69]
  • the invention also comprises the above sequences suitably modified in order to obtain conjugates that are more stable in biological media or conjugates that are less immunogenic or conjugates that are more soluble or less prone to aggregation.
  • conjugates can be obtained by well known chemical or enzymatic reactions which make use of appropriate substituents with well known actions on the molecule.
  • derivatives or conjugates of the antibodies of the present invention are functional derivatives, that is chemically or enzymatically modified derivatives which, in functional assays, retain the same biological function - or a similar biological function - of the unmodified products, however exhibiting one or more of the above described properties.
  • functional derivatives of the described antibodies may be antibodies with identical sequence however bearing changes introduced by chemical or enzymatic reactions: amidation, glycosylation, carbamoylation, acylation (eg, acetylation), sulfation, phosphorylation, lipidization, pegylation, biotinylation, fluoresceination.
  • canonical, divalent, trivalent and tetravalent ScFv can be prepared as fusion products with other proteins.
  • Any protein or polypeptide can be used as fusion partner, however preferred fusion partners should not have undesirable biological effects in vivo.
  • Such fusion partners can be fused either at the C-terminal or at the N-terminal end of ScFv and, optionally, a short polypeptide segment can be inserted between the polypeptide sequences of ScFv and of fusion partners.
  • a short polypeptide segment can be inserted between the polypeptide sequences of ScFv and of fusion partners.
  • such segments can be recognition sequences for endogenous proteases, thus allowing removal of ScFv from their fusion partner, in vitro or in vivo.
  • Examples of such segments include, but are not limited to: D-D-D-D-Y (SEQ. ID NO. 75), G-P-R, V-P-R, A-G-G e H-P-F-H-L (SEQ. ID NO. 76), which may be hydrolysed by enterokinase, thrombin, ubiquitin cleaving enzyme and renin, respectively (see U.S. Patent No. 6,410,707).
  • a chemically or enzymatically modified functional derivative may be a pegylated derivative of monoclonal antibodies or of their respective ScFv.
  • Pegylated compounds may provide additional benefits, such as for example improved solubility, increased stability, longer circulation persistence and reduced immunogenicity (see U.S. Patent No. 4,179,337).
  • a polymer to be used for derivatization may have any molecular weight and may be linear or branched. Polymers with different molecular weight can be employed based on the desired therapeutic profile, for example, circulation persistence or release time, or any other property modifying their biological properties. The choice of the polymer and its characteristics may also depend on the ease of use, the degree or lack of immunogenicity or other known effects on the therapeutic properties of monoclonal antibodies or ScFv.
  • PEG may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 1 1 ,000, 1 1 ,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
  • the monoclonal antibodies of the present invention are human or humanized monoclonal antibodies.
  • Antibody fragments retaining the same antigen binding properties are also comprised in the present invention.
  • Examples of such functional fragments are the Fab and (Fab)2 or polypeptides corresponding to the complementarity determining region (CDR) considered in isolated form, hence not considered in the context of an Ig-like domain, and retaining the same ability or a similar ability to bind the antigens, namely the entire E2 protein or its fragments or its homodimers, or heterodimers of E1 and E2 proteins.
  • This example includes synthetic polypeptides or the same polypeptides obtained by recombinant DNA technique, with sequence [SEQ. ID. No 1 - SEQ. ID. No 6].
  • amino acids can be replaced with asparagine residues and vice versa
  • glutamic acid residues can be replaced with aspartic acid residues and vice versa
  • lysine residues can be replaced with arginine or histidine residues and vice versa
  • serine residues can be replaced with threonine residues and vice versa
  • aromatic residues can be replaced with other aromatic compounds.
  • Integral part of this first aspect of the invention are also all the compositions containing the antibodies of the present invention or their fragments or functional derivatives, alone or in binary or ternary mixtures, or in general multiple mixtures capable of binding and neutralizing viral entry to varying extent, or capable of producing experimental artifacts that reproduce their activity in cellular model systems such as, for example, those described in EP1551960A2.
  • compositions are used to neutralize the action of the virus also in recently described animal models of the disease.
