Patent Application
20220315645
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 9, 2020, is named 076091_00092_SL.txt and is 307,317 bytes in size.
Despite the existence of effective vaccines, hepatitis B virus (HBV) infection remains a major global health problem with an estimated 257 million people living with the infection. Whereas 95% of adults and 50-75% of children between the ages of 1 and 5 years spontaneously control HBV, only 10% of infants recover naturally. The remainder develop a chronic infection that can lead to liver cirrhosis and hepatocellular carcinoma. Although chronic infection can be suppressed with antiviral medications, there is no effective curative therapy (Dienstag, 2008; Revill et al., 2016; Thomas, 2019).
HBV is an enveloped double-stranded DNA virus of the Hepadnaviridae family. Its genome is the smallest genome among pathogenic human DNA viruses, with only four open reading frames. Infected liver cells produce both infectious HBV virions (Dane particles) and non-infectious subviral particles (Australia antigen) (Dane et al., 1970; Hu and Liu, 2017). The virion is a 42 nm-diameter particle containing the viral genome and HBV core antigen (HBcAg) encapsidated by a lipid membrane containing the hepatitis B surface antigen (HBsAg) (Blumberg, 1964; Venkatakrishnan and Zlotnick, 2016). Subviral particles lack the viral genome.
HBV strains were originally grouped into four HBsAg serotypes (adr, adw, ayw, and ayr). Genetic analysis revealed several highly conserved domains and defined eight genotypes A-H, which are highly correlated with the 4 serotypes (Norder et al., 2004). The HBV surface protein, HBsAg, has 4 putative transmembrane domains and can be subdivided into PreS1-, PreS2- and S-regions. The S domain is a cysteine-rich protein consisting of 226 amino acids that contain all 4 of the transmembrane domains (Abou-Jaoude and Sureau, 2007). In addition, the S-protein can be glycosylated at asparagine residue 146 (Julithe et al., 2014).
Antibodies to HBsAg (anti-HBs) are associated with successful vaccination and recovery from acute infection, while antibodies to HBcAg (anti-HBc) are indicative of past or current HBV infection (Ganem, 1982). Indeed, the most significant difference between chronically infected and naturally recovered individuals is a robust antibody response to HBsAg (Ganem, 1982). Conversely, the inability to produce these antibodies during acute infection is associated with chronicity (Trepo et al., 2014). Whether these associations reflect an etiologic role for anti-HBs antibodies in protecting from or clearing established infection is not known. However, depletion of antibody-producing B lymphocytes in exposed humans by anti-CD20 therapies (e.g. rituximab) is associated with HBV reactivation, indicating that B cells and/or their antibody products play a significant role in controlling the infection (Loomba and Liang, 2017).
Several human antibodies against HBsAg have been obtained using a variety of methods including: phage display (Kim and Park, 2002; Li et al., 2017; Sankhyan et al., 2016; Wang et al., 2016); humanized mice (Eren et al., 1998); Epstein-Barr virus-induced B cell transformation (Heijtink et al., 2002; Heijtink et al., 1995; Sa'adu et al., 1992); hybridoma technology (Colucci et al., 1986); human B cell cultures (Cerino et al., 2015); and microwell array chips (Jin et al., 2009; Tajiri et al., 2010). However, the donors in these studies were not selected for serum neutralizing activity. Thus, there remains a need for improved approaches and compositions of combatting HBV infection. The present disclosure is pertinent to this need.
The disclosure provides in part a description of the human humoral immune response to HBsAg in immunized and spontaneously recovered individuals that had been selected for high levels of serum neutralizing activity. The disclosure demonstrates that these individuals develop closely related bNAbs that target shared non-overlapping epitopes in HBsAg. The crystal structure of one of the antibodies with its peptide target reveals a loop that helps to explain why certain amino acid residues are frequently mutated in escape viruses and why combinations of bNAbs may be needed to control infection. In vivo experiments in humanized mice demonstrate that the bNAbs are protective and can be therapeutic when used in combination.
Any antibody described herein can comprise at least one modification of its constant region. The modification may be made for any one or more amino acids. The modification can have any of a number of desirable effects. In certain approaches, the modification increases in vivo half-life of the antibody, or alters the ability of the antibody to bind to Fc receptors, or alters the ability of the antibody to cross placenta or to cross a blood-brain barrier or to cross a blood-testes barrier, or inhibits aggregation of the antibodies, or a combination of said modifications, or wherein the antibody is attached to a label or a substrate. In embodiments, the modification improves the manufacturability of the antibody. In embodiments, any antibody or combination thereof described herein can be present in an immunological assay, such as an enzyme-linked immunosorbent assay (ELISA) assay, or an ELISA assay control. The ELISA assay can be any of a direct ELISA assay, an indirect ELISA assay, a sandwich ELISA assay, or a competition ELISA assay.
In another aspect the disclosure provides a method for prophylaxis or therapy of a hepatitis viral infection comprising administering to an individual in need thereof an effective amount of at least one antibody described herein, or an antigen binding fragment thereof. The antibody may comprise at least one modification of the constant region. In embodiments, the composition is administered to an individual who is infected with or is at risk of being infected with a hepatitis B virus. In one approach, at least two antibodies are administered, wherein optionally the two antibodies recognize distinct HBV epitopes. In an embodiment, administering at least two distinct antibodies suppresses formation of viruses that are resistant to the antibodies.
In another aspect the disclosure provides vaccine formulations. In an embodiment a vaccine formulation comprises an isolated or recombinant peptide or a polynucleotide encoding the peptide, wherein the peptide is derived from an epitope that is frequently targeted by HepB immune resistance, and which is located in a loop anchored by oppositely charged residues, as further described herein.
In another aspect the disclosure provides one or more recombinant expression vectors, and kits comprising the expression vectors. The expression vectors encode at least the heavy chain and the light chain CDRs of any of the antibodies of described herein. Cells comprising the recombinant expression vectors are included, as are methods of making antibodies by culturing cells that comprise expression vectors that express the antibodies, and separating antibodies from the cells. Cell culture media containing such cells and/or antibodies is also included.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein.
This disclosure includes every nucleotide sequence described herein, and in the tables and figures, and all sequences that are complementary to them, RNA equivalents of DNA sequences, all amino acid sequences described herein, and all polynucleotide sequences encoding the amino acid sequences. Every antibody sequence and functional fragments of them are included. Polynucleotide and amino acid sequences having from 80-99% similarity, inclusive, and including ranges of numbers there between, with the sequences provided here are included in the invention. All of the amino acid sequences described herein can include amino acid substitutions, such as conservative substitutions, that do not adversely affect the function of the protein or polypeptide that comprises the amino acid sequences. It will be recognized that when reference herein is made to an “antibody” it does not necessarily mean a single antibody molecule. For example, “administering an antibody” includes administering a plurality of the same antibodies. Likewise, a composition comprising an “antibody” can comprise a plurality of the same antibodies.
For amino acid and polynucleotide sequences of this disclosure, contiguous segments of the sequences are included, and can range from 2 amino acids, up to full-length protein sequences. Polynucleotide sequences encoding such segments are also included.
The disclosure includes DNA and RNA sequences encoding the antibodies and antigen fragments thereof, and any virus peptides described herein for use in prophylactic and therapeutic approaches as protein or DNA and/or RNA vaccines, which may be formulated and/or delivered according to known approaches, given the benefit of this disclosure. The disclosure includes a cDNA sequences encoding the antibodies, antigen binding fragments thereof, and any viral proteins or peptides described herein. Expression vectors that contain cDNAs are also included, and encode said antibodies, antigen binding fragments thereof, and viral proteins and peptides.
All sequences from the figures, text, and tables of this application or patent include every amino acid sequence associated with every Donor ID, and all possible combinations of the amino acid sequences given for all complementarity determining regions (CDRs), e.g., all combinations of heavy chain CDR1, CDR2, CDR3 sequences, and all combinations of light chain CDR1, CDR2, and CDR3 sequences, including heavy chain sequences, and light chain sequences that are either lambda or kappa light chain sequences.
The disclosure includes all combinations of antibodies described herein. One or more antibodies may also be excluded from any combination of antibodies.
The disclosure includes antibodies described herein, which are present in an in vitro complex with one or more hepatitis B proteins.
In embodiments, the disclosure provides an isolated or recombinant antibody that binds with specificity to a hepatitis B virus epitope, and wherein the antibody optionally comprises a modification of its amino acid sequence, including but not limited to a modification of its constant region.
In embodiments, one or more antibodies described herein bind with specificity to an epitope present in the HBsAg protein or the S-protein in the unmutated, or mutated form.
In embodiments, the antibodies described herein bind to a hepatitis B protein that comprises one or more HepB escape mutations. In embodiments, the antibodies bind to a hepatitis B virus protein that comprises a mutation that is a substitution of a large positively charged residue for a small neutral residue. In embodiments, the mutation is present in the so-called “a” determinant area, which is known in the art. In embodiments, the epitope is present in the major hydrophilic region of the HBsAg protein. In embodiments, the epitope to which the antibodies bind is present in the S-protein, including but not necessarily limited to the predicted or actual extracellular domain of the S-protein.
In embodiments, the epitope to which the described antibodies bind is common to HBsAg L-protein, M-protein, or S-protein. In embodiments, the antibodies bind to an epitope present in the L-protein version of HBsAg, which comprises the amino acid sequence that is accessible via Accession number: AAL66340.1 as that amino acid sequence exists in the database as of the filing date of this application or patent. In an embodiment, this amino acid sequence is:
In embodiments, the disclosure includes use of only two proteins, or at least two proteins. In an embodiment, the S proteins may be used as bait to sort B cells purified from Chinese hamster ovary (CHO) cells, or any other suitable cell type, including but not limited to human cell cultures. In embodiments, the S protein comprises or consists of the amino acid sequence available under Uniprot ID P30019, the amino acid sequence of which is incorporated herein as it exists in the database at the filing date of this application or patent. In an embodiment, the S protein comprises the sequence:
In non-limiting embodiments, the S polynucleotide sequence used for alanine scanning comprises:
The amino acid sequence encoded by the DNA sequence immediately above is:
In embodiments, antibodies of this disclosure bind to an epitope present in any of the foregoing amino sequences, including linear and confirmation epitopes that may be formed by proteins comprising or consisting of said sequences.
In an embodiment, the isolated or recombinant antibody or antigen binding fragment thereof binds with specificity to an epitope comprised by a structurally defined peptide loop, as further described herein. In embodiments, the loop is as generally depicted in
In embodiments, antibodies described herein bind with specificity to an amino acid sequence comprised by any peptide sequence described herein. In embodiments, the peptide comprises the sequence KPSDG (SEQ ID NO: 6), or mutants thereof. In embodiments, antibodies described herein bind with specificity to an epitope in an amino acid sequence that comprises the sequence PSSSSTKPSDGNSTS (SEQ ID NO: 7), or mutants thereof. Additional and non-limiting examples of peptides of this disclosure include those shown on
In embodiments, the disclosure comprises compositions and methods that involve use of more than one distinct antibody or antigen binding fragment thereof. In embodiments, the methods of this disclosure comprise administering a combination of antibodies or antigen binding fragment thereof which bind distinct hepatitis B epitopes. In embodiments, distinct antibodies recognize epitopes present in two dominant non-overlapping antigenic sites on the HBsAg, or epitopes present on the S-protein. In embodiments, the disclosure provides for use of a combination of the Group-I and Group-II antibodies described herein. Thus, the disclosure comprises co-administration or sequential administration of a combination of antibodies. In an embodiment, administration of a combination of distinct antibodies suppresses formation of viruses that are resistant to the effects of any one of the antibodies alone. In embodiments, the disclosure includes administering a combination comprising at least one Group I antibody and at least one Group II antibody, wherein at least one of the antibodies is G145R mutation resistant. In non-limiting embodiments, antibodies that are provided by the present disclosure, and which can be administered to an individual in need thereof, comprise at least one of H006, H007, H0017, H0019, or H020. Further, H005, H008 and H009 are similar to H006, and therefore may be used as alternatives to H006.
All combinations of H and L chains described herein are included, including all kappa and lambda light chains. In embodiments, a single antibody of this disclosure may comprise an H+L chain from one antibody, and an H+L chain from another antibody. In embodiments, the antibodies comprise modifications that are not coded for in any B cells obtained from an individual, and/or the antibodies are not produced by immune cells in an individual from which a biological sample from the individual is used at least in part to identify and/or generate and/or characterize the antibodies of this disclosure. In embodiments, antibodies provided by this disclosure can be made recombinantly, and can be expressed with a constant region of choice, which may be different from a constant region that was coded for in any sample from which the amino acid sequences of the antibodies were deduced.
As discussed above, in embodiments, the disclosure includes a combination of antibodies or antigen binding fragments thereof, or a composition comprising or consisting of said antibodies or antigen binding fragments thereof. In embodiments, a combination of antibodies of this disclosure are effective in preventing viral escape by mutation. In this regard, the disclosure includes data demonstrating that not all antibody combinations are effective in preventing escape by mutation, such as the combination of H006 and H007, which are ineffective. Thus, in embodiments, a combinations of antibodies or antigen binding fragments collectively target more than one commonly occurring escape mutation, examples of which escape mutations are known in the art and are described herein. Accordingly, combinations of antibodies and antigen binding fragments thereof of this disclosure may target non-overlapping groups of common escape mutations. In embodiments, the disclosure thus includes a proviso that excludes any combination of antibodies that collectively only target separate epitopes but have overlapping sensitivity to commonly occurring escape mutations.
In embodiments, at least one antibody or antigen binding fragment thereof included in this disclosure, and in the combinations and methods of this disclosure, has greater virus neutralizing activity than a control neutralizing activity value, such as the neutralizing capability of libivirumab. In embodiments, at least one antibody or antigen binding fragment of this disclosure exhibits a viral neutralizing activity with an IC50 values that is less than 128 ng/ml, or less than 35 ng/ml, or less than 5 ng/ml, and including all integers and ranges of integers between 128 and 5 ng/ml. Such neutralizing activity can be determined using known approaches, such as by ELISA or immunofluorescence assays, and as further described in Example 5 of this disclosure. In embodiments, an antibody or antigen binding fragment thereof that is encompassed by this disclosure includes but is not limited to antibodies or antigen binding fragments selected from the H016, H017 and H019 antibodies, as defined by their CDRs. In an embodiment, the disclosure includes combinations of these antibodies, and can include antigen binding fragments thereof. In embodiments, the combination of antibodies comprises the H017 and H019 antibodies, and/or antigen binding fragments thereof. In an embodiment, the combination optionally further comprises the H016 antibody or an antigen binding fragment thereof. In embodiments, a combination of the disclosure comprises a combination that consists of only the H017 and H019 antibodies or antigen binding fragments thereof. In embodiments, a combination of the disclosure comprises a combination that consists of only the H016, H017, and H019 antibodies or antigen binding fragments thereof. Methods of administration of the described antibody combinations, and all other antibodies and antigen binding fragments thereof described herein, sequentially and concurrently are included within the scope of this disclosure. Thus, the disclosure includes administering to an individual in need concurrently or sequentially a combination of antibodies or antigen binding fragments thereof, which in certain embodiments comprise or consist of H017 and H019, or H016, H017, and H019 and antigen binding fragments thereof. Additional antibodies and antibody combinations, including antigen binding fragments thereof, include but are not limited to antibodies and antigen binding fragments thereof that comprise the heavy and light chain CDRs of H004, H005, and H009, and H020.
With respect to the H016, H017, and H019 antibodies, as can been seen from Table S2, the H016 antibody comprises a heavy chain CDR1 with the amino acid sequence GFTFPSHT (SEQ ID NO: 8), a heavy chain CDR2 with the amino acid sequence ISTTSEAI (SEQ ID NO: 9), and a heavy chain CDR3 with the amino acid sequence ARVGLALTISGYWYFDL (SEQ ID NO: 10). The H016 antibody comprises a kappa light chain CDR1 with the amino acid sequence QSISSN (SEQ ID NO: 11), a kappa light chain with the CDR2 amino acid sequence RAS, and a kappa light chain with the CDR3 amino acid sequence QQYDHWPLT (SEQ ID NO: 12).
As can be seen from Table S2, the H017 antibody comprises a heavy chain CDR1 with the amino acid sequence GFTFSNYW (SEQ ID NO: 13), a heavy chain CDR2 with the amino acid sequence ISTDGSST (SEQ ID NO: 14), and a heavy chain CDR3 with the amino acid sequence ARGSTYYFGSGSVDY (SEQ ID NO: 15). The H017 antibody comprises a lambda light chain with the CDR1 sequence SSDIGVYNY (SEQ ID NO: 16), a lambda light chain with the CDR2 sequence DVT, and a lambda light chain with the CDR3 sequence SSYRGSSTPYV (SEQ ID NO: 17).
As can be seen from Table S2, the H019 antibody comprises a heavy chain CDR1 with the amino acid sequence GGSITTGDYY (SEQ ID NO: 18), a heavy chain CDR2 with the amino acid sequence IYYSGST (SEQ ID NO: 19), and a heavy chain CDR3 with the amino acid sequence AIYMDEAWAFE (SEQ ID NO: 20). The H019 antibody comprises a lambda light chain CDR1 with the amino acid sequence QSIGNY (SEQ ID NO: 21), a lambda light chain with the CDR2 amino acid sequence AVS, and a lambda light chain with the CDR3 amino acid sequence QQSYTISLFT (SEQ ID NO: 22).
In certain embodiments, the antibodies contain one or more modifications, such as non-naturally occurring mutations. As non-limiting examples, in certain approaches the Fc region of the antibodies can be changed, and may be of any isotype, including but not limited to any IgG type, or an IgA type, etc. Antibodies of this disclosure can be modified to improve certain biological properties of the antibody, e.g., to improve stability, to modify effector functions, to improve or prevent interaction with cell-mediated immunity and transfer across tissues (placenta, blood-brain barrier, blood-testes barrier), and for improved recycling, half-life and other effects, such as manufacturability and delivery.
In embodiments, an antibody of this disclosure can be modified by using techniques known in the art, such as those described in Buchanan, et al., Engineering a therapeutic IgG molecule to address cysteinylation, aggregation and enhance thermal stability and expression mAbs 5:2, 255-262; March/April 2013, and in Zalevsky J. et al., (2010) Nature Biotechnology, Vol. 28, No. 2, p 157-159, and Ko, S-Y, et al., (2014) Nature, Vol. 514, p 642-647, and Horton, H. et al., Cancer Res 2008; 68: (19), Oct. 1, 2008, from which the descriptions are incorporated herein by reference. In certain embodiments an antibody modification increases in vivo half-life of the antibody (e.g. LS mutations), or alters the ability of the antibody to bind to Fc receptors (e.g. GRLR mutations), or alters the ability to cross the placenta or to cross the blood-brain barrier or to cross the blood-testes barrier. Thus, in certain embodiments an antibody modification comprises a change of G to R, L to R, M to L, or N to S, or any combination thereof.
In embodiments bi-specific antibodies are provided by modifying and/or combining segments of antibodies as described herein, such as by combining heavy and light chain pairs from distinct antibodies into a single antibody. Suitable methods of making bispecific antibodies are known in the art, such as in Kontermann, E. et al., Bispecific antibodies, Drug Discovery Today, Volume 20, Issue 7, July 2015, Pages 838-847, the description of which is incorporated herein by reference.
In embodiments, any antibody described herein comprises a modified heavy chain, a modified light chain, a modified constant region, or a combination thereof, thus rendering them distinct from antibodies produced by humans. In embodiments, the modification is made in a hypervariable region, and/or in a framework region (FR).
In embodiments, mutations to an antibody described herein, including but not limited to the antibodies described, comprise modifications relative to the antibodies originally produced in humans. Such modifications include but are not necessarily limited to the heavy chain to increase the antibody half-life.
In embodiments, antibodies of this disclosure have variable regions that are described herein, and may comprise or consist of any of these sequences, and may include sequences that have from 80-99% similarity, inclusive, and including ranges of numbers there between, with the sequences expressly disclosed herein, provided antibodies that have differing sequences retain the same or similar binding affinity as an antibody with an unmodified sequence. In embodiments, the sequences are at least 95%, 96%, 97%, 98% or 99% similar to an expressly disclosed sequence herein.
Antibodies comprising the sequences described in Table S2 have been isolated and characterized for at least binding affinity, and as otherwise described herein, such as for virus neutralizing activity. Thus, in embodiments the disclosure provides neutralizing antibodies. The term “neutralizing antibody” refers to an antibody or a plurality of antibodies that inhibits, reduces or completely prevents viral infection. Whether any particular antibody is a neutralizing antibody can be determined by in vitro assays described in the examples below, and as is otherwise known in the art. The term “broadly neutralizing” antibody refers to an antibody that can neutralize more than one strain or serotype of a virus.
Antibodies of this disclosure can be provided as intact immunoglobulins, or as antigen binding fragments of immunoglobulins, including but not necessarily limited to antigen-binding (Fab) fragments, Fab′ fragments, (Fab′)2 fragments, Fd (N-terminal part of the heavy chain) fragments, Fv fragments (the two variable domains), dAb fragments, single domain fragments or single monomeric variable antibody domains, isolated CDR regions, single-chain variable fragment (scFv), and other antibody fragments that retain virus-binding capability and preferably virus neutralizing activity as further described below. In embodiments, the variable regions, including but not necessarily limited to the described CDRs, may be used as a component of a Bi-specific T-cell engager (BiTE), bispecific killer cell engager (BiKE), or a chimeric antigen receptor (CAR), such as for producing chimeric antigen receptor T cells (e.g. CAR T cells). In embodiments, the disclosure includes tri-valent antibodies, which can bind with specificity to three different epitopes.
Antibodies and antigens of this disclosure can be provided in pharmaceutical formulations. It is considered that administering a DNA or RNA polynucleotide encoding any protein described herein (including peptides and polypeptides), such as antibodies and antigens described herein, is also a method of delivering such proteins to an individual, provided the protein is expressed in the individual. Methods of delivering DNA and RNAs encoding proteins are known in the art and can be adapted to deliver the protein, particularly the described antigens, given the benefit of the present disclosure. Similarly, the antibodies of this disclosure can be administered as DNA molecules encoding for such antibodies using any suitable expression vector(s), or as RNA molecules encoding the antibodies.
Pharmaceutical formulations containing antibodies or viral antigens or polynucleotides encoding them can be prepared by mixing them with pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers include solvents, dispersion media, isotonic agents and the like. The carrier can be liquid, semi-solid, e.g. pastes, or solid carriers. Examples of carriers include water, saline solutions or other buffers (such as phosphate, citrate buffers), oil, alcohol, proteins (such as serum albumin, gelatin), carbohydrates (such as monosaccharides, disaccharides, and other carbohydrates including glucose, sucrose, trehalose, mannose, mannitol, sorbitol or dextrins), gel, lipids, liposomes, resins, porous matrices, binders, fillers, coatings, stabilizers, preservatives, liposomes, antioxidants, chelating agents such as EDTA; salt forming counter-ions such as sodium; non-ionic surfactants such as TWEEN, PLURONICS or polyethylene glycol (PEG), or combinations thereof. In embodiments, a pharmaceutical/vaccine formulation exhibits an improved activity relative to a control, such as antibodies that are delivered without adding additional agents, or a particular added agent improves the activity of the antibodies.
The formulation can contain more than one antibody type or antigen, and thus mixtures of antibodies, and mixtures of antigens, and combinations thereof as described herein can be included. These components can be combined with a carrier in any suitable manner, e.g., by admixture, solution, suspension, emulsification, encapsulation, absorption and the like, and can be made in formulations such as tablets, capsules, powder (including lyophilized powder), syrup, suspensions that are suitable for injections, ingestions, infusion, or the like. Sustained-release preparations can also be prepared.
The antibodies and vaccine components of this disclosure are employed for the treatment and/or prevention of hepatitis B virus infection in a subject, as well as for inhibition and/or prevention of their transmission from one individual to another.
The term “treatment” of viral infection refers to effective inhibition of the viral infection so as to delay the onset, slow down the progression, reduce viral load, and/or ameliorate the symptoms caused by the infection.
The term “prevention” of viral infection means the onset of the infection is delayed, and/or the incidence or likelihood of contracting the infection is reduced or eliminated.
In embodiments, to treat and/or prevent viral infection, a therapeutic amount of an antibody or antigen vaccine disclosed herein is administered to a subject in need thereof. The term “therapeutically effective amount” means the dose required to effect an inhibition of infection so as to treat and/or prevent the infection.
The dosage of an antibody or antigen vaccine depends on the disease state and other clinical factors, such as weight and condition of the subject, the subject's response to the therapy, the type of formulations and the route of administration. The precise dosage to be therapeutically effective and non-detrimental can be determined by those skilled in the art. As a general rule, a suitable dose of an antibody for the administration to adult humans parenterally is in the range of about 0.1 to 20 mg/kg of patient body weight per day, once a week, or even once a month, with the typical initial range used being in the range of about 2 to 10 mg/kg. Since the antibodies will eventually be cleared from the bloodstream, re-administration may be required. Alternatively, implantation or injection of the antibodies provided in a controlled release matrix can be employed.
The antibodies can be administered to the subject by standard routes, including oral, transdermal, and parenteral (e.g., intravenous, intraperitoneal, intradermal, subcutaneous or intramuscular). In addition, the antibodies and/or the antigen vaccines can be introduced into the body, by injection or by surgical implantation or attachment such that a significant amount of an antibody or the vaccine is able to enter blood stream in a controlled release fashion. In certain embodiments antibodies described herein are incorporated into one or more prophylactic compositions or devices to, for instance, neutralize a virus before it enters cells of the recipient's body. For example, in certain embodiments a composition and/or device comprises a polymeric matrix that may be formed as a gel, and comprises at least one of hydrophilic polymers, hydrophobic polymers, poly(acrylic acids) (PAA), poly(lactic acids) (PLA), carageenans, polystyrene sulfonate, polyamides, polyethylene oxides, cellulose, poly(vinylpyrrolidone) (PVP), poly(vinyl alcohol) (PVA), chitosan, poly(ethylacrylate), methylmethacrylate, chlorotrimethyl ammonium methylmethacrylate, hydroxyapatite, pectin, porcine gastric mucin, poly(sebacic acid) (PSA), hydroxypropyl methylcellulose (HPMC), cellulose acetate phthalate (CAP), magnesium stearate (MS), polyethylene glycol, gum-based polymers and variants thereof, poly (D,L)-lactide (PDLL), polyvinyl acetate and povidone, carboxypolymethylene, and derivatives thereof. In certain aspects the disclosure comprises including antibodies in micro- or nano-particles formed from any suitable biocompatible material, including but not necessarily limited to poly(lactic-co-glycolic acid) (PLGA). Liposomal and microsomal compositions are also included. In certain aspects a gel of this disclosure comprises a carbomer, methylparaben, propylparaben, propylene glycol, sodium carboxymethylcellulose, sorbic acid, dimethicone, a sorbitol solution, or a combination thereof. In embodiments a gel of this disclosure comprises one or a combination of benzoic acid, BHA, mineral oil, peglicol 5 oleate, pegoxol 7 stearate, and purified water, and can include any combination of these compositions.
Antibodies of this disclosure can be produced by utilizing techniques available to those skilled in the art. For example, one or distinct DNA molecules encoding one or both of the H and L chains of the antibodies can be constructed based on the coding sequence using standard molecular cloning techniques. The resulting DNAs can be placed into a variety of suitable expression vectors known in the art, which are then transfected into host cells, which are preferably human cells cultured in vitro, but may include E. coli or yeast cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, and human embryonic kidney 293 cells, etc. Antibodies can be produced from a single, or separate expression vectors, including but not limited to separate vectors for heavy and light chains, and may include separate vectors for kappa and lambda light chains as appropriate.
In embodiments, the antibodies may be isolated from cells. In embodiments, the antibodies are recombinant antibodies. “Recombinant” antibodies mean the antibodies are produced by expression within cells from one or more expression vectors.
In certain approaches the disclosure includes neutralizing antibodies as discussed above, and methods of stimulating the production of such antibodies.
In certain approaches the disclosure includes vaccinating an individual using a composition described herein, and determining the presence, absence, and/or an amount of neutralizing antibodies produced in response to the vaccination. Thus, methods of determining and monitoring efficacy of a vaccination at least in terms of neutralizing antibody production are included. In an embodiment, subsequent to determining an absence of neutralizing antibodies, and/or an amount of neutralizing antibodies below a suitable reference value, the invention includes administering a composition disclosed herein to the individual. Subsequent administrations and measurements can be made to track the treatment efficacy and make further adjustments to treatment accordingly.
Antibodies and proteins of this disclosure can be detectably labeled and/or attached to a substrate. Any substrate and detectable label conventionally used in immunological assays and/or devices is included. In embodiments the substrate comprises biotin, or a similar agent that binds specifically with another binding partner to facilitate immobilization and/or detection and/or quantification of antibodies and/or viral proteins.
In embodiments any type of enzyme-linked immunosorbent (ELISA) assay can be used, and can be performed using polypeptides and/or antibodies of this disclosure for diagnostic purposes, and can include direct, indirect, and competitive ELISA assays, and adaptations thereof that will be apparent to those skilled in the art given the benefit of this disclosure.
Any diagnostic result described herein can be compared to any suitable control. Further, any diagnostic result can be fixed in a tangible medium of expression and communicated to a health care provider, or any other recipient. In one aspect the disclosure comprises diagnosing an individual as infected with hepatitis B virus and administering a composition of this invention to the individual.
In certain embodiments the disclosure includes one or more recombinant expression vectors encoding at least H and L chains of an antibody or antigen binding fragment of this disclosure, cells and cell cultures comprising the expression vectors, methods comprising culturing such cells and separating antibodies from the cell culture, the cell culture media that comprises the antibodies, antibodies that are separated from the cell culture, and kits comprising the expression vectors encoding an antibody and/or a polypeptide of this disclosure. Products containing the antibodies and/or the polypeptides are provided, wherein the antibodies and/or the polypeptides are provided as a pharmaceutical formulation contained in one or more sealed containers, which may be sterile and arranged in any manner by which such agents would be suitable for administration to a human or non-human subject. The products/kits may further comprise one or more articles for use in administering the compositions.
The following Examples are intended to illustrate but not limit the invention.
Serologic Responses Against HBV
To select individuals with outstanding antibody responses to HBsAg, we performed ELISA assays on serum obtained from 159 volunteers. These included 15 uninfected and unvaccinated controls (HBsAg−, anti-HBs−, anti-HBc−), 20 infected and spontaneously recovered (HBsAg−, anti-HBs+/−, anti-HBO, and 124 vaccinated (HBsAg−, anti-HBs+/−, anti-HBc−) volunteers. These individuals displayed a broad spectrum of anti-HBs titers (x-axis in
The HBV surface protein, HBsAg can be subdivided into PreS1-, PreS2- and S-regions (
Human Monoclonal Antibodies to HBV
To characterize the antibodies responsible for neutralizing activity in the selected individuals, we purified S-protein binding class-switched memory B cells (Escolano et al., 2019; Scheid et al., 2009a). Unexposed naïve controls and vaccinated individuals with low anti-HBs ELISA titers showed background levels of S-protein specific memory B cells (
Immunoglobulin heavy (IGH) and light (IGL or IGK) chain genes were amplified from single memory B cells by PCR (Robbiani et al., 2017; Scheid et al., 2009b; von Boehmer et al., 2016). Overall, we obtained 244 paired heavy and light chain variable regions from S-protein-binding memory B cells from eight volunteers with high anti-HBs ELISA titers (
Breadth of Reactivity
Twenty representative antibodies from 5 individuals, designated as H001 to H020, were selected for expression and further testing (
Four major serotypes of HBV exist as defined by a constant “a” determinant and two variable and mutually exclusive determinants “d/y” and “w/r” (Bancroft et al., 1972; Le Bouvier, 1971) with a highly statistically significant association between serotypes and genotypes (Kramvis et al., 2008; Norder et al., 2004). To determine whether our antibodies cross-react to different HBsAg serotypes, we performed ELISAs with 5 additional HBsAg antigens: yeast-expressed serotype “adr”, “adw”, and “ayw”, as well as “ad” and “ay” antigen purified from human blood (
Antigenic Epitopes on S-Protein
To determine whether the selected antibodies bind to overlapping or non-overlapping epitopes, we performed competition ELISA assays, in which the S-protein was pre-incubated with a selected antibody followed by a second biotinylated antibody. Antibodies that showed weak levels of binding in ELISA (H002, H012, H013, H014, H018) were excluded. As expected, all of the antibodies tested blocked the binding of the autologous biotinylated monoclonal (yellow boxes in
To further define these epitopes, we produced a series of alanine mutants spanning most of the predicted extracellular domain of the S-protein with the exception of cysteines, alanines, and amino acid residues critical for S-protein production (Salisse and Sureau, 2009) (
However alanine scanning suggested that some residues such as D144 and G145 are critical for binding of monoclonals in both Group-I and Group-II despite their inability to compete with each other for binding to the native antigen (
In addition to alanine scanning, we also produced 44 common naturally occurring escape variants found in chronically infected individuals (Hsu et al., 2015; Ijaz et al., 2012; Ma and Wang, 2012; Salpini et al., 2015). Whereas alanine scanning showed that some of the antibodies in Group-I and -II were resistant to G145A, the corresponding naturally occurring mutations at the same position, G145E and G145R, revealed decreased binding by most antibodies (
In Vitro Neutralizing Activity
To determine whether the new monoclonals neutralize HBV in vitro, we performed neutralization assays using HepG2-NTCP cells (
Structure of the H015 Antibody/Peptide Complex
H015 differed from other antibodies in that its binding was inhibited by 5 consecutive alanine mutations spanning positions K141-G145 indicating the existence of a linear epitope. This idea was verified by ELISA against a series of overlapping peptides comprising the predicted extracellular domain of S-protein (
To examine the molecular basis for H015 binding, its Fab fragment was co-crystallized with the target peptide epitope PSSSSTKPSDGNSTS (SEQ ID NO: 24), where all cysteine residues that flank the recognition sequence were substituted with serine to avoid non-physiological cross-linking. The 1.78 Å structure (
The residues that form the hairpin are important for anti-HBs antibody recognition as determined by alanine scanning (
HBsAg can be glycosylated at N146 and this site is also strictly conserved. However, some studies have suggested that this glycosylation site is never fully occupied, resulting in a nearly 1:1 ratio of glycosylated and non-glycosylated isoforms on the surface of viral envelope (Julithe et al., 2014). The glycosylation may be either NAG-NAG-MAN or NAG-(FUC)-NAG-MAN (Hyakumura et al., 2015). We have modeled both fucosylated and non-fucosylated options by grafting a 7mer and 11mer glycan conjugated at N146 of peptide in the presence of the Fab. We found that both glycosylation forms are tolerated at that location with only minimal torsional adaptations without clashes with the Fab, though the fucosylated (branched) glycan required some additional torsional angle changes to the Fab, as well.
Protection and Therapy in Humanized Mice
HBV infection is limited to humans, chimpanzees, tree shrews, and human liver chimeric mice (Sun and Li, 2017). To determine whether our anti-HBs bNAbs prevent infection in vivo we produced human liver chimeric Fah−/− NODRag1−/− IL2rgnull (huFNRG) mice (de Jong et al., 2014) and injected them with control or H020 (Group-I) or H007 (Group-II) antibodies before infection with HBV (
To determine whether bNAbs can also control established infections, we infused control antibody or bNAb H020 (Group-I) or H007 (Group-II) into huFNRG mice with HBV viral loads of 106-108 copies/ml of serum (
Animals that received the control antibodies further increased viremia to as high as ˜1011 DNA copies/ml (
To determine whether the animals that showed increased HBV DNA levels during antibody monotherapy developed escape mutations, we sequenced the viral DNA recovered from mouse blood. All three mice that escaped H020 (Group-I) or H007 (Group-II) monotherapy developed viruses that carried a G145R mutation in the S-protein (arrow-1/3 in
To determine whether a combination of bNAbs targeting 2 separate epitopes would interfere with the emergence of resistant strains, we co-administered H006+H007 (Group-I and -II, respectively) to 8 HBV-infected huFNRG mice (
To attempt to block the emergence of escape mutations, we combined H017+H019 (Group-III and -I, respectively) bNAbs because they displayed complementary sensitivity to commonly occurring natural mutations (
This Example provides a description of materials, methods, and subjects used to obtain the foregoing results.
Experimental Models and Subjects
Human Subjects
Volunteer recruitment and blood draws were performed at the Rockefeller University Hospital under a protocol approved by the institutional review board (IRB QWA-0947). Study participants ranged in age from 22-65 with a mean of 43, the female:male ratio was 81:78 (
Animals
Fah−/− NODRag1−/− IL2rgnull (FNRG) female mice were produced as reported (de Jong et al., 2014) and maintained in the AAALAC-certified facility of the Rockefeller University. Animal protocols were in accordance with NIH guidelines and approved by the Rockefeller University Institutional Animal Care and Use Committee under protocol #18063. Female littermates were randomly assigned to experimental groups.
Cell Lines
HepG2-NTCP cells (Michailidis et al., 2017) and HepDE19 cells (Cai et al., 2012) were maintained in collagen-coated flasks in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% or 3% fetal bovine serum (FBS) and 0.1 mM non-essential amino acids (NEAA). Huh7.5-NTCP cells were maintained in DMEM supplemented with 10% FBS and 0.1 mM NEAA. All liver cell lines were cultured at 37° C. in 5% CO2. Human embryonic kidney HEK293-6E suspension cells were cultured at 37° C. in 8% CO2 with shaking at 120 rpm.
Viruses
HBV-containing supernatant from HepDE19 cells was collected and concentrated as previously described (Michailidis et al., 2017). The concentrated virus stock was aliquoted and stored at −80° C. For in vivo experiments one aliquot of mouse-passaged genotype C HBV virus, originally launched from patient serum (Billerbeck et al., 2016), was stored at −80° C. and thawed for mouse infection experiments. For protection and treatment experiments, animals were challenged intravenously using 1×104 DNA copies per mouse.
Bacteria
E. coli DH5-alpha were cultured at 37° C. with shaking at 230 rpm.
Methods
Collection of Human Samples
Samples of peripheral blood were collected from volunteers at the Rockefeller University Hospital. Serum was isolated by centrifugation of coagulated whole blood, and aliquoted for storage at −80° C. PBMCs were isolated using a cell separation tube with frit barrier and cryopreserved in liquid nitrogen in 90% heat-inactivated FBS supplemented with 10% dimethylsulfoxide (DMSO).
HBV Stock
HepDE19 cells (Cai et al., 2012) were cultured in the absence of tetracycline to induce HBV replication. After seven days, supernatant was collected every other day for two weeks and fresh medium was added. After each collection, medium was spun down to remove cell debris, passed through a 0.22 μm filter, and kept at 4° C. Collected medium was concentrated 100-fold via centrifugation using Centricon Plus-70 centrifugal filter devices (Millipore-Sigma, Billerica, Mass.). Mouse-passaged genotype C HBV virus (Billerbeck et al., 2016) was used for in vivo mouse experiment.
In Vitro HBV Neutralization Assay
In vitro HBV infection was performed as previously described (Michailidis et al., 2017). Briefly, HepG2-NTCP cells were seeded in 96-well collagen-coated plates in DMEM supplemented with 10% FBS and 0.1 mM NEAA. The medium was changed to DMEM with 3% FBS, 0.1 mM NEAA, and 2% DMSO the next day and cultured for an additional 24 hours before infection. The inoculation was in DMEM supplemented with 3% FBS and 0.1 mM NEAA 4% PEG and 2% DMSO. Antibodies or serum samples were incubated with the virus in the inoculation medium for one hour at 37° C. before adding to cells. Serum neutralization capacity (y-axis in
Chemiluminescence Immunoassay
For quantitative analysis of secreted antigen HBsAg or HBeAg, 50 μl of the collected supernatant was loaded into 96-well plates of a chemiluminescence immunoassay (CLIA) kit (Autobio Diagnostics Co., Zhengzhou, China) according to the manufacturer's instructions. Plates were read using a FLUOstar Omega luminometer (BMG Labtech). The absolute concentrations were measured and the relative values were calculated by normalizing to the virus-only control well in the same lane. For example, the absolute HBsAg/HBeAg level in virus-only control well (considered as reference) was 20 NCU/ml (national clinical units per milliliter), while adding one neutralizing serum sample might reduce this to 5 NCU/ml. Therefore, after normalization, the relative HBsAg/HBeAg level were calculated as 100% in control and 25% for this neutralizing serum. Since many factors (virus concentration, cell concentration, immunofluorescence reading, etc.) vary between different plates or different rounds of experiments, normalization is necessary for combining data for comparison.
Immunofluorescence
Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, washed with PBS and permeabilized with 0.1% Triton X-100 in PBS. After blocking with 5% goat serum, the cells were incubated with rabbit anti-HBV core antibody (AUSTRAL Biologicals) overnight at 4° C. and visualized with goat anti-rabbit Alexa Fluor 594 (Thermo Fisher Scientific). Nuclei were stained with DAPI. Cells were imaged using a Nikon Eclipse TE300 fluorescent microscope and processed with ImageJ. For high-content imaging analysis ImageXpress Micro XLS (Molecular Devices, Sunnyvale, Calif.) was used. The absolute HBc+ percentages were obtained and the relative percentage of HBc+ cells was calculated by normalizing to the virus-only control well in the same lane. For example, the absolute HBc+ cell percentage in virus-only control well (considered as reference) was 40%, while adding one neutralizing serum sample might reduce this to 10%. Therefore, after normalization, the relative percentages of HBc+ cells were calculated as 100% in control well and 25% for this neutralizing serum sample. Since many factors (virus concentration, cell concentration, immunofluorescence reading, etc.) vary between different plates or different rounds of experiments, normalization is necessary for combining data for comparison.
ELISA Assays
Blood samples were submitted to Memorial Sloan Kettering Cancer Center for clinical testing. The presence of HBsAg protein and anti-HBc antibody, as well as anti-HBs titers, were determined by ELISA (Abbott Laboratories) as per the manufacturer's instructions.
The binding of serum or recombinant IgG antibodies to HBsAg proteins (see KEY RESOURCES TABLE) was measured by coating ELISA plates with 10 μg/ml of antigen in PBS. Plates were blocked with 2% BSA in PBS and incubated with antibody for one hour at room temperature. Visualization was with HRP-conjugated goat anti-human IgG (Thermo Fisher Scientific). The 50% effective concentration (EC50) needed for maximal binding was determined by non-linear regression analysis in software PRISM.
For competition ELISAs plates were coated with 0.12 μg/m1HBsAg (adr CHO) and incubated with 16.7 μg/ml primary antibody for two hours, followed by directly adding 0.25 μg/ml biotinylated secondary antibody and incubation for 30 minutes all at room temperature. Detection was with streptavidin-HRP (BD Biosciences).
Autoreactivity and Polyreactivity
Autoreactivity and polyreactivity assays were performed as described (Gitlin et al., 2016; Mayer et al., 2017; Robbiani et al., 2017). For the autoreactivity assays, monoclonal antibodies were tested with the Antinuclear antibodies (HEp-2) Kit (MBL International). Antibodies were incubated at 100 μg/ml and were detected with Alexa Fluor 488 AffiniPure F(ab′)2 Fragment Goat Anti-Human IgG (H+L) (Jackson ImmunoResearch) at 10 μg/ml. Fluorescence images were taken with a wide-field fluorescence microscope (Axioplan 2, Zeiss), a 40× dry objective and a Hamamatsu Orca ER B/W digital camera. Images were analyzed with Image J. Human serum containing antinuclear antibodies (MBL International) was used as a positive control. For the polyreactivity ELISA assays, antibody binding to five different antigens, double-stranded DNA (dsDNA), insulin, keyhole limpet hemocyanin (KLH), lipopolysaccharides (LPS), and single-stranded DNA (ssDNA), were measured. ED38 (Wardemann et al., 2003) and mG053 (Yurasov et al., 2005) antibodies were used as positive and negative controls, respectively.
Synthetic Peptides
Eighteen peptides spanning the antigenic loop region of S-protein antigen were synthesized at the Proteomics Resource Center of The Rockefeller University. For peptide ELISAs plates were coated with 10 μg/ml peptide in PBS.
HBsAg-Binding Memory B Cells
S-protein (adr serotype) expressed and purified from Chinese hamster ovary (CHO) cells (ProSpec) and ovalbumin (Sigma-Aldrich) were biotinylated using EZ-Link™ Micro NHS-PEG4-Biotinylation kit (Thermo Fisher Scientific). S-protein-PE and S-protein-APC were prepared by incubating 2-3 μg of biotin-S-protein with streptavidin-PE (eBioscience) or streptavidin-APC (BD Biosciences) in PBS respectively overnight at 4° C. in the dark. Ovalbumin-Alexa Fluor 488 was generated by incubating biotin-ovalbumin with streptavidin-Alexa Fluor 488 (Thermo Fisher Scientific).
B cell purification, labeling, and sorting were as previously described (Escolano et al., 2019; Robbiani et al., 2017; Tiller et al., 2008; von Boehmer et al., 2016). Briefly, PBMCs were thawed and washed with RPMI medium at 37° C. B lymphocytes were positively selected using CD19 MicroBeads (Miltenyi Biotec) followed by incubation with human Fc block (BD Biosciences) and anti-CD20-PECy7 (BD Biosciences), anti-IgG-Bv421 (BD Biosciences), S-protein-PE at 10 μg/ml, S-protein-APC at 10 μg/ml, and ovalbumin-Alexa Fluor 488 at 10 μg/ml at 4° C. for 20 minutes. Single CD20+ IgG+ S-protein-PE+ S-protein-APC+ Ova-Alexa Fluor 488− memory B cells were sorted into 96-well plates using a FACSAriaII (Becton Dickinson) and stored at −80° C.
Antibody Cloning, Sequencing and Production
Antibody cloning, sequencing and production were done as previously reported (Robbiani et al., 2017; Tiller et al., 2008; von Boehmer et al., 2016). Primers are listed in Table S3. Unmutated common ancestor (UCA) antibody sequences of H006, H019 and H020 were synthesized by gBlock IDT (Table S3) and were inserted into antibody vectors for expression. V(D)J gene segment and CDR3 sequences were determined by IgBlast (Ye et al., 2013) and/or IMGT/V-QUEST (Brochet et al., 2008).
S-Protein Mutagenesis
Oligonucleotides fragments with the target point mutations were synthesized by gBlock IDT (Table S3), and were substituted into the antigenic loop region in plasmid p1.3×HBV-WT by Sequence and Ligation-Independent Cloning (SLIC) (Jeong et al., 2012). Mutant plasmids were transfected into Huh-7.5-NTCP cells using X-tremeGENE 9 DNA Transfection Reagent (Sigma-Aldrich) and the culture medium was changed to serum-free DMEM after 24 hours. Supernatants were collected 2 days later and stored at −80° C. Serum-free medium (50 μl) was directly used to coat ELISA plates.
Crystallization, X-Ray Data Collection, Structure Determination and Refinement
Antibody Fab (25 mg/ml) in 50 mM Tris 8.0, 50 mM NaCl was mixed with peptide (5 mg/ml) in the same buffer at 5:1 v/v. Molar ratio of Fab:peptide is around 1:2. Crystals were obtained upon substitution of all peptide-11 cysteine residues with serine in the peptide synthesis (Proteomics Resource Center, RU). The crystallization condition for Fab15/peptide-11Ser was identified from a commercial screen (Morpheus by Molecular Dimensions) by the sitting-drop vapor-diffusion method at room temperature. The crystal used for data collection was obtained directly from the initial setup (position E1) in a precipitant solution consisting of 0.12 M Ethylene glycols (Di, Tri, Tetra and Penta-ethylene glycol), 0.1 M Buffer Mix 1 (Imidazole/MES) at pH 6.5 and 30% Precipitant Mix 1 (20% v/v PEG 500* MME; 10% w/v PEG 20000). The crystals were flash-cooled in liquid nitrogen directly from the mother liquor without additional cryoprotectant. X-ray diffraction data were collected from a single crystal on the Advanced Photon Source (APS) beamline 24-ID-E to 1.78 Å resolution. The data were integrated and scaled with the program XDS (Kabsch, 2010a, b) and other data processing utilities from the CCP4 suite (Collaborative Computational Project, 1994) using RAPD, the software available at the beam-line. Initial phase estimates and electron-density maps were obtained by molecular replacement with Phaser (McCoy et al., 2007) using a single FAB molecule from (PDB: SGGU) as an initial search model in Phenix (Adams et al., 2010). Iterative model building and structural refinement were manually performed using COOT (Emsley et al., 2010) and Phenix, respectively. The peptide density was well defined, and refined to 90% occupancy, for residues STKPSDGNST (SEQ ID NO: 25). All other residues were not visible and the area where they would be is fully solvent, with no crystal contacts involving any of the peptide atoms. The quality of the final model was good as noted in a Ramachandran of 96% of the observed residues within the allowable region. Data-collection and refinement statistics are summarized (
Humanized Mice and In Vivo Studies
Six to eight week old Fah−/− NODRag1−/− IL2rgnull (FNRG) female mice were transplanted with one million human hepatocytes from a pediatric female donor HUM4188 (Lonza Bioscience) as previously described (de Jong et al., 2014). Briefly, during isoflurane anesthesia mice underwent skin and peritoneal incision, exposing the spleen. One million hepatocytes were injected in the spleen using a 28-gauge needle. The peritoneum was then approximated using 4.0 VICRYL sutures (Johnson & Johnson), and skin was closed using MikRon Autoclip surgical clips (Becton Dickinson). Mice were cycled off the drug nitisinone (Yecuris) on the basis of weight loss and overall health. Humanization was monitored by human albumin quantification in mouse serum using a human-specific ELISA (Bethyl Labs). Humanized FNRG mice with human albumin values greater than 1 mg/ml were used for infection experiments. The human liver chimeric (huFNRG) mice are extremely immunodeficient. The Rag1−/− renders the mice B and T cell deficient and the IL2rgnull mutation prevents cytokine signaling through multiple receptors, leading to a deficiency in functional NK cells. Moreover, the genetic background is NOD background, with suboptimal antigen presentation, defects in T and NK cell function, reduced macrophage cytokine production, suppressed wound healing, and C5 complement deficiency. Thus the mice would be unable to produce antibody-dependent effector functions, including antibody-dependent cell-mediated cytotoxicity (ADCC), or passive antibody-enhanced adaptive immunity.
Mice were challenged intravenously with 1×104 genome equivalent (GE) of mouse-passaged genotype C HBV viruses diluted in PBS. For prophylaxis experiments, 500 μg of monoclonal antibody was administered intraperitoneally at 20 and again at 6 hours before infection. For therapy experiments, huFNRG mice with established HBV infections (<108 DNA copies/ml of serum) were injected with 500 μg of each monoclonal antibody intraperitoneally 3 times per week.
DNA in mouse serum collected weekly was extracted using a QIAamp DNA Blood Mini Kit (Qiagen). Total HBV DNA was determined by quantitative PCR (Michailidis et al., 2017). PCR was performed using a TaqMan Universal PCR Master Mix (Applied Biosystems), primers and probe (Table S3).
To obtain HBV DNA from serum for sequence analysis the S domain was amplified using primers (Table S3), and Phusion DNA polymerase (Thermo Fisher Scientific). Initial denaturation was at 98° C. for 30 s, followed by 40 amplification cycles (98° C. for 10 s, 60° C. for 30 s, and 72° C. for 30 s), followed by one cycle at 72° C. for 5 min. A ˜700 bp fragment was gel extracted for Sanger sequencing. Sequence alignments were performed using MacVector.
Quantification and Statistical Analysis
The detailed information of statistical analysis could be found in the Result and Figure Legends. Correlation was evaluated by Spearman's rank correlation method (
Previous studies have identified several anti-HBs neutralizing antibodies from a small number of otherwise unselected spontaneously recovered or vaccinated individuals (Cerino et al., 2015; Colucci et al., 1986; Eren et al., 1998; Heijtink et al., 2002; Heijtink et al., 1995; Jin et al., 2009; Kim and Park, 2002; Li et al., 2017; Sa'adu et al., 1992; Sankhyan et al., 2016; Tajiri et al., 2007; Tokimitsu et al., 2007; Wang et al., 2016). In contrast, in the present disclosure, sera from 144 exposed volunteers was screened to identify elite neutralizers. Serologic activity varied greatly among the donors with a small number of individuals demonstrating high levels of neutralizing activity. To understand this activity, we isolated 244 anti-HBs antibodies from single B cells obtained from the top donors. Each of the elite donors tested showed expanded clones of memory B cells expressing bNAbs that targeted 3 non-overlapping sites on the S-protein. Moreover, the amino acid sequence of several of the bNAbs was highly similar in different individuals. These closely related antibodies target the same epitope.
The near identity of clones of HBV bNAbs in unrelated elite individuals is akin to reports for elite responders to HIV-1 (Scheid et al., 2011; West et al., 2012), influenza (Laursen and Wilson, 2013; Pappas et al., 2014; Wrammert et al., 2011), Zika (Robbiani et al., 2017), and malaria (Tan et al., 2018). However, none of the elite anti-HBs bNAbs shares both IgH and IgL with previously reported HBV neutralizing antibodies, the best of which have been tested in the clinic but are less potent than some of the bNAbs of this disclosure (libivirumab IC50: 35 ng/ml, tuvirumab IC50: ˜100 ng/ml) (Galun et al., 2002; Heijtink et al., 2001; van Nunen et al., 2001).
The described alanine scanning and competition binding analyses are consistent with the existence of at least 3 domains that can be recognized concomitantly by bNAbs (Gao et al., 2017; Tajiri et al., 2010; Zhang et al., 2016). However, the domains do not appear to be limited to either of two previously defined circular peptide epitopes, 123-137 and 139-148 (Tajiri et al., 2010; Zhang et al., 2016). Instead, residues spanning most of the external domain can contribute to binding by both Group-I and -II antibodies. For example, alanine scanning indicates that Group-I H020 binding is dependent on I110, K141, D144, G145 and T148, while Group-II H016 binding depends on T123, D144, and G145. Thus, despite having non-overlapping binding sites some of the essential residues are shared by Group-I and II suggesting that the epitopes are conformational. Moreover, the antibody epitopes on S-protein identified using mouse and human antibodies may be distinct (Chen et al., 1996; Ijaz et al., 2003; Paulij et al., 1999; Zhang et al., 2019; Zhang et al., 2016). Finally, G145, a residue that is frequently mutated in infected humans (Ma and Wang, 2012; Tong et al., 2013), is believed to be essential for binding by all the Group-II but not all Group-I or -III antibodies tested.
Crystallization of the Group-II bNAb H015 and its linear epitope revealed a loop that includes P142, S/T143, D144, and G145, all of which are frequently mutated during natural infection to produce well-documented immune escape variants (Hsu et al., 2015; Ijaz et al., 2012; Ma and Wang, 2012; Salpini et al., 2015). In addition to immune escape, the residues that form this structure are also essential for infectivity, possibly by facilitating virus interactions with cell surface glycosaminoglycans (Sureau and Salisse, 2013). Mutations in K141, P142 as well as C139 and C147, all of which contribute to the stability of the structure, decrease viral infectivity (Salisse and Sureau, 2009). Without intending to be bound by any particular theory, it is considered that drugs that destabilize the newly elucidated H015-peptide loop structure may also interfere with infectivity.
The G145R mutation, which is among the most frequent immune escape variants, replaces a small neutral residue with a bulky charged residue that would likely interfere with antigenicity by destroying the salt bridge between K141 and D144 that anchors the peptide loop. However, this drastic structural change does not alter infectivity (Salisse and Sureau, 2009), possibly because the additional charge compensates for otherwise altered interactions between HBV and cell surface glycosaminoglycans (Sureau and Salisse, 2013). Thus, the additional charge may allow G145R to function as a dominant immune escape variant while preserving infectivity.
The present disclosure describes antibodies directed at S-protein antigen in part because this is the antigen used in the currently FDA-approved vaccines, and because purified S-protein blocked nearly all of the neutralizing activity in the serum of the elite neutralizers irrespective of whether they were vaccinated or spontaneously recovered. Nevertheless, individuals who recover from infection also produce antibodies to the PreS1 domain of HBsAg (Li et al., 2017; Sankhyan et al., 2016). The PreS1 domain is essential for the virus to interact with the entry factor NCTP on hepatocytes and potent neutralizing antibodies to PreS1 have been described (Li et al., 2017). However, these are not naturally occurring antibodies but rather randomly paired IgH and IgL chains derived from phage libraries obtained from unexposed or vaccinated healthy donors (Li et al., 2017). Moreover, the phage antibodies required further engineering to enhance their neutralizing activity (Li et al., 2017). Thus, whether the human immune system also produces potent anti-PreS1 bNAbs has not been determined.
Chronic HBV infection remains a major global public health problem in need of an effective curative strategy (Graber-Stiehl, 2018; Lazarus et al., 2018; Revill et al., 2016). Chronically infected individuals produce an overwhelming amount of HBsAg that is postulated to incapacitate the immune system. Consequently, immune cells, which might normally clear the virus, are unable to react to antigen, a phenomenon referred to as exhaustion or anergy (Ye et al., 2015). The appearance of anti-HBs antibodies is associated with spontaneous recovery from the disease, perhaps because they can clear the antigen and facilitate the emergence of a productive immune response (Celis and Chang, 1984; Zhang et al., 2016; Zhu et al., 2016). These findings led to the hypothesis that passively administered antibodies might be used in conjunction with antiviral drugs to further decrease the antigenic burden while enhancing immune responses that maintain long-term control of the disease. The presently described results in huFNRG mice infected with HBV indicate that antibody monotherapy with a potent bNAb can lead to the emergence of the very same escape mutations commonly found in chronically infected individuals. Moreover, not all bNAb combinations are effective in preventing escape by mutation. Combinations that target separate epitopes but have overlapping sensitivity to commonly occurring escape mutations such as H006 and H007 are ineffective. In contrast, combinations with complementary sensitivity to common escape mutations prevent the emergence of escape mutations in huFNRG mice infected with HBV. Thus, as described above, the present disclosure provides immunotherapy for HBV infection with combinations of antibodies with complementary activity to avert this potential problem.
The following reference listing is not an indication that any particular reference(s) is material to patentability.
It will be recognized from the foregoing that the present disclosure describes screening individuals who were either vaccinated or had spontaneously recovered from HBV infection. Antibody cloning from memory B cells revealed that all 5 of the top individuals produced clