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EP4493586A1 - Breit neutralisierende antikörper gegen hepatitis-e-virus - Google Patents

Breit neutralisierende antikörper gegen hepatitis-e-virus

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Publication number
EP4493586A1
EP4493586A1 EP23710899.8A EP23710899A EP4493586A1 EP 4493586 A1 EP4493586 A1 EP 4493586A1 EP 23710899 A EP23710899 A EP 23710899A EP 4493586 A1 EP4493586 A1 EP 4493586A1
Authority
EP
European Patent Office
Prior art keywords
sequence identity
seq
antibody
construct
hev
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23710899.8A
Other languages
English (en)
French (fr)
Inventor
Thomas Krey
George SSEBYATIKA
Katja DINKELBORG
Patrick BEHRENDT
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Twincore Zentrum Fuer Experimentelle und Klinische Infektionsforschung GmbH
Universitaet zu Luebeck
Original Assignee
Twincore Zentrum Fuer Experimentelle und Klinische Infektionsforschung GmbH
Universitaet zu Luebeck
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Twincore Zentrum Fuer Experimentelle und Klinische Infektionsforschung GmbH, Universitaet zu Luebeck filed Critical Twincore Zentrum Fuer Experimentelle und Klinische Infektionsforschung GmbH
Publication of EP4493586A1 publication Critical patent/EP4493586A1/de
Pending legal-status Critical Current

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Classifications

    • 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
    • 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
    • C07K2317/34—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to broadly neutralizing monoclonal antibodies against infectious hepatitis E virus particles.
  • the present invention relates to antibody constructs capable of specifically binding to a conformational epitope of a non-glycosylated polypeptide encoded by hepatitis E virus ORF2, e.g., to monoclonal antibody p60.1 or monoclonal antibody p60.12 or single chains, derivatives or antigen-binding fragments thereof, as specified herein. It also provides heavy chain constructs and/or a light chain constructs of said antibody constructs.
  • the antibody constructs, expression vectors encoding them and host cells capable of expressing them are useful in medicine, e.g., for preventing, treating and diagnosing hepatitis E virus infection.
  • Hepatitis E virus is the most common cause of acute viral hepatitis worldwide and is an emerging problem in industrialized countries. Approximately 2 billion people live in areas endemic for HEV and are at risk of infection. It is estimated that one third of the population in the world may be infected with HEV during their lifetime. This virus infects approximately 20 million people every year and is responsible for an estimated 3.4 million symptomatic cases and 70,000 deaths, mainly in developing countries. While HEV infection is asymptomatic for most patients, some human populations including pregnant women and immunocompromised patients have higher risk to develop severe forms and chronic infections, respectively. Currently, there is neither a specific treatment nor a universal vaccine against HEV.
  • HEV strains infecting humans have been classified into 4 main distinct genotypes (gt) belonging to a single serotype. Genotypes gt1 and gt2 that infect humans only, are primarily transmitted through contaminated drinking water and are responsible for waterborne hepatitis outbreaks in developing countries. In contrast, gt3 and gt4 are zoonotic and are largely circulating in industrialized countries. They are mainly transmitted by contact with swine and consumption of inadequately heated pork products. Recently, further HEV genotypes, subgenotypes and strains have been identified in rabbit, hare, wild boar, moose, camels, and other animals.
  • rat HEV In addition to human pathogenic HEV, related viruses have been identified in fowl, rats, bats, carnivores and even in fish. After its initial description in Germany, rat HEV has been detected in many regions of the world suggesting a worldwide distribution. Recently, several rat HEV-caused human disease cases have been reported, and rat HEV is thought to pose a considerable zoonotic threat.
  • HEV is a quasi-enveloped, positive-sense RNA virus expressing three open reading frames (ORFs): ORF1 , ORF2 and ORF3.
  • ORF1 encodes the non-structural polyprotein that is the viral replicase.
  • ORF2 encodes the viral capsid protein which is involved in particle assembly, binding to host cells and eliciting neutralizing antibodies.
  • ORF3 encodes a small protein that is involved in virion morphogenesis and egress.
  • ORF2 is the best characterized of the three ORFs. Its protein, pORF2, is the main structural component of the virion, carrying the receptor binding domain, the major target for neutralizing antibodies. Three potential N-linked glycosylation sites at positions N137, N310, and N562 have been identified in pORF2, but N-linked glycosylation has only been confirmed to occur at positions N137 and N562. During its lifecycle, HEV produces at least 3 forms of the pORF2 capsid protein: infectious pORF2 (pORF2i), glycosylated pORF2 (pORF2g), and cleaved pORF2 (pORF2c).
  • the pORF2i protein is the structural component of infectious particles.
  • pORF2g and pORF2c are highly glycosylated, and are not associated with the infectious particle. Notably, these secreted forms are very stable, exist as dimers, and are the most abundant forms of pORF2 in sera of HEV infected patients. These proteins probably do not play an important role in the life cycle of HEV, but may inhibit antibody mediated neutralization, thus serving as an immune evasion mechanism for HEV.
  • pORF2g and pORF2c likely act as a humoral immune decoy that inhibits antibody-mediated neutralization.
  • WO2018138344A1 Hepatitis E virus ORF2 capsid polypeptides and uses thereof’ concerns hepatitis E virus pORF2 polypeptides which may be both glycosylated or not and antibodies specific for either the non-glycosylated pORF2i or the glycosylated pORF2g polypeptides for use in HEV diagnosis.
  • WO2018138344A1 does not teach the therapeutic use of antibodies specific for the non-glycosylated pORF2i.
  • W02020011755A1 Antibodies having specificity for the ORF2i protein of hepatitis E virus and uses thereof for diagnostic purposes” concern antibodies which specifically bind to pORF2i and not to pORF2g or pORF2c, to a linear epitope around glycosylation site N3 562NTT.
  • W02020011755A1 does not teach the therapeutic use of antibodies specific for the non-glycosylated pORF2i.
  • detection of infectious virus particles under non-denaturing and non-reducing conditions e.g., in stool or plasma samples from HEV infected individuals
  • ELISA detection of infectious virus particles under non-denaturing and non-reducing conditions (e.g., in stool or plasma samples from HEV infected individuals) by ELISA using antibodies described in W02020011755A1 has not been shown. This is likely due to the linear character of the targeted epitope described therein that is not accessible in natively folded pORF2 dimers constituting infectious particles
  • the present invention in one aspect relates to a heavy chain construct and/or a light chain construct of an antibody construct capable of specifically binding to a conformational epitope of a non-glycosylated polypeptide encoded by hepatitis E virus ORF2, a) wherein the heavy chain construct comprises a CDR1 having at least 90% sequence identity to SEQ ID NO: 2, a CDR2 having at least 88% sequence identity to SEQ ID NO: 3, and a CDR3 having at least 90% sequence identity to SEQ ID NO: 4, and/or the light chain construct comprises a CDR1 having at least 83% sequence identity to SEQ ID NO: 6, a CDR2 having at least 66% sequence identity to the sequence AAS, and a CDR3 having at least 85% sequence identity to SEQ ID NO: 8; or b) wherein the heavy chain construct comprises a CDR1 having at least 87% sequence identity to SEQ ID NO: 10, a CDR2 having at least 87% sequence identity to SEQ ID NO: 11 , and a CDR3
  • said heavy chain construct and/or light chain construct of the invention comprise a) a heavy chain variable region having at least 90% sequence identity to amino acids 1-
  • the antibody construct may be a Fab fragment, a Fab 2 fragment or an scFv or a single chain antibody.
  • the antibody construct may be a human antibody, optionally, a human antibody comprising a constant region.
  • sequence identity in the CDR1 , CDR2, and/or CDR3 may be 100% (to the respective defined CDRs), wherein, preferably, all sequence identity in the CDRs is 100%.
  • sequence identity in the variable regions also is 100% to the respective defined variable regions.
  • the invention provides an antibody construct comprising the heavy chain construct and light chain construct of the invention, i.e. , an antibody construct capable of specifically binding to a conformational epitope of a non-glycosylated polypeptide encoded by hepatitis E virus ORF2.
  • the present invention also relates to a nucleic acid molecule encoding a heavy chain construct and/or light chain construct of the invention or an antibody construct of the invention, as well as to a recombinant expression vector comprising said nucleic acid molecule, preferably, under the control of a heterologous promoter, and to a host cell comprising said recombinant expression vector, which is able to express the heavy chain construct and/or light chain construct or the antibody construct of the invention.
  • the cell may be, e.g., a B cell, a T cell or an NK cell.
  • the present invention also relates to said antibody construct for use in the diagnosis of hepatitis E virus infection.
  • the present invention also relates to a diagnostic kit for the diagnosis of hepatitis E virus infection, comprising at least one of the antibody constructs of the invention, , or any combination thereof, in an amount effective for diagnosis, and, optionally, detection agents suitable for detection of the interaction of antigen and antibody construct.
  • the present invention also relates to the use of said antibody constructs as a medicament for prophylaxis and/or treatment of hepatitis E virus infection.
  • composition comprising a) the antibody construct of the invention, b) the expression vector of the invention, wherein the nucleic acid encodes the antibody construct of the invention and/or c) the host cell of the invention, wherein the nucleic acid encodes the antibody construct of the invention, and, optionally, a pharmaceutically acceptable vehicle and/or excipient.
  • the present invention also relates to said pharmaceutical composition for use in preventing or treating hepatitis E virus infection.
  • FIG 1 is a schematic illustration of HEV pORF2 domains (A) and expression constructs for the P domain fused to either mNeon or mRuby (B).
  • pGS38 intracellular P domain
  • pGS39 secreted P domain.
  • the secreted construct carried a BiP signal sequence to allow translocation into the endoplasmic reticulum. Numbers indicate amino acid positions within HEV gt3 pORF2 (SEQ ID NO: 17).
  • Figure 2 shows the results of neutralization assays of HEV Kernow C1 p6 G1634R viral strain. Neutralization of naked viral particles by p60.1 (A) and p60.12 (B) as well as neutralization of pseudo-enveloped viral particles by p60.1 (C) and p60.12 (D) is shown. Each panel shows the titration results (left) and the respective dose response fitted curve used to calculate the IC50 (right). Panel E shows neutralization results of p60.1 and p60.12 against the gt3 HEV viral strain 83-2-27. FFU/well normalized to the control are depicted.
  • Figure 3 illustrates monoclonal antibody expression and purification:
  • A Size exclusion chromatography profiles representing the general behavior of the antibodies p60.1 and p60.12 after separation on SD200 increase 10/300 GL column equilibrated in 1x PBS at a flow rate of 0.5 ml/min.
  • B A 12 % Coomassie-stained SDS-PAGE gel with the antibodies p60.1 and p60.12 loaded under reducing conditions. The estimated molecular weight for the heavy chain is ca. 55 kDa and ca. 25 kDa for the light chain.
  • Panel B shows the results of binding of the depicted antibody to proteins present in hepatoma cells transfected with the indicated viral strain.
  • the GLUC reporter replicon was used as negative control as it does not contain ORF2.
  • convalescent patient serum was used.
  • p60.1 shows binding to the gt1 viral strain SAR55.
  • Figure 6 shows ELISA results using the anti-HEV monoclonal antibodies p60.1 or p60.12 together with sera from infected patients.
  • the indicated antibodies were used to capture antigens present in HEV RNA positive patient plasma.
  • Figure 7 illustrates the differential binding activity of the anti-HEV monoclonal antibodies of the invention as tested by size exclusion chromatography.
  • the elution profiles demonstrate that scFv-p60.1 and scFv-p60.12 only bind to the non-secreted P domain (pGS99) and not to the secreted P domain (pGS100) from HEV gt3.
  • Figure 8 illustrates the determination of P domain-monoclonal antibody binding properties:
  • A Sensorgrams showing association and dissociation phases of the interaction between non-secreted HEV gt3 P domain and the neutralizing antibodies of the invention.
  • B Sensorgrams showing association and dissociation phases of the interaction between secreted HEV gt3 P domain and the neutralizing antibodies of the invention.
  • Analyte concentrations (mAbs) were 5, 10, 25, 50 and 75 nM. Binding data are shown as black lines and fitting is shown overlaid in gray lines.
  • Figure 9 shows the size exclusion chromatography profiles obtained from complexes of anti-HEV scFvs with the HEV gt3 P domain for scFv-p60.1 and pGS99 and scFv-p60.12 and pGS99.
  • Figure 10 shows the crystals obtained from complexes of anti-HEV scFvs with the HEV gt3 P domain (pGS99) in complex with the neutralizing mAbs (A) scFv-p60.1 and (B) Fab- p60.12.
  • Figure 11 shows the crystal structure of the HEV gt3 P domain (pGS99) in complex with the neutralizing mAb scFv-p60.1 , shown in cartoon representation with the heavy (VH) and light chains (VL).
  • the P domain dimer has one protomer colored black and the other grey.
  • One complex in the asymmetric unit shows the P domain with surface representation, while the other is a cartoon representation.
  • Figure 12 depicts an overview (top panels) and close-up view (lower panels) of antibodyantigen interfaces of p60.1 (A) and p60.12 (B) in complex with the dimeric HEV gt3 P domain.
  • the antibody dasheavy chain in dark grey, light chain in light grey
  • contacts the two protomers of the P domain grey, shown at the bottom
  • the asparagine side chain of N562 shown in sticks
  • Figure 13 shows a comparison of the complexes between the HEV gt3 P domain dimer with human (scFv-p60.1) and mouse (scFv from Fab 8C11 and scFv from Fab 8G12) anti- HEV antibodies.
  • Figure 14 illustrates the fitting of the crystal structures of the HEV gt3 P domain in complex with mouse and human scFvs to HEV virus-like particle (VLP) structure.
  • the VLP (PDB 2ztn) is shown as surface representation while the scFvs are shown as cartoons.
  • the P domain was used to fit the complexes to the VLP.
  • Figure 15 shows the amino acid sequence alignment of the P domains from the five HEV genotypes studied (SEQ ID NO: 18-22).
  • Contact residues for the mAbs are indicated as follows: ⁇ : contact residues shared between p60.1 and p60.12 epitope. •: contact residues for p60.12 and ⁇ : contact residues for p60.1.
  • Neutralizing antibodies can be classified into two groups, the first group comprising antibodies that inhibit only the infecting viral variant and not any other virus strains or variants. These antibodies are known as autologous antibodies and their neutralization is considered transient and highly affected by viral evolution.
  • the second group of antibodies comprises broadly neutralizing antibodies (bNAbs). These have the ability to neutralize several variants of the same virus or members of the same viral family. Such antibodies often target epitopes that are highly conserved across diverse strains, virus subtypes and families.
  • the present invention provides highly potent bNAbs, which may be used for the diagnosis, the prophylaxis and/or the therapy of HEV, showing high binding affinity, potent neutralization activity, as well as broad reactivity across HEV genotypes, including human and rat HEV.
  • Said antibodies or antibody constructs specifically target a glycan-dependent conformational neutralization epitope in pORF2 that is only accessible in the infectious (non-glycosylated) HEV particle, so that their neutralization activity is specific for the non- glycosylated form of pORF2, and cannot be evaded by the soluble secreted (glycosylated) forms of pORF2.
  • the first object of the present invention is a heavy chain construct and/or a light chain construct of an antibody construct capable of specifically binding to a conformational epitope of a non-glycosylated polypeptide encoded by hepatitis E virus ORF2, a) wherein the heavy chain construct comprises a CDR1 having at least 90% sequence identity to SEQ ID NO: 2, a CDR2 having at least 88% sequence identity to SEQ ID NO: 3, and a CDR3 having at least 90% sequence identity to SEQ ID NO: 4, and/or the light chain construct comprises a CDR1 having at least 83% sequence identity to SEQ ID NO: 6, a CDR2 having at least 66% sequence identity to the sequence AAS, and a CDR3 having at least 85% sequence identity to SEQ ID NO: 8 (i.e., the CDRs of monoclonal antibody p60.1); or b) wherein the heavy chain construct comprises a CDR1 having at least 87% sequence identity to SEQ ID NO: 10, a CDR2 having
  • said heavy chain construct and/or light chain construct of the invention comprise a) a heavy chain variable region having at least 90% sequence identity to amino acids 1-
  • the invention provides a heavy chain construct and/or light chain construct of the invention, wherein a) the heavy chain construct comprises a CDR1 having at least 90% sequence identity to SEQ ID NO: 2, a CDR2 having at least 88% sequence identity to SEQ ID NO: 3, and a CDR3 having at least 90% sequence identity to SEQ ID NO: 4, and/or the light chain construct comprises a CDR1 having at least 83% sequence identity to SEQ ID NO: 6, a CDR2 having at least 66% sequence identity to the sequence AAS, and a CDR3 having at least 85% sequence identity to SEQ ID NO: 8.
  • the heavy chain variable region has at least 90% sequence identity to amino acids 1-121 of SEQ ID NO: 1 and/or and the light chain variable region has at least 90% sequence identity to amino acids 1-106 of SEQ ID NO: 5, and, optionally, the heavy chain has at least 90% sequence identity to SEQ ID NO: 1 and/or and light chain has at least 90% sequence identity to SEQ ID NO: 5.
  • Antibody constructs comprising said CDRs, said variable regions or said heavy and light chain constructs are designated p60.1.
  • the preferred scFv p.60.1 comprises a heavy chain construct of SEQ ID NO: 1 and a light chain construct of SEQ ID NO: 5.
  • the invention provides a heavy chain construct and/or light chain construct of the invention, wherein a) the heavy chain construct comprises a CDR1 having at least 87% sequence identity to SEQ ID NO: 10, a CDR2 having at least 87% sequence identity to SEQ ID NO: 11 , and a CDR3 having at least 93% sequence identity to SEQ ID NO: 12 and/or the light chain construct comprises a CDR1 having at least 88% sequence identity to SEQ ID NO: 14, a CDR2 having at least 66% sequence identity to the sequence DVT, and a CDR3 having at least 90% sequence identity to SEQ ID NO: 16.
  • the heavy chain variable region has at least 90% sequence identity to amino acids 1-122 of SEQ ID NO: 9 and/or and the light chain variable region has at least 90% sequence identity to amino acids 1-111 of SEQ ID NO: 13, and, optionally, the heavy chain has at least 90% sequence identity to SEQ ID NO: 9 and/or and light chain has at least 90% sequence identity to SEQ ID NO: 13.
  • Antibody constructs comprising said CDRs, said variable regions or said heavy and light chain constructs are designated p60.12.
  • the preferred scFv p.60.12 comprises a heavy chain construct of SEQ ID NO: 9 and a light chain construct of SEQ ID NO: 13.
  • antibody construct or “antibody”, also “immunoglobulin”, has its general meaning in the art and refers to any antibody-like molecule that has an antigen binding region, and this term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, and single domain antibodies (DABs). Chimeric antigen receptors (CARs) are also considered antibody constructs.
  • Antibody constructs may, as in natural antibodies, e.g., of the IgG class, comprise two heavy chains linked to each other by disulfide bonds, wherein each heavy chain is linked to a light chain by a disulfide bond.
  • Antibody constructs of the invention may comprise constant regions, and may belong to each isotype.
  • antibodies of the invention may be of the IgG isotype, e.g. IgGi or lgG4. Each chain contains distinct sequence domains.
  • the light chain typically includes two domains, a variable domain (VL) and a constant domain (CL).
  • the heavy chain typically includes four domains, a variable domain (VH) and three constant domains (CH1 , CH2 and CH3, collectively referred to as CH).
  • VL variable domain
  • VH variable domain
  • CH constant domain
  • Fab antigen-binding fragments
  • Fc crystallizable fragment
  • the constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors.
  • the Fv fragment is the N-terminal part of the Fab fragment of an antibody and consists of the variable portions of one light chain and one heavy chain.
  • the specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant.
  • Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FR, meaning amino acid sequences interposed between CDRs) influence the overall domain structure.
  • CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native antibody binding site.
  • the light and heavy chains of an antibody each have three CDRs, designated L-CDR1 , L-CDR2, L- CDR3 and H-CDR1 , H- CDR2, H-CDR3, respectively.
  • An antigen-binding site therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain variable region.
  • antibody fragment refers to at least one portion of an intact antibody, preferably the antigen binding region or variable region of the intact antibody, that retains the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, single chain antibody molecules, in particular scFv (single chain variable fragments), disulfide-l inked Fvs (sdFv), a Fd fragment consisting of the VH and CH1 domains, linear antibodies, single domain antibodies such as, for example, sdAb (either VL or VH), camelid VHH domains, multi-specific antibodies formed from antibody fragments such as, for example, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody.
  • An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv.
  • Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III.
  • the antibody construct of the invention may be a Fab fragment or a single chain antibody.
  • the antibody construct is a monoclonal antibody.
  • monoclonal antibody refers to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody refers to a population of antibody molecules that contain only one species of antibody combining site capable of immunoreacting with a particular epitope.
  • a monoclonal antibody thus typically displays a single binding affinity for any epitope with which it immunoreacts.
  • a monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different epitope, e.g., a bispecific monoclonal antibody.
  • a monoclonal antibody was produced by immortalization of a clonally pure immunoglobulin secreting cell line, a monoclonally pure population of antibody molecules can also be prepared by other methods known in the art.
  • a heavy chain construct or a light chain construct of the invention is able to form an antibody construct of the invention, together with the respective other chain.
  • the exact structure of the heavy and light chain constructs thus depends on the desired structure of the antibody construct, e.g., as detailed herein.
  • the heavy chain construct and the light chain construct can also be part of the same amino acid chain, e.g., in a single chain antibody such as an scFv.
  • a neutralizing antibody or “broadly neutralizing antibody” is one that can neutralize the ability of that pathogen to initiate and/or perpetuate an infection in a host.
  • the antibodies produced in accordance with the present invention have neutralizing activity, where the antibody can neutralize at a concentration of 10’ 8 M or lower (e.g., 10’ 9 M, 10’ 10 M, 10’ 11 M, 10’ 12 M or lower).
  • binding refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges.
  • the antibody construct of the present invention has specificity for the ORF2i protein.
  • specificity refers to the ability of an antibody to detectably bind to a conformational epitope presented on the ORF2i protein, while having relatively little detectable reactivity with the ORF2g protein and the ORF2c protein. Specificity can be relatively determined by binding or competitive binding assays as known from the art. Specificity can be exhibited by, e.g., an about 10: 1 , about 20: 1 , about 50: 1 , about 100: 1 , 10.000: 1 or greater ratio of affinity/avidity in binding to the specific antigen versus nonspecific binding to other irrelevant molecules.
  • affinity means the strength of the binding of an antibody to an epitope. Binding affinity can be described by an antibody's equilibrium dissociation constant (KD), which is defined as the ratio Kd/Ka at equilibrium. Ka is the antibody's association rate constant and Kd is the antibody's dissociation rate constant. Binding affinity is determined by both the association and the dissociation and alone neither high association nor low dissociation can ensure high affinity.
  • Ka equilibrium dissociation constant
  • Kon on-rate constant
  • the association rate constant is expressed in M -1 s -1 , and is symbolized as follows: [Ab]x[Ag]xKon.
  • the dissociation rate constant (Kd), or off-rate constant (Koff) measures the number of dissociation events per unit time propensity of an antibody-antigen complex to separate (dissociate) reversibly into its component molecules, namely the antibody and the antigen.
  • the dissociation rate constant is expressed in s -1 , and is symbolized as follows: [Ab+Ag]xKoff.
  • the equilibrium dissociation constant measures the rate at which new antibody-antigen complexes formed equals the rate at which antibody-antigen complexes dissociate at equilibrium.
  • the antibody construct of the invention has an equilibrium dissociation constant KD for non-glycosylated form of pORF2 of less than 100 nM, less than 50 nM less than 10 nM, less than 5 nM, less than 2 nM, less than 1 nM or preferably less than 0.5 nM.
  • epitope refers to a specific arrangement of amino acids located on a protein to which an antibody binds. Epitopes often consist of a chemically active surface grouping of molecules such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics, as well as specific charge characteristics. Epitopes can be linear or conformational, i.e. , involving two or more sequences of amino acids in various regions of the antigen that may not necessarily be contiguous.
  • formational epitope refers to amino acid residues that are, at least in part, discontinuous in the epitope protein sequence yet come within close proximity to form an antigenic surface on the protein's three-dimensional structure.
  • polypeptide refers to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a polypeptide is not limited to a specific length: it must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a polypeptide's sequence.
  • Peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • the term “peptides” refers to a linear polymer of amino acids linked together by peptide bonds, preferably having a chain length of less than about 50 amino acids residues; a "polypeptide” refers to a linear polymer of at least 50 amino acids linked together by peptide bonds, and a protein specifically refers to a functional entity formed of one or more peptides or polypeptides, optionally of non-polypeptides cofactors.
  • polypeptide may be an entire protein, or a subsequence thereof.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • a polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.
  • glycosylated with respect to a protein means that a carbohydrate moiety is present at one or more sites of the protein molecule.
  • a glycosylated protein refers to a protein that is typically modified by N-glycan or O-glycan addition.
  • ORF2 refers to the open reading frame encoding the HEV ORF2 viral capsid protein (“pORF2”).
  • the ORF2 protein sequence contains 660 amino acids, has a signal peptide at its N-terminus (that can shuttle it to the extracellular compartment, depending on the start codon), and is further organized into three domains designated as shell (S, amino acids 129 - 319), middle (M, amino acids 320 - 455), and protruding domain (P, amino acids 456 - 606).
  • N-X-S/T Three highly conserved potential N-glycosylation sites represented by the sequon Asn-X-Ser/Thr (N-X-S/T) have been identified in pORF2, but N-linked glycosylation has only been confirmed to occur at positions N137 and N562.
  • HEV infection leads to the production of at least 3 forms of pORF2 capsid protein: infectious ORF2 (ORF2i), glycosylated ORF2 (ORF2g), and cleaved ORF2 (ORF2c), as shown by adjoin et al. (2018. “Hepatitis E virus lifecycle and identification of 3 forms of the ORF2 capsid protein” Gastroenterology 154 (1): 211-223).
  • the ORF2i protein is the structural component of infectious particles, and it is not glycosylated.
  • ORF2g and ORF2c proteins are secreted in large amounts in culture supernatant and sera of infected patients, are sialylated, N- and O-glycosylated but are not associated with infectious virions.
  • ORF2g and ORF2c proteins the most abundant antigens detected in patient sera, might inhibit antibody-mediated neutralization.
  • the present invention provides antigen-binding proteins, antibody constructs, such as antibodies and antigen-binding fragments thereof, preferably monoclonal antibodies, that specifically bind to a non-glycosylated (infectious) HEV particle, or an antigenic fragment thereof, but not to a glycosylated (non-infectious) HEV particle.
  • the present invention provides antibody constructs, such as antibodies and antigen-binding fragments thereof, preferably monoclonal antibodies, that specifically bind to a conformational epitope of non-glycosylated pORF2, preferably that specifically bind to a non-glycosylated conformational epitope at the dimerization interface at the tip of the P domain dimer of pORF2 containing glycosylation site N562.
  • antibody constructs such as antibodies and antigen-binding fragments thereof, preferably monoclonal antibodies, that specifically bind to a conformational epitope of non-glycosylated pORF2, preferably that specifically bind to a non-glycosylated conformational epitope at the dimerization interface at the tip of the P domain dimer of pORF2 containing glycosylation site N562.
  • said antibody construct e.g., said monoclonal antibody is monoclonal antibody p60.1 against HEV, which can specifically bind and neutralize non-glycosylated (infectious) HEV particles. It can also be a derivative thereof, preferably, sharing the CDRs and, optionally, the variable regions thereof .
  • said antibody constructs e.g., said monoclonal antibody is monoclonal antibody p60.12 against HEV, which can specifically bind and neutralize non-glycosylated (infectious) HEV particles. It can also be a derivative thereof, preferably, sharing the CDRs and, optionally, the variable regions thereof .
  • the antibody constructs of the invention recognize a quaternary epitope that assembles at the dimerization interface at the tip of the P domain dimer of pORF2.
  • the broad neutralizing antibodies of the invention bind predominantly to one protomer, but make contacts also with the other one, resulting in an asymmetric binding mode.
  • a correctly folded pORF2 dimer is required for antigen-antibody interaction of said antibodies with HEV according to the invention.
  • the antibodies of the invention recognize conformation-sensitive epitopes comprising three segments distant in the amino acid sequence of pORF2: 1) amino acids 480-490, 2) amino acids 555-565 and 3) amino acids 580-590.
  • N562 of both protomers which serve as attachment points for the N-linked glycans in the secreted pORF2 dimer, form hydrogen bonds with both antibodies, so that such an attached glycan would preclude binding of these bnAbs via steric clashes and thereby causes glycan sensitivity of the antibodies.
  • the CDRs of the heavy chain variable region correspond to the amino acid sequences as set forth in SEQ ID NO:2 (p60.1-HC CDR1), SEQ ID NO:3 (p60.1-HC CDR2) and SEQ ID NO:4 (p60.1 -HC CDR3).
  • the CDRs of the light chain variable region correspond to the amino acid sequences as set forth in SEQ ID NO:6 (p60.1-LC CDR1), the sequence AAS (p60.1-LC CDR2) and SEQ ID NO:8 (p60.1-LC CDR3).
  • the CDRs of the heavy chain variable region correspond to the amino acid sequences as set forth in SEQ ID NO:10 (p60.12-HC CDR1), SEQ ID NO:11 (p60.12-HC CDR2) and SEQ ID NO:12 (p60.12-HC CDR3).
  • the CDRs of the light chain variable region correspond to the amino acid sequences as set forth in SEQ ID NO: 14 (p60.1-LC CDR1), the sequence DVT (p60.12-LC CDR2) and SEQ ID NO: 16 (p60.12-LC CDR3).
  • the antibodies of the invention may be human antibodies.
  • human antibodies includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences whether in a human cell or grafted into a non-human cell, e.g., a mouse cell.
  • the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • the term includes antibodies recombinantly produced in a non-human mammal or in cells of a non-human mammal.
  • human antibody is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (e.g., humanized antibodies).
  • the antibodies may alternatively be non-human antibodies, e.g., murinized antibodies or rat antibodies, e.g., for in vivo experiments in mice or rats.
  • the present invention also relates to conserved variants or active fragments of any one of the antibody constructs of the invention sharing the CDRs of p60.1 or p60.12, wherein one or more of amino acid residues in said variant or active fragment thereof have been conservatively substituted, added or deleted, wherein the variant or fragment still retains the capability of specifically binding to a conformational epitope of a non-glycosylated polypeptide encoded by hepatitis E virus ORF2.
  • conserved variants used herein means that the variants substantially retain the parent's properties, such as basic immunological properties, structural properties, regulating properties or biochemical properties.
  • amino acid sequence of the conserved variants of the polypeptide is limitedly different from the parent polypeptide such that the conserved variants and the parent polypeptide are closely similar as a whole and are identical in a lot of regions.
  • the difference of amino acid sequence between the conserved variants and parent polypeptide can be replacement, addition and deletion of one or more amino acid residues or any combination thereof.
  • the replaced or added amino acid residues may or may not be encoded by genetic code.
  • the conserved variants of the polypeptide may be variants produced spontaneously or not spontaneously.
  • polypeptide's conserved variants produced not spontaneously may be produced by induced mutation techniques or by direct synthesis. According to the disclosed content of the invention, a person skilled in the art would appreciate that the fragment of said antibody of the invention may be modified to substantially preserve the property of specifically binding to hepatitis E virus of the generated antibody variants.
  • any deviations from the defined SEQ ID NO: may be conservative substitutions, wherein, preferably, there is at most one conservative substitution per chain.
  • Conservative substitutions are known in the art, wherein an amino acid is exchanged for a different amino acid with similar biochemical properties, e.g., charge, hydrophobicity and/or size.
  • substitutions are selected from the same class of amino acids, as defined herein:
  • the sequence identity in the CDR1 , CDR2, and/or CDR3 may be 100% (to the respective defined CDRs).
  • sequence identity in all CDRs or a heavy chain consruct and/or a light chain construct is 100%.
  • sequence identity in the variable regions also is 100%, i.e., the variable regions of the antibody construct or the inventions correspond to the variable regions of p60.1 or p60.2.
  • the invention does not only provide the heavy and light chain constructs, but also an antibody construct comprising the heavy chain construct and light chain construct of the invention, i.e., an antibody construct capable of specifically binding to a conformational epitope of a non-glycosylated polypeptide encoded by hepatitis E virus ORF2.
  • an antibody construct capable of specifically binding to a conformational epitope of a non-glycosylated polypeptide encoded by hepatitis E virus ORF2.
  • said antibody constructs have been found to be particularly advantageous for diagnostic, therapeutic and prophylactic uses.
  • nucleic acid molecule encoding a heavy chain construct and/or light chain construct of the invention or an antibody construct of the invention.
  • a is understood to mean "at least one", i.e., a nucleic acid molecule of the invention may also encode a heavy chain construct of the invention and a light chain construct of the invention, and preferably encodes an antibody constructs of the invention.
  • Said nucleic acid molecule may be a recombinant expression vector comprising said nucleic acid molecule, preferably, under the control of a heterologous promoter.
  • the promotor preferably is able to mediate expression in a host cell, e.g., a bacterial cell, a yeast cell, a eukaryotic cell, an insect cell, a mammalian cell such as a CHO cell or a human cell, e.g., a B cell, a T cell or an NK cell.
  • a host cell e.g., a bacterial cell, a yeast cell, a eukaryotic cell, an insect cell, a mammalian cell such as a CHO cell or a human cell, e.g., a B cell, a T cell or an NK cell.
  • the promotor can be a constitutive promotor or an inducible promotor.
  • the skilled in the art can get the nucleotide sequences encoding the same and get their variants according to codon degeneracy.
  • the nucleic acid is codon-optimized for expression in the desired host cell.
  • the skilled in the art can get recombinant expression vectors comprising the nucleotide sequences described above, and host cells transformed with the expression vectors by many methods. Selection of host cells and transformation technologies that can be used to express said antibodies or their active fragments are well known from the art.
  • the present invention also relates to a recombinant expression vector comprising the nucleic acid molecule encoding said antibodies or active fragments.
  • vector any genetic element, such as a plasmid, minicircle, YAC, phage, transposon, e.g., suitable for being transposed by Sleeping Beauty, cosmid, chromosome, a viral vector or virus, e.g., a retroviral or adenoviral vector etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells.
  • the term includes cloning and expression vectors, as well as viral vectors.
  • the antibodies of the invention and/or active fragments thereof can be expressed in proper host cells by genetic engineering methods known in the art.
  • the invention further relates to host cells transformed with said recombinant expression vectors.
  • Many expression host cells can be used in the present invention, for example, prokaryotic cells including but not limited to Escherichia coli, Bacillus, Streptomyces, eukaryotic cells including but not limited to Aspergillus, Saccharomycetes, as well as mammalian cells, plant cells and so on.
  • the expression of the antibody constructs of the invention is not limited to any specific expression vectors or host cells, as long as they can be used to express said antibodies.
  • the invention thus also provides a host cell comprising said recombinant expression vector, which is able to express the heavy chain construct and/or light chain construct or the antibody construct of the invention.
  • the host cell expresses said construct, e.g., said antibody construct, on its cell surface (e.g., in case of a CAR or a transmembrane immunoglobulin), or it secretes said construct (typically, in the case of an antibody, e.g., a single chain antibody).
  • the cell may be a human cell, e.g., a B cell, a T cell or an NK cell.
  • the antibody constructs of the present invention bind to antigens present in sera of patients infected with HEV.
  • the present invention also relates to said antibody constructs for use in diagnosis of hepatitis E virus infection.
  • a further object of the present invention relates to a method for detecting the presence of native, non-glycosylated HEV pORF2 in a sample, e.g., a sample from a patient, comprising contacting the sample with the antibody constructs of the present invention under conditions that allow an immunocomplex of the protein and the antibodies to form, wherein detection of the immunocomplex indicates the presence of native, nonglycosylated HEV pORF2 in the sample (for instance: immunoprecipitation, immunofluorescence or western blotting).
  • infectious HEV particles may be detected using the antibody constructs of the present invention because these antibody constructs, e.g., monoclonal antibodies, recognize specific antigenic determinants for infectious HEV particles located on the surface of HEV.
  • a further object of the present invention relates to a method for detecting the presence of infectious HEV particles in a sample comprising contacting the sample with the antibodies of the invention under conditions that allow an immunocomplex of the antibody and the infectious particles to form, wherein detection of the immunocomplex indicates the presence of the infectious particles in the sample.
  • sample includes any solid or fluid sample, liable to contain infectious particles of hepatitis E virus.
  • the sample is selected from the group consisting of ascites, urine, saliva, sweat, milk, synovial fluid, peritoneal fluid, amniotic fluid, percerebrospinal fluid, lymph fluid, lung embolism, cerebrospinal fluid, and pericardial fluid.
  • the sample is a feces sample.
  • the sample is a urine sample.
  • the sample is a saliva sample.
  • the sample is a blood sample.
  • blood sample means any blood sample derived from a subject.
  • the detecting methods of the present invention are particularly suitable for diagnosing acute HEV infection, recent HEV infection, chronic HEV infection, weak active HEV infection or cleared HEV infection.
  • Assays and conditions for the detection of immunocomplexes are known to those of skill in the art. Such assays include, for example, competition assays, direct reaction assays sandwich-type assays and immunoassays (e.g., ELISA).
  • the assays may be quantitative or qualitative. There are a number of different conventional assays for detecting formation of an antibody-peptide complex comprising an antibody of the present invention.
  • the detecting step can comprise performing an ELISA assay, performing a lateral flow immunoassay, performing an agglutination assay, analyzing the sample in an analytical rotor, or analyzing the sample with an electrochemical, optical, oropto-electronic sensor.
  • the antibody constructs of the present invention may be used in highly sensitive methods for screening and identifying individuals carrying HEV and/or infected with HEV, as well as for screening for HEV-contaminated samples.
  • the antibody constructs of the present invention may also be used in assays for monitoring the progress of anti-HEV therapies in treated individuals, or for monitoring the growth rate of HEV cultures used in research and investigation of the HEV agent.
  • kits for the diagnosis of hepatitis E virus infection comprising at least one of the antibody constructs of the invention, or any combination thereof (e.g., an antibody construct based on p60.1 and an antibody construct based on p60.12).
  • the kit may comprise the antibody construct in an amount effective for diagnosis. It optionally comprises detection agents suitable for detection of the interaction of antigen and antibody construct corresponding to the adopted detection method.
  • the kit may also comprise suitable buffers.
  • the term "amount effective for diagnosis” is intended to mean the antibody construct of the invention, conserved variants or active fragments thereof at the amount effective for detection of HEV in a biological sample.
  • the amount of the above-mentioned material would be variable depending on the different immunochemical methods used.
  • the diagnostic kit where appropriate, should further include suitable absorbent carriers, buffer reagent/solutions, reagents used to produce visible signals fortesting and instructions for use. Examples of kits include but are not limited to ELISA assay kits, and kits comprising test strips and dipsticks.
  • the antibody constructs of the invention specifically recognize antigenic determinants located on the surface of infectious HEV particles, showing high binding affinity, potent neutralization activity, as well as broad reactivity across HEV genotypes.
  • the antibodies of the invention efficiently neutralize naked and quasi-enveloped viral particles. Said antibodies may therefore be used for the prophylaxis and/or for the passive immunization treatment of HEV infections, especially for chronical and immunocompromised HEV patients.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a) an antibody construct of the invention, b) an expression vector of the invention, wherein the nucleic acid encodes the antibody construct of the invention and/or c) a host cell of the invention, wherein the nucleic acid encodes the antibody construct of the invention, and, optionally, a pharmaceutically acceptable vehicle and/or excipient.
  • Said pharmaceutical composition may be for use in preventing or treating hepatitis E virus infection. They may comprise a pharmaceutically effective amount of said antibody construct.
  • the present invention relates to the antibody constructs of the invention for use as a medicament for prophylaxis and/or treatment of HEV infection.
  • the pharmaceutical composition of the invention When supplied prophylactically , the pharmaceutical composition of the invention is provided in advance of any exposure to any one or more of the HEV strains or in advance of any symptoms due to infection of the viruses.
  • the prophylactic administration of the pharmaceutical composition of the invention serves to prevent, reduce the risk or likelihood of or attenuate any subsequent infection of these viruses in a mammal, preferably in a human subject.
  • the pharmaceutical composition of the invention is provided at or after (preferably, shortly after) the onset of infection or at the onset of any symptom of infection or any disease or deleterious effects caused by these viruses.
  • the therapeutic administration of the pharmaceutical composition of the invention serves to attenuate the infection or disease.
  • the pharmaceutical composition of the present invention may, thus, be provided either prior to the anticipated exposure to HEV or after the initiation of infection. According to the specific conditions of the subject to be treated, the skilled in the art know how to select proper doses and administration routes.
  • the pharmaceutical composition of the invention may also comprise a therapeutically effective amount of the antibodies of the invention combined with at least one other anti-viral agent as an additional active ingredient.
  • agents may include but are not limited to interferons, other anti HEV monoclonal antibodies, anti HEV polyclonal antibodies, RNA polymerase inhibitors, protease inhibitors, IRES inhibitors, helicase inhibitors, immunomodulators, antisense compounds and ribozymes.
  • a method of treating a subject infected with HEV or of reducing the likelihood of infection of a subject at risk of contracting HEV comprising delivering to said subject a prophylactically effective amount or a therapeutically effective amount of at least one of the antibody constructs of the invention.
  • prophylactically effective amount can be replaced with the term “immunologically effective amount", which means the amount sufficient to elicit immune prevention for a subject. It is well known that the “prophylactically effective amount” may vary according for example to the administration way, the characteristics of the individual subject, and the antibody construct used. According to the published references, teachings and corresponding clinical criteria in the art, the “prophylactically effective amount” of an antibody construct can be determined.
  • the preferable prophylactically I immunologically effective amount is 0.0001 mg - 0.1 mg per dose.
  • the term "therapeutically effective amount” means the amount sufficient to elicit effective protection for the subjects and to neutralize HEV, or an amount effective in alleviating the symptoms of the HEV infection or reducing the number of circulating viral particles in an individual. It is well known that a therapeutically effective amount of an antibody may vary depending on different treatment schemes, illness courses, characteristics of the individual subject, and the antibodies used. According to the published references, teachings and corresponding clinical criteria in the art, a clinician can determine the "therapeutically effective amount" of an antibody construct based on their own experience. The preferable therapeutically effective amount is 0.001 mg - 20 mg per kg weight.
  • the antibody constructs of the invention based on p60.1 are particularly effective in neutralizing HEV g3, g4 and g2, and the constructs based on p60.12 are particularly effective in neutralizing HEV g3, g4 and rat-HEV, they are preferably used for diagnosis, treatment or prevention of said virus subtypes.
  • the P domain serves as the receptor-binding domain of HEV and is thus a primary target for neutralizing antibodies.
  • pORF2 exhibits both secreted and non-secreted forms during infections. The former are present as glycosylated dimers in sera of infected patients, while the later are components of the infectious particle.
  • constructs for all genotypes were designed to represent the non-secreted form of the P domain.
  • a construct representing the secreted form was designed using HEV genotype 3 P domain (Table 1).
  • the pGS plasmids according to Table 1 verified by sequencing to carry the desired inserts were transfected into Drosophila S2 cells essentially as described previously (Johansson et al. 2012. Production of Recombinant Antibodies in Drosophila mela nogaster S2 Cells. Patrick Chames (ed.), Antibody Engineering: Methods and Protocols, Second Edition, Methods in Molecular Biology, 907). Briefly, stable cell lines were generated by cotransfecting the plasmids coding for the gene of interest with a puromycin-resistance selection plasmid. Following selection with puromycin and adaptation of the cells to insect- Xpress media, cells were induced with CdCh for large-scale protein production. On day five post-induction, the intracellular and the secreted P domains were affinity purified from cell lysates and culture supernatants, respectively. Protein purity assessment was performed by SDS-PAGE analysis.
  • antigen-specific memory B cells were isolated from peripheral blood mononuclear cells (PBMCs) from two convalescent HEV patients (N1960 (p60), 4100869889, 8.5 x 10 7 cells and N1961 (p61), 1707407510, 1.44 x 10 8 cells) by magnetic activated cell sorting (MACs). MACs-sorted cells were then stained with fluorescently labeled P domains and analyzed for the frequency and phenotype of antigen-specific B cells by flow cytometry prior to sorting of antigen-specific B cells. This was then followed by single B cell RNA sequencing as described in the next sections.
  • PBMCs peripheral blood mononuclear cells
  • MACs-sorted cells were then stained with fluorescently labeled P domains and analyzed for the frequency and phenotype of antigen-specific B cells by flow cytometry prior to sorting of antigen-specific B cells. This was then followed by single B cell RNA sequencing as described in the next sections.
  • PBMCs from two convalescent HEV patients were isolated using density gradient separation medium (Histopaque, Sigma Aldrich) according to the manufacturer’s instructions and stored at -150 °C in 10% DMSO and 90% (v/v) fetal bovine serum.
  • B cells were obtained from PBMCs by magnetic separation using CD19 microbeads and stained with anti-human CD20-Alexa Fluor 700, anti-human IgG-APC, LIVE/DEADTM Fixable Near-IR Dead Cell Stain, and recombinant differentially fluorescently labelled HEV gt3 P domains (pGS38 and pGS39) with the aim of obtaining antibodies that bind the nonglycosylated form but not the glycosylated form.
  • Approximately 4,170 and 19,760 live cells were sorted for p60 and p61 , respectively, using band pass filters R 780/60, R 730/45, R 670/30, YG 610/20 and B 530/30.
  • Antigen-specific memory B cells were single-cell sorted using 10xGenomics technology and B cell receptor sequences obtained by Next Generation Sequencing using Illumina technology. The obtained FASTQ files were used to generate single cell V(D)J sequences and annotations using Cell ranger vdj (version 3.1.0). Output files were loaded and analyzed with the Loupe V(D)J Browser (10x Genomics).
  • variable regions of antibodies were selected after analysis of the sequences using the Loupe V(D)J browser based on a criterion described in the result section below.
  • the variable region of the heavy chain was covalently linked via its C-terminus to the N-terminus of the respective light chain variable region using a 3x glycine-serine linker (GS-linker).
  • Codon-optimized synthetic genes of the covalently linked paired sequences cloned into a pMT vector were purchased from Twist biosciences. All constructs carried a Drosophila BiP signal sequence at the N-terminus as well as an EK cleavage site and a double strep-tag at the C-terminus.
  • the name of the individual scFvs was defined by scFv as a prefix, followed by the donor ID and the clone number. Expression and purification of all scFvs followed the protocols in sections 1.1.3.
  • the P domains and TEV protease were mixed in a 1 :4 ratio and digestion done in SEC buffer at 28 °C for 4 hours.
  • a reaction mix for EK digestion was set up containing 2 mg protein, 300pl 10x EK buffer (500 mM NaCI, 20 mM CaCh, 200 mM Tris pH 8), and 100 pl EKMaxTM enterokinase (Thermo Scientific) diluted 1 :25 in 1x EK buffer. This was then topped up to 3 ml with Milli-Q water and incubated at 28 °C overnight, after which 30 pl 100 mM PMSF was added to inactivate the enterokinase. Since crystallization requires large amounts of protein, the quantity of protein digested was adjusted accordingly.
  • the digestion product was flown through a strep-tactin column, thereby retaining the undigested protein still carrying the strep tag.
  • the digestion product was applied onto a 1 ml HisTrapTM excel column (Cytiva), thereby retaining the TEV in the column.
  • the flow-through and wash fractions that contained the digested proteins were pooled and further purified by size exclusion chromatography.
  • the elution fractions containing the proteins of interest were pooled, concentrated, and stored at -80°C.
  • Crystallization experiments of HEV gt3 P domain in complex with the best neutralizing scFvs involved mixing pGS99 with scFvs (1 :1.2 molar ratio, respectively) and incubating the mixture overnight at 16 °C.
  • the complexes were purified on a superdex 200 increase 10/300 GL column (Cytiva) equilibrated in gel filtration buffer.
  • the purified complexes were then concentrated to 9.3 mg/ml (scFv-p60.1-pGS99) and 6.7 mg/ml (Fab-p60.12-pGS99).
  • Paired heavy and light chain variable regions of antibodies were amplified from the respective top neutralizing scFvs. These were inserted into a pcDNA3.1 expression vector.
  • the heavy chain variable regions were cloned into the IgG 1 expression vector, while their respective kappa or lambda pairs were cloned into expression vectors carrying the corresponding constant regions. All genes were cloned downstream a CD5 signal peptide to allow the secretion of mature IgGs. Plasmid DNA was amplified, isolated and sequenced.
  • HEK Expi293FTM cell lines were thawed and maintained in Expi293TM expression medium (Gibco) according to the supplier’s manual and cultured at 37 °C, 8 % CO2, with shaking at 125 rpm.
  • the ExpiFectamineTM 293 transfection kit (Gibco) was used with slight modifications. A day before transfection, 2.5 x 10 6 viable cells/ml were seeded and cultured overnight in a 500 ml Erlenmeyer flask. Cell density and viability were determined by trypan blue exclusion on a Countess II FL cell counter (Life technologies).
  • the required volume of cells was centrifuged for 5 minutes at 300x g before carefully resuspending the cells in fresh media.
  • the overnight culture with a viability > 90 % was diluted to 2 x 10 6 viable cells/ml in 85 ml pre-warmed medium, and cells were returned to the incubator.
  • the plasmid DNA cocktail was prepared in 5 ml Opti-MEM (Gibco) at ratios listed in Table 3, resulting in total plasmid DNA of 1 pg per ml of culture volume transfected.
  • addition of p21 , p27 and SV40 serves to enhance protein expression in HEK cells.
  • the ExpiFectamineTM 293 transfection reagent was then prepared by mixing 266.7 pl of the reagent with 4733.3 pl Opti-MEMTM and allowed to incubate for 5 minutes at room temperature. Following the 5-minute incubation, the diluted reagent was mixed with the plasmid DNA cocktail by inversion. The ExpiFectamineTM 293/DNA complexes were incubated for 20 minutes at room temperature, after which they were added to the cells dropwise, and the cells were subsequently incubated for 16-22 hours. This was followed by dropwise addition of 500 pl ExpiFectamineTM 293 transfection Enhancer 1 and 5 ml ExpiFectamineTM 293 transfection Enhancer 2 to the transfected cells, and the cells were returned to the incubator.
  • Plasmids Amount (pq/ml of transfection) pq/100 ml transfection volume
  • the cells were harvested by adding 10 ml 10x PBS and centrifugation at 70,000x g for 20 minutes. The supernatant was filtered through a 0.22 pm syringe filter prior to loading onto a 1 ml HiTrap Protein-G column (Cytiva) connected to the AKTATM pure 25 system. Unbound proteins were washed off with 1x PBS and IgGs eluted in 0.1 M Glycine pH 2.7, which was then neutralized by adding 100 pl 1 M Tris-HCI pH 8. The eluted neutralized IgGs were buffer exchanged to PBS using a superdex 200 increase 10/300 GL column. The elution fractions containing the protein of interest were pooled, concentrated and stored at 4 °C until needed for further experiments (neutralization assays, ELISA and Biacore).
  • Strep-TactinXT (I BA Life sciences) was amine coupled to a carboxyl-derivatized CM-5 sensor chip (Cytiva) using a Biacore 3000 (GE Healthcare) following the protocol from the Twin-Strep-tag capture kit for surface plasmon resonance (I BA Life sciences).
  • the chip surfaces were prepared by pre-treatment with three consecutive 1 -minute pulse injections of 50 mM NaOH. The surfaces were then activated for 10 minutes with a 1 :1 mixture of NHS and EDC at a flow rate of 10 pl/min. 35 pg/ml Strep-TactinXT in 10 mM sodium acetate pH 4.5 was immobilized for 10 minutes at a density of 3922 RU on all flow cells.
  • HBS-EP buffer 25 nM ligand (26 kDa, HEV gt3 secreted and non-secreted P domains) in HBS-EP buffer was injected for 1 minute over flow cell 2 at a flow rate of 10 pl/min. Then, the analytes (lgG-p60.1 and p60.12) in HBS-EP buffer were injected over two flow cells (1 and 2) at concentrations of 75, 50, 25, 10 and 5 nM at a flow rate of 30 pl/min and a temperature of 25 °C. The complex was allowed to associate and dissociate for 3.33 and 7 minutes, respectively. The surfaces were then regenerated with a 45-second injection of 3 M guanidine hydrochloride. Measurements were performed twice per sample, and the individual curves were used to produce the mean affinity constant by global fitting to a 1 :1 binding model using the BiaEvaluation software.
  • HEV gt3 P domain comprising amino acid 456-660 (UniProtKB accession number: C4B4T9; SEQ ID NO: 17) fused to fluorescent proteins
  • the constructs carried either mNeonGreen or mRuby at the N-terminus for the non-secreted (pGS38) and secreted form (pGS39), respectively ( Figure 1 B).
  • the secreted form was cloned into a pMT vector carrying a BiP signal sequence while the non-secreted form was inserted into the same vector without the signal peptide for expression in Drosophila S2 cells as described above.
  • a stable S2 transfectant was established per construct and the proteins produced as described above.
  • the retention volume of both proteins was approximately 190 ml on a HiLoad 26/600 superdex 200 pg (Cytiva), but the profile of the secreted P domain showed an additional peak eluting slightly earlier. Since the P domains form dimers, the elution volume was expected, but the additional peak observed for the secreted P domain suggested a tendency to form higher oligomers.
  • Both intracellular and secreted proteins showed > 90 % purity as judged from analysis by SDS-PAGE using a 12 % gel under reducing conditions followed by Coomassie staining. Interestingly, the apparent molecular weight of the secreted P domain was slightly higher than the intracellular protein, a difference that can be attributed to the attached glycan at position 562 in the secreted construct.
  • sequence pairs 50 from donor p60 and 41 from donor p61 , were selected for expression in Drosophila S2 cells.
  • the sequences represented 6 germline genes, and varied in heavy-chain complementarity determining region-3 (CDRH3) amino acid lengths (8-25 aa long), according to IMGT numbering, as well as somatic hypermutations (2-37 nucleotide substitutions per VH sequence).
  • CDRH3 heavy-chain complementarity determining region-3
  • Synthetic genes of the selected 91 -paired sequences, cloned into a pMT vector as singlechain fragments of the variable region (scFv) were purchased from Twist biosciences. The name was defined by scFv as a prefix, followed by the donor ID and the clone number. All scFvs carried a C-terminal EK site and a double strep-tag to aid purification. Furthermore, they were cloned downstream a BiP signal sequence to allow expression through the secretory pathway. A stable S2 cell line was generated per scFv as described above, and a similar protocol was followed for expression and purification of the scFvs.
  • HEV gt3 Kemow-C1 p6 clone produced as previously described (Todt et al. 2020. Robust hepatitis E virus infection and transcriptional response in human hepatocytes PNAS 117 (3): 1731-1741) were performed.
  • HepG2 cells were transfected with the viral genome by electroporation. Four days after transfection, virus was harvested. To generate viral stocks containing naked viral particles, transfected cells were lysed by three freeze-thaw cycles using liquid nitrogen. For virus stocks containing pseudo-enveloped particles, the transfected cells supernatants were used after filtration (0.45pm).
  • Indicated virus stocks were incubated with different mAb concentrations for 1 hour at room temperature before infecting HepG2/C3a Hepatoma cell lines. Cells were then incubated for 24 hours before the addition of new media. After four days, cells were fixed with paraformaldehyde and treated with 0.2 % Triton X-100 solution for 5 minutes at room temperature for permeabilization. Subsequently, samples were washed and stained overnight with an anti-ORF2 rabbit polyclonal antibody. Infected cells were visualized following staining with Alexa Fluor 488-labeled goat anti-rabbit antibody, and focus forming units (FFU) counted using the Elispot CTL system (Immunospot).
  • FFU focus forming units
  • IC50 half-maximal inhibitory concentration
  • Figures 2 C and D show neutralization results with pseudo-enveloped viral particles (IC50 values for p60.1 and p60.12 are 0.27 pg/ml and 0.12 pg/ml).
  • IC50 values for p60.1 and p60.12 are 0.27 pg/ml and 0.12 pg/ml.
  • Neutralization of naked viral particles of another genotype 3 viral strain (HEV83-2-27) at a concentration of 10pg/ml is shown in figure 2 E.
  • mAb p60.1 showed neutralization of more than 80% whereas p60.12 neutralized more than 50% of the virus.
  • variable region genes for 15 scFvs with IC50 ⁇ 0.5pg/ml were cloned into plasmids for expression of human lgG1 , and expressed in Expi293F cells. All purified antibodies eluted as homogenous single heterodimeric peaks ( Figure 3 A) with > 95 % purity upon analysis on a Coomassie-stained reducing SDS-PAGE gel ( Figure 3 B).
  • genes encoding the P domains (amino acid residues 456-660) from the four human pathogenic genotypes and a rat-HEV isolate (Table 2) were cloned into a pMT vector for expression in Drosophila S2 cells.
  • the proteins carried both a TEV and EK protease cleavage sites in tandem, in addition to an N-terminal double strep-tag.
  • the intracellular form of the P domains was used except in the case of genotype 3.
  • both the secreted and intracellular forms were cloned.
  • Stably transfected S2 cells were generated, protein expression induced, and purified as described above.
  • the retention volume for the dimers was approximately 200 ml, however, the rat intracellular (pGS135) and genotype 3 secreted (pGS100) P domains showed a similar tendency to form higher oligomers. Nevertheless, all proteins showed high purity upon analysis on a Coomassie- stained 15 % SDS-PAGE gel loaded under reducing conditions.
  • the HEV gt3 secreted P domain (pGS100) migrated slightly slower than the intracellular domains. Fractions considered in this study were obtained from the dimeric peak.
  • Human pathogenic HEV genotypes have a single serotype and the level of amino acid sequence variation ranges between 89 and 93 % across the four genotypes, suggesting high level of conservation.
  • the amino acid conservation between the rat-HEV P domain and the human genotypes reaches approximately 31.5 %.
  • the level of conservation suggests that it is possible to isolate cross-binding and potentially cross-neutralizing mAbs. Therefore, to explore the cross-genotype binding activity of the mAbs, the purified P domains from section 2.5.2 were used in an indirect ELISA assay as described above.
  • the antibodies were tested for their ability to distinguish between the secreted and intracellular P domains.
  • the goal was to identify antibodies that targeted only the non-secreted form, a component of the infectious particle, but not a secreted soluble pORF2 dimer that is present in the serum at high levels.
  • an indirect ELISA was set-up using HEV gt3 secreted (pGS100) and non-secreted (pGS99) P domains as antigens.
  • a sandwich ELISA confirmed binding of both mAb p60.1 and p60.12 to antigens present in sera of three different patients, chronically infected with HEV (Figure 6). All positive samples were highly HEV-RNA positive (between 1x10 5 and 9x10 6 lU/ml) as determined by the central laboratory of Hannover Medical School. Presence of HEV antigens was confirmed by using the commercial WANTAI HEV Ag ELISA. As control, a negative serum as determined by HEV RNA PCR and WANTAI HEV Ag ELISA was used.
  • SEC size exclusion chromatography
  • the two mAbs were considered for further analysis in SEC because of their IC50 0.01 g/ml.
  • the antigen was incubated overnight at 16 °C with a 1 :3 molar ratio (pGS100: scFv) of the respective scFvs. The mixture was centrifuged at maximum speed in a tabletop centrifuge (Eppendorf) for 10 min at 4 °C to remove any precipitate.
  • the antibodies were further characterized by surface plasmon resonance (SPR) using a Biacore 3000 (GE Healthcare). Experiments were performed at 25 °C using a Strep- TactinXT CM-5 chip prepared as described above. Briefly, the ligand (HEV gt3 P domains, pGS99 or pGS100) was injected over one flow cell followed by injecting different concentrations of the analytes (mAbs p60.1 , p60.12) over two flow cells. Measurements were fitted to a 1 :1 binding model using the BiaEvaluation software resulting in the sensorgrams of Figure 8 A and B. SPR data showed that the binding of the antibodies towards the intracellular P domain was in the picomolar range, with p60.1 showing the highest affinity binding (Table 5).
  • SPR surface plasmon resonance
  • association (Ka) and dissociation (Kd) rates for all antibodies using the non-secreted P domain (pGS99) were similar, but the absolute binding (as judged from the number of response units) obtained for p60.1 and p60.12 were lower than for the other antibodies.
  • the kinetic parameters for p60.1 and p60.12 using the secreted P domain (pGS100) were not calculated because data was below the threshold recommended in the kit manual (I BA Lifesciences twin-strep-Tag capture kit). Overall, the data show that the antibodies of the invention isolated from HEV-infected patients interact with high affinity with the HEV P domains.
  • Protein crystallization was carried out using pGS99 in complex with scFvs or Fabs derived from the mAbs of the invention in order to structurally characterize the antibody-antigen interaction.
  • strep-tags Prior to crystallization, strep-tags were proteolytically removed from pGS99 and the scFvs/Fabs using TEV protease and enterokinase, respectively. Thereafter, complex formation was performed by mixing pGS99 and the antibodies (scFv p60.1 and Fab p60.12) in 1 :1.2 molar ratio, and incubating at 16°C overnight. Purification of the complexes by gel filtration showed that the antibody fragments formed complexes with pGS99 ( Figure 9, black arrows), with excess of the antibody fragments observed in both complexes ( Figure 9).
  • Crystallization drops were set-up using the purified complexes described above. Crystals for complexes with scFv-p60.1 and Fab-p60.12 were obtained at 293K using sitting drop vapor diffusion method (Figure 10). The conditions that produced diffraction quality crystals per complex are presented in Table 6. The crystals of the complexes were obtained from initial screening conditions and required no further optimization. For data collection, all crystals were flush frozen in mother liquor containing 30 % ethylene glycol. The diffraction capacity of the crystals is listed in Table 6. Table 6: Crystallographic data collection and statistics
  • the complex pGS99_scFv-p60.1 crystallized as two P domain dimers each bound by the scFv at the dimer interface per asymmetric unit ( Figure 11). Since the P domain is known to form dimers, the presence of these dimers in the structures of scFv-p60.1 was expected.
  • the electron density map of the P domain was well defined and superposition of the complex to the E2s dimer (PDB 3rkc) yielded a root mean square deviation of 0.234 A for all Ca atoms of the E2s domain. This suggests that the conformation of the P domain remains unaltered upon binding of the scFv.
  • the linker region connecting the variable heavy and light chains of the scFv was not defined in the electron density map as expected, since this region is known to be intrinsically disordered.
  • Both p60.1 and p60.12 recognize a quaternary epitope that assembles at the dimerization interface at the tip of the P domain dimer ( Figure 12).
  • Both broad neutralizing antibodies bind predominantly to one protomer, but make contacts also with the other one, resulting in an asymmetric binding mode and indicating that a correctly folded pORF2 dimer is required for antigen-antibody interaction.
  • Scrutiny of the interfaces reveals three segments distant in the amino acid sequence that constitute the two epitopes including 1) aa 480- 490, 2) aa 555-565 and 3) aa 580-590, confirming that both antibodies recognize conformation-sensitive epitopes.
  • N562 of both protomers which serve as attachment points for the N-linked glycans in the secreted pORF2 dimer, form hydrogen bonds with both antibodies, demonstrating that such an attached glycan would preclude binding of these bnAbs via steric clashes and thereby causing the observed glycan sensitivity.
  • scFv-p60.1 In order to gain insight into the biological relevance of the interactions observed in the complexes of the present study, the crystal structure of scFv-p60.1 was superposed onto HEV VLP (PDB 2ztn) ( Figure 13). The superpositions shows that scFv-p60.1 occupies the apex of the dimer. Thus, scFv-p60.1 may act by blocking attachment to the receptor, since its epitope is located in the region proposed to contain the receptor binding site (Mori and Matsuura, 2011. Structure of hepatitis E viral particle. Virus Research 161 (1): 59-64; Xu et al., 2016. Role of asparagine at position 562 in dimerization and immunogenicity of the hepatitis E virus capsid protein. Infection, Genetics and Evolution 37: 99-107).
  • HEV gt3 P domains fused to fluorescent proteins for antigen-specific single B cell sorting and sequencing sequences of roughly 3800 antibodies from two HEV convalescent patients were obtained. All the most efficient antibodies in the neutralization assay except one were obtained from patient p60 suggesting differences in response to infection. The variation between patients is not unusual as a number of factors such as age, sex, and comorbidities are known to influence response to infection and disease.
  • the isolated antibodies were highly potent in the neutralization assays with HEV gt3, blocking viral infection at concentrations as low as ⁇ 0.01 pg/ml and therefore provide a potential option for treatment of HEV infection.
  • the non-secreted form is the component of the infectious particle (Ankavay et al., 2019. New insights into the ORF2 capsid protein, a key player of the hepatitis E virus lifecycle. Scientific Reports, 9(1)), whereas the secreted form is a soluble dimer that is present in the serum at high levels even after infection is cleared (Behrendt etal., 2016. Hepatitis e Virus (HEV) ORF2 Antigen Levels Differentiate between Acute and Chronic HEV Infection. Journal of Infectious Diseases 214 (3): 361-368).
  • HEV Hepatitis e Virus
  • the secreted form is glycosylated at the conserved glycan position N562 (Xu et al., 2016. Role of asparagine at position 562 in dimerization and immunogenicity of the hepatitis E virus capsid protein. Infection, Genetics and Evolution 37: 99-107; Ankavay et al., 2019). This position is located at the apex of the P domain, which is the receptor-binding region and a binding site for some neutralizing antibodies (Guu et al., 2009. Structure of the hepatitis E virus-like particle suggests mechanisms for virus assembly and receptor binding. PNAS 106 (31): 12992-12997; Mori and Matsuura, 2011. Structure of hepatitis E viral particle.
  • mAb 8G12 is a mouse derived cross-genotype antibody against HEV (Gu et al., 2015. Structural basis for the neutralization of hepatitis E virus by a cross-genotype antibody. Cell Research 25 (5): 604-620) that binds at the dimer interface. Residues T563 and T564 are among the contact residues of 8G12 and are located next to N562.
  • the close proximity of the glycan therefore may sterically block the binding of 8G12.
  • structural studies of the best neutralizing antibodies of the present study revealed that p60.1 and p60.12 bind at the apex of the P domain, directly making contact with the two-asparagine sidechain that serve as attachment site for N-linked glycans at the dimer interface.
  • p60.1 makes contact with residues T489, N560, Y561 , N562, T564, T585, and T586 which have been implicated in HEV receptor-interaction (Mori and Matsuura, 2011. Structure of hepatitis E viral particle. In Virus Research 161 (1):59-64.
  • C1-C6 Six antigenic cluster sites (C1-C6) targeted by anti-HEV antibodies have been mapped on the P domain by alanine scanning and antibody competition assays (Zhao et al., 2015; Wen et al., 2020).
  • a comparison of the epitopes targeted by p60.1 and p60.12 shows that both antibodies occupy antigenic site C3, which is located at the apex of the P domain and is the cell attachment region.
  • C2 and C6 are described as the target for the majority of the neutralizing mAbs, with the C6- directed mAbs being the most potent.
  • C6 is associated with high neutralization potency and limited or no cross-genotype reactivity, whereas clusters C5 and C3 demonstrate weak or no neutralization potential. This is in contrast to the present invention in which it is shown that surprisingly antibodies targeting antigenic cluster 03, p60.1 and p60.12, are the most potent neutralizers. p60.1 and p60.12 are highly potent neutralizers, demonstrate cross-genotype binding potential and cross-neutralization and may act via blocking virusreceptor interactions and a direct binding competition with the receptor.
  • potent neutralizing anti-HEV antibodies from HEV convalescent patients have been isolated. These antibodies bind with picomolar affinities to HEV gt3 P domain, show potent neutralization activity in in vitro assays, and demonstrate broad reactivity across HEV genotypes. The most potent antibodies target only the non-secreted form of the P domain, which is the component of the infectious particle. So far, it is hypothesized that the antibodies of the present study likely act by blocking receptor interaction. Currently, the best antibodies are being tested in an HEV infection model to evaluate their potency in vivo.
  • HEV circulates as quasi-enveloped virions in the blood and such virions have been so far reported to be resistant to antibody neutralization (Yin et al., 2016. Distinct Entry Mechanisms for Nonenveloped and Quasi-Enveloped Hepatitis E Viruses. Journal of Virology 90 (8): 4232-4242).
  • the antibodies isolated in the present invention efficiently neutralize also quasi-enveloped virions and therefore demonstrate their potential therapeutic benefits for chronical and immunocompromised HEV patients.

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