  • compositions may contain at least one antibody object of the present invention or a functional fragment or derivative thereof and at least one carrier or adjuvant or diluent.
  • compositions may contain at least one monoclonal antibody or its functional fragments, possibly chemically or enzymatically modified as described.
  • the antibodies and/or their fragments, whether they are modified or not, and their preparations described above can be used for several applications related to prevention and treatment of hepatitis C virus infections.
  • the described monoclonal antibodies or their fragments or functional derivatives, as described above, can be used in analytical methods in order to identify new compounds capable of neutralizing HCV infection.
  • step (iii) assessment in step (iii) as to whether the compound added in step (ii) is capable of interfering with the binding of antibodies or their fragments or functional derivatives with the E2 protein.
  • compositions can be used that contain at least one antibody or its fragment or functional derivative thereof and at least one carrier or adjuvant or diluent.
  • anti-HCV monoclonal antibodies can be used in diagnostic kits for the purpose of identifying and quantifying the E2 protein and its variants in biological samples.
  • Monoclonal anti-HCV antibodies, and their fragments or functional derivatives, as described above, can be prepared and purified by known techniques.
  • this method includes the following steps:
  • a further aspect of the present invention are the antigens used to generate antibodies as described above.
  • the main epitope of such antigens is represented by the amino acid sequence corresponding to the region 412-422 of the E2 protein (QLINTNGSWHI) [SEQ.ID. 77] or its variants in which two other residues are added to the two ends, thus allowing generation of cyclic structures with the same number of atoms or a number of atoms equal to:
  • N number of atoms present in the structure as defined above (41 atoms), in which said added residues are two cysteines and i varies from 1 to 10 and wherein two lysine residues are possibly inserted at the C- terminus and at the N-terminus.
  • Epitope 1 one example of epitope according to the invention has the sequence:
  • amino acid sequence is shown according to the single letter code, well known to the experts in the field, and refers to amino acids in L configuration.
  • this sequence can be reduced in order to obtain new epitopes of smaller size, up to a minimum of 5 amino acids.
  • Epitope 1 refers to the sequence in between the two cysteine residues.
  • GLINTNGSWH SEQ. ID. No. 22
  • QLINTGGSWH SEQ. ID. No. 23
  • HLVNSNGSWH [SEQ. ID. No. 37]
  • NLVNTNGSWH [SEQ. ID. No. 38]
  • NLIKTNGSWH [SEQ. ID. No. 43]
  • HLVNSNGSWH [SEQ. ID. No. 44]
  • Epitope 1 can be prepared and used also in a form that lacks the two N-terminal lysines.
  • Integral part of this aspect of the present invention are epitopes wherein amino acids of Epitope 1 can be in D configuration.
  • the same peptide molecules can be generated in retro or retro-inverse form, since it is known that antibodies directed at a parent peptide can recognize also variants with amino acids in D configuration, retro and retro-inverse variants.
  • Epitope 1 with sequence [SEQ. ID. No. 7], its methylated variant and 4 mutants were designed, as indicated in Table III, in order to study the antigen-binding site of the antibodies.
  • the syntheses were performed using a SYRO I synthesizer purchased from the company Multisyntech (Germany).
  • Reagents for solid phase peptide synthesis (resin, Fmoc-protected amino acids and condensing agents) were purchased from the companies Inbios (Napoli), GL Biochem (Shanghai, China) and Novabiochem (Laufelfingen, Switzerland).
  • the solvents for the synthesis, dimethylformamide (DMF), dichloromethane (DCM), isopropanol (Iso-Pro) and ether (Et 2 O) were purchased from the company DelChimica (Napoli) and Sigma-Aldrich (Milano).
  • Piperidine which is used to remove the Fmoc group
  • TSA trifluoroacetic acid
  • TIS triisopropylsilane
  • ACN Acetonitrile
  • Reverse phase analytical and preparative columns for analysis and purification of peptides were purchased by the company Phenomenex (Bologna).
  • Preparative chromatographic systems are products obtained from Shimadzu (Milano).
  • the LC-MS system for characterization of crude and purified peptides is a product of ThermoFisher (Milan).
  • Peptides were prepared by chemical synthesis using a solid phase technique on RINK AMIDE resin that makes possible to have the C-terminal amidated peptides. Synthesis of each peptide was performed on a 50 pmole scale. Condensation reactions between amino acids were performed for 25 minutes at room temperature, employing HATU and DIPEA (HATU: 2-(1 H-7-Azabenzotriazol-1-yl)- -1 ,1 ,3,3-tetramethyl uronium hexafluorophosphate Methanaminium; DIPEA: N, N- Diisopropylethylamine) using 10-fold amino acid excess (500 pmoles).
  • HATU 2-(1 H-7-Azabenzotriazol-1-yl)- -1 ,1 ,3,3-tetramethyl uronium hexafluorophosphate Methanaminium
  • DIPEA N, N- Diisopropylethylamine
  • Deprotection reactions for the Fmoc group were carried out for 10 minutes at room temperature, using 30% of PIP solutions in DMF.
  • the resins obtained at the end of synthesis were manually washed with DMF (3 times, 1 ml_), with DCM (3 times, 1 ml_), with Iso-Pro (3 times, 1 mL) and with Et. 2 0 (5 times, 1 ml_).
  • Resins were dried under vacuum and treated with a mixture of TFA:H 2 0:TIS (3 mL for about 100 mg of resin) for 5 hours at room temperature.
  • Products obtained in acid solutions were precipitated by addition of cold ethyl ether, separated by centrifugation and freeze- dried from a 50% H2O/CAN solution. Crude products were characterized by LC- MS in terms of purity and molecular weight (ESTIMATED AVERAGE PURITIES WERE APPROXIMATELY 65%).
  • peptide were dissolved in 0.5 ml_ of 50 mM phosphate buffer, pH 7.0.
  • the solution was mixed with a solution containing 1.5 mg of KLH (product obtained from SIGMA H7017, Milano) in 0.5 mL phosphate buffer, pH 7.0, followed by addition of 0.5 mL 0.2% glutaraldehyde solution in water (Sigma-Aldrich, Milan).
  • KLH product obtained from SIGMA H7017, Milano
  • phosphate buffer pH 7.0
  • glutaraldehyde solution in water Sigma-Aldrich, Milan
  • the mixture was incubated for 2 hours at room temperature, followed by addition of 1 mL 1 M glycine solution in phosphate buffer, and was left to react for 1 additional hour at room temperature.
  • the solution was finally dialyzed 2 times against 50 mM phosphate buffer pH 7.0 and finally lyophilized.
  • the starting linear peptide was methylated in solution on cysteine thiol groups in order to have a stable molecule in solution during the testing phase of monoclonal antibodies and, at the same time, containing the least possible modifications.
  • the methylation reaction was carried out on 3 mg of crude peptide (about 1.7 moles, about 3.4 moles of SH groups) dissolved in 500 ⁇ of ACN containing 1 % DIPEA. 100 pL of a methyl iodide solution (CH 3 I, Sigma-Aldrich, Milano, Italy) in 10% ACN v/v (10 mg, about 170 pmoles) were added to the solution, incubating the reaction for 1 minute at room temperature. The reaction was stopped by ice- cooling followed by addition of 100 L H 2 0/TFA 9:1.
  • the product was taken up in 2 mL of 9:1 H 2 O/ACN, 0.1 % TFA and was purified by HPLC on reverse phase column, obtaining, after purification, approximately 1.5 mg of purified product. Successful methylation was verified by LC-MS analysis of an aliquot of purified sample.
  • Peptide I conjugated with KLH was used as antigen for production of monoclonal antibodies.
  • TiterMax Gold Adjuvant (Sigma) was used as adjuvant, thus ensuring an optimal activation of innate immunity which is essential for better antibody production. Particular attention has been paid to mixing the adjuvant and the peptide in order to create a perfect emulsion. For this, one syringe was loaded with 500 ⁇ _ of adjuvant and a second syringe was loaded with 500 ⁇ _ of antigen at a concentration of 4 mg/mL in PBS buffer (phosphate 50 mM, NaCI 150 mM).
  • the adjuvant was mixed with the antigen using a three-way connector making sure that the aqueous phase (antigen) flowed into the oil phase (adjuvant) and not vice versa. The mixing operation is continued until the emulsion became homogeneous.
  • Peptide I or linear Peptide II conjugated to BSA were used as antigens in ELISA assays. This allows determination of antibodies actually produced against Peptide I, avoiding the interference of anti-KLH antibodies.
  • the two mice which showed the best antibody titer were sacrificed by cervical dislocation.
  • the spleen was taken and placed in a 50 mL Falcon tube containing 10 mL of RPMI-GM medium (whose composition is shown in the section below) pre-warmed at 37°C.
  • the spleen was transferred into a 100 mm plate containing 10 mL of medium and was disrupted by using a cell strainer (BD-Falcon, Milano).
  • the solution containing the splenocytes was centrifuged at 800 rpm for 5 minutes at room temperature. After centrifugation the supernatant was removed and cells were resuspended in 5 mL RPMI-GM medium and counted.
  • the following dilutions were prepared for cell counting:
  • 10 pL of 0.4% Trypan blue dye (Sigma- Aldrich, Milan, Italy) were added to 10 pL of the various splenocyte dilutions.
  • a number of myeloma cells was used that was 1/5 of spleen cells resuspended in 10 mL of RPMI-GM medium.
  • Myeloma cells were added to splenocytes, followed by addition of 15 mL of RPMI-GM medium (30 mL final volume). Cells were centrifuged at 600 rpm for 5 minutes, at room temperature.
  • the supernatant was discarded and the cell pellet was resuspended in 1 mL RPMI-GM medium containing 1300-1600 PEG (hybri-Max, Sigma-Aldrich, Milano) and 75 pL DMSO (Sigma-Aldrich, Milano).
  • the cell pellet was gently disrupted with a pipette tip, followed by slow addition of 9 mL RPMI-GM medium supplemented with 10% FBS (Foetal Bovine Serum, Sigma-Aldrich, Milano), taking care to continually mix the cell suspension.
  • FBS Fetal Bovine Serum
  • Cells were centrifuged to 500 rpm for 7 minutes at room temperature and the pellet was resuspended in 200 mL of selective RPMI-HAT medium (Sigma-Aldrich, Milano). Cells were plated in twenty 96-well plates and left in the incubator for at least 10 days.
  • the 30 clones selected were isolated and sub-cloned. After this step, eight clones lost productivity and the remaining 22 clones were subjected to a stabilization process.
  • FBS Foetal Bovine Serum
  • PSG Penicillin-streptomycin-glutamine
  • Figure 1 B shows instead a dose-response ELISA assay carried out with Peptide I immobilized at a concentration of 0.5 ug/mL and with increasing concentrations of all 7 antibodies l-VII used at concentrations comprised between 0 and about 3.3 nM, without prior purification.
  • a control with linear Peptide II, lacking detectable binding, is shown also in this assay.
  • Figure 1 C shows the result of a dose-response ELISA assay which was carried out again with 0.5 pg/mL immobilized peptide and with increasing concentrations of purified MAb l-VII (0 - 3.5 nM).
  • the 7 selected antibodies were covalently immobilized on the surface of the biochip by using the EDC/NHS chemistry [Ethyl-3-(3-dimethylaminopropyl) carbodiimide/(N-hydroxy-succinimide, GE Healthcare, Milano], following manufacturer's instructions [GE Healthcare, Milano].
  • Immobilization was carried out with antibody solutions at a concentration of 5.0 pg/mL, obtaining for all a level of immobilization comprised between 6500 and 8000 RU (Resonance Units).
  • Binding assays were performed at a flow rate of 30 pL/min for 2 minutes in HBS, injecting Peptide I solutions at increasing concentrations ranging from 6.25 nM and 5 ⁇ , above which curves show saturation. Regeneration was performed with 50 mM NaOH, injected for 10 sec.
  • KD values were calculated using the BiaEvaluation software, vers. 4.1 (GE Healthcare, Milano), implemented in the instrument software. Calculation was performed by determining Ko n and k 0 ff values for each individual experiment, and the and K D was obtained from the Koff/k 0 n ratio. Values were then averaged for all concentrations.
  • Figures 2A-G show sensorgrams obtained after processing and subtraction of reference curves. Graphs of RUmax vs concentration documenting the dose- response profile of binding and its saturability are also shown in the insets.
  • Table VII instead shows the K D values determined by SPR analysis compared to K D values for the same antibodies determined by ELISA assays. There is some discrepancy between the values obtained with the two methods. However the values determined by the SPR technique are considered to be much more accurate.
  • Binding experiments were carried out with monoclonal antibodies II and VI and with Peptides lll-VI, as shown in Table III, to further evaluate the specificity in antibody recognition of Peptide I. Experiments were conducted under the same conditions used for analysis of Peptide I and linear Peptide II (30 uL/min flow rate, 25 °C temperature). In several experiments (shown in the figures and the respective legends), peptides were evaluated at variable concentrations however comprised between 125 nM and 5000 nM. Illustrative sensorgrams regarding the binding of Peptides III, IV and V with Mab II and Mab VI are shown in Figures 4A-G, while Tables Villa and Vlllb show K D values determined for all peptides.
  • - monoclonal antibody II does not bind the cyclic antigen in the vicinity of amino acids NTN, however recognizes residues in the vicinity of the disulfide bridge, in particular residues QLI and GSWHI (SEQ ID NO. 81 ).
  • mutants makes possible to rule out non-specific recognition of portions of the molecule in the vicinity of the disulfide bridge or of the two N- terminal lysines.
  • the 293T cell line consisting of human embryonic kidney epithelial cells transformed with the Simian Virus 40 (SV40) T antigen, was cultured in DMEM Hi- Glucose medium (Dulbecco's Modified Eagle's Medium Hi Glucose, Sigma- Aldrich, Milano) supplemented with 10% FBS (Gibco, Invitrogen, Carlsbad, CA) that was inactivated at 56 °C for 30 minutes, 2 mM L-glutamine (Sigma-Aldrich), 1000U/mL penicillin (Sigma-Aldrich), 100 g/mL streptomycin (Sigma-Aldrich), 1 % nonessential amino acids (Sigma-Aldrich).
  • DMEM Hi- Glucose medium Dulbecco's Modified Eagle's Medium Hi Glucose, Sigma- Aldrich, Milano
  • FBS Gibco, Invitrogen, Carlsbad, CA
  • the HuH-7 cell line consisting of human hepato-carcinoma cells susceptible to the entry of HCV pseudoparticles, was cultured in DMEM medium (Dulbecco's Modified Eagle's Medium, Sigma-Aldrich) supplemented with 10% FBS (EuroClone Ltd, Torquay, UK) that was inactivated at 56 °C for 30 minutes, 2mM L-glutamine (Sigma-Aldrich), 1000U/mL penicillin (Sigma-Aldrich), 100 pg/mL streptomycin streptomycin (Sigma-Aldrich), 1 % nonessential amino acids (Sigma- Aldrich).
  • DMEM medium Dulbecco's Modified Eagle's Medium, Sigma-Aldrich
  • FBS EuroClone Ltd, Torquay, UK
  • Cells were cultured in T75 flasks (BD Falcon, Bedford, MA, USA) in a cell culture incubator (Sanyo Incubator, Japan) at 37 °C and 5% CO 2 .
  • Feline immunodeficiency virus (FlV)-derived constructs made in the Centro Retrovirus dell'Universita di Pisa were used to produce particles with a FIV-derived vector, which were pseudotyped with E1 -E2 and express GFP as reporter gene.
  • the constructs are based on a split component system that makes use of three constructs: packaging, designated pAenvl (derived from p34TF10, a FIV molecular clone containing the whole genome of Petaluma isolate); vector, indicated with the abbreviation LAW-GFP carrying GFP as reporter gene; and three alternatively used envelope constructs.
  • the first, pE1 E2gen1a contains sequences encoding E1 and E2 surface proteins of the HCV 1a genotype
  • the second, pE1 E2gen3a contains sequences encoding E1 and E2 of genotype 3a
  • the third designated pVSV-G encodes the G protein of vesicular stomatitis virus (VSV-G).
  • Plasmid constructs expressing E1/E2 of 1a and 3a genotype were produced from clinical HCV isolates, using plasma from peripheral blood as source. The cloning of E1/E2 from HCV RNA extracted from plasma was performed as described by Cosset and collaborators (Bartosch et al., J. Exp Med 197: 633-642, 2003).
  • Viral FIV-derived particles pseudotyped with E1-E2 of genotype 1a or 3a were designated FIV- E1 E2/1a and FIV-E1 E2/3a while those pseudotyped with protein G were designated FIV-VSV.
  • Plasmids were transformed in bacterial MAX Efficiency Stbl Cells (Invitrogen, Milano) by heat shock at 42 °C for 45 seconds. Transformed cells were then incubated on ice for 2 minutes, supplemented with SOC medium and incubated for 1 hour with shaking at 37 °C.
  • PRODUCTION AND FUNCTIONAL ANALYSIS OF HCV PSEUDOPARTICLES AND VSV-G CONTROL PSEUDOPARTICLES Pseudoparticles were produced in HEK293T cells. Twenty-four hours before transfection, 3x10 6 cells were seeded in 10 cm Petri dishes (BD Biosciences, Milano). On the next day, cells were transfected with 20 ug of LAW-GFP vector construct, 10 ug of pAenvl packaging construct and 5 ug of envelope construct (pE1 E2gen1a, pE1 E2gen3a or VSV-G).
  • Transfection was with 10 ⁇ polyethyleneimine polymer (PEI, Sigma-Aldrich); briefly, DNA was mixed with 150 mM NaCI solution up to 700 ⁇ volume; likewise 100 ⁇ _ of PEI were brought to 700 pl_ with 150 mM NaCI. The two solutions were mixed and incubated at room temperature for 15 minutes and the new solution was gently added stepwise onto the cells. Prior to transfection, the cell culture medium was replaced with 5 mL of Hi-Glucose DMEM containing 2 mM L-glutamine, while 6 hours after transfection the medium was replaced with 10 mL of complete Hi-Glu DMEM.
  • PEI polyethyleneimine polymer
  • the supernatant containing vector particles was collected 48 hours after transfection, clarified at 1800 RPM for 10 minutes at room temperature and then stored in aliquots at -80 °C until use.
  • Antibodies were stored at -20°C. When necessary, they were thawed and two aliquots were prepared for each antibody, using PBS as diluent, corresponding to 10 ug/mL and 100 ug/mL dilutions. Aliquots were frozen at -20°C until use.
  • Western blot analysis for recognition of anti- HCV E2 protein of 1a and 3 genotype A western blot analysis on transfected cells and their produced pseudotyped vectors was performed in order to evaluate the actual binding of anti-HCV antibodies to the E2 protein.
  • HEK293T cells transfected, as above described, with LAW-GFP, packaging pAenvl and envelope (pE1 E2gen1a, pE1 E2gen3a or VSV- G) constructs were lysed 2 days after transfection and the supernatant containing vector particles was collected for western blot analysis.
  • Cell lysis was by cold incubation for 1 hour under mild stirring in the following lysis buffer: 50 mM Tris pH 7.5, 150 mM NaCI, 1% Triton, 1 mM EDTA, 0.1% protease inhibitor, 5 mg/mL MG123 proteasome inhibitor [Merck, Milano, Italy]).
  • the cell lysate was frozen in aliquots at -20 °C until use.
  • the supernatant (10 mL) was ultracentrifuged at 70000 g at 4°C for 2 hours (Beckman L-70 ultracentrifuge) and the pellet was resuspended in 50 pL lysis buffer (10 mM Tris, 2 mM EDTA, 0.15 mM NaCI, 0.5% Nonidet P-40).
  • the cell lysate containing total protein concentrations of 60 and 600 ug and the virus resuspended in lysis buffer were supplemented with 20 ⁇ _ of 2X SDS sample buffer dye (0.5 M Tris HCI pH 6.8, 10% glycerol, 10% SDS, 0.05% ⁇ -mercaptoethanol, 0.05% bromo phenol blue).
  • 2X SDS sample buffer dye 0.5 M Tris HCI pH 6.8, 10% glycerol, 10% SDS, 0.05% ⁇ -mercaptoethanol, 0.05% bromo phenol blue.
  • the samples thus prepared were boiled for 7 minutes and half sample was loaded onto 10% acrylamide/bis- acrylamide gel consisting of a stacking gel (30% Acrylamide/Bis Solution 29:1 [Bio-Rad Italy, Milano]; 0.5 mM Tris HCI pH 6.8, 10% SDS, 10% APS, 10% TEMED) and a resolving gel (30% Acrylamide/Bis Solution 29:1 , 1.5 mM Tris HCI pH 8.8, 10% SDS, 10% APS, 10 % TEMED). Electrophoresis was performed in X running buffer (25 mM Tris, 0.1 % SDS, 1.44% glycine, pH 9.0) at a potential difference of 80- 00 volts for 1 -2 hours.
  • X running buffer 25 mM Tris, 0.1 % SDS, 1.44% glycine, pH 9.0
  • Monoclonal antibody AP33 defines a broadly neutralizing epitope on the hepatitis C virus E2 envelope glycoprotein. J. Virol. 79:11095-11104].
  • Each antibody was tested at two different concentrations of 0.5 ug/mL and 5.0 ug/mL. Moreover, for a triplicate test, three wells were tested for each antibody. Monoclonal antibody AP33, which recognizes a linear epitope in E2 which partially overlaps the epitope present in Peptide I, was used as positive control. AP33 was also used, at concentrations of 0.5 ug/mL and 5.0 ug/mL, for more direct comparison of the neutralizing activity.
  • Each test was performed using both VSV-G pseudotyped particles (as positive control of cell transduction) and particles pseudotyped with E1/E2 of genotype 1a and genotype 3a in order to evaluate the neutralizing efficacy of the antibodies tested against these two different genotypes.
  • VSV-G pseudotyped particles as positive control of cell transduction
  • particles pseudotyped with E1/E2 of genotype 1a and genotype 3a in order to evaluate the neutralizing efficacy of the antibodies tested against these two different genotypes.
  • 2x10 4 HuH-7 cells were seeded in 48-well multiwall plates. One multiwell was seeded for each vector preparation under use.
  • the neutralization assay was set the day after cell seeding: For each well that was seeded with cells, 200 ⁇ _ of each vector (FIV-E1 E2/1a, FIV-E1 E2/3a, FIV- E1 E2 VSV-G) were incubated in small eppendorf tubes with 10 ⁇ of antibody prediluted at dilutions of 10 ⁇ g/mL and 100 ⁇ g/mL in order to obtain samples where antibody concentrations are 0.5 ug/mL and 5.0 ug/mL, respectively. After one hour incubation of samples at 37°C, cell medium was removed and replaced with the mixture of pseudoparticles and antibodies. Multiwell plates were then incubated at 37 °C and 5% CO 2 for 6 hours.
  • the wells were washed 48 hours later with 200 ⁇ _ PBS, treated with 100 ⁇ _ trypsin/ EDTA (Lonza, Walkersville, MD, USA) and incubated for 5 minutes at 37 °C, after which 150 ⁇ _ complete DMEM was added to the cells and each well was collected in a FACS test tube (Sarsted).
  • Sample reading for fluorescence evaluation was with the FACScan model flow cytometer (BD Biosciences) and sample analysis with CellQuest software (BD Biosciences) was used to assess the reduced number of fluorescent cells pre- incubated with the antibody samples as compared with controls (Kc and Kv).
  • the neutralizing ability of the sample was expressed as percent reduction of virus entry. A 40% reduction of infectivity is considered as cut-off for neutralizing activity.
  • VSV-G pseudotyped particles that use a ubiquitous plasmamembrane phospholipid receptor were used as negative control in tests. Possibly, significant reduction of the infectivity of VSV-G pseudotyped particles would imply that anti-E2 monoclonal antibodies bind different portions of the particle or otherwise prevent the infection process by mechanisms not dependent on blockade of the interaction between HCV E2 protein and its cellular receptor (nonspecific neutralization).
  • the monoclonal antibody AP33As As positive control in the various neutralization test, the monoclonal antibody AP33As was used that is capable of neutralizing a broad spectrum of HCV isolates from different genotypes and in a high range of concentrations [Owsianka, A., A. W. Tarr, V. S. Juttla, D. Lavillette, B. Bartosch, F.-L. Cosset, J. K. Ball, and A. H. Patel. 2005. Monoclonal antibody AP33 defines a broadly neutralizing epitope on the hepatitis C virus E2 envelope glycoprotein. J. Virol. 79:11095-11104].
  • Figure 5A shows an analysis of E2 expression in transfected cells.
  • the protein expression level is unknown because of its high degree of glycosylation and multiple forms (variously glycosylated precursors and products)
  • 600 and 60 ug of total proteins of the lysate from cells transfected with vector construct, packaging construct and constructs for E1/E2 with genotypes 1a and 3 were assayed with AP33 and the MAbl-MAbVII mixture.
  • E2 detection was rather blurred, most probably because of the several glycosylation patterns [Owsianka, A., A. W. Tarr, V. S. Juttla, D. Lavillette, B.
  • Monoclonal antibody AP33 defines a broadly neutralizing epitope on the hepatitis C virus E2 envelope glycoprotein. J. Virol. 79:11095-11104], Figure 5A shows that the MAbl-MAbVII mixture of anti-E2 monoclonal antibodies recognizes the protein of genotype 1a and 3 essentially like AP33 which is, as mentioned above, the monoclonal antibody which recognizes a linear epitope of E2 that is highly conserved across HCV genotypes.
  • the MAbl-MAbVII mixture specifically recognizes E2 glycoproteins of genotype 1a and 3 on pseudotyped viral particles generated from transfected cells and purified.
  • the higher molecular weight band is the E1/E2 glycoprotein, non-glycosylated or partially glycosylated, occurring in immature form that is cleaved to produce E1 and E2 during the maturation process which is the final stage of the virus replication cycle that takes place at the extracellular level.
  • the antibodies with greater neutralizing activity are MAb II, MAb VI and MAbVII which reduce infectivity of particles pseudotyped with E1/E2 of genotype 1a by about 40% and 30%, using in the assay 5.0 and 0.5 ug/mL of antibody respectively.
  • This neutralizing activity is quite comparable to that shown for AP33 and is considered to be clinically significant [Keck ZY, Machida K, Lai MM, Ball JK, Patel AH, Foung SK. Therapeutic control of hepatitis C virus: the role of neutralizing monoclonal antibodies.Curr Top Microbiol Immunol. 2008;317:1-38, Stamataki Z, Grove J, Balfe P, McKeating JA.
  • MAb II, MAb VI and MAb VII were used in binary combination between them and all together.
  • the total antibody concentration in the assay was kept 0.5 and 5.0 ug/mL. Therefore, the concentration of each individual MAb is halved in the case of a combination of two antibodies. It is instead reduced to one third in the case of a combination of the three antibodies. Regardless of the association employed, no increase of neutralizing activity was observed compared to the same antibodies assayed individually ( Figure 8). On the contrary, the combination of three antibodies considerably reduces their effectiveness, thus suggesting a possible antibody competition for epitope binding or steric hindrance.

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Abstract

L'invention concerne des anticorps monoclonaux d'origine humaine ou murine qui sont aptes à se lier et à neutraliser l'entrée cellulaire du virus du VHC dans une certaine mesure, et par conséquent sont utiles pour l'immunisation passive de mammifères sains ou infectés par le VHC et pour des procédés analytiques dans l'identification de nouveaux composés aptes à neutraliser une infection par le VHC ou en tant que témoins positifs dans des procédés de détermination des propriétés de neutralisation de nouveaux composés.
PCT/IB2013/054884 2012-06-15 2013-06-14 Anticorps monoclonaux aptes à se lier à la protéine e2 virale du vhc, leur préparation et leur utilisation WO2013186752A1 (fr)

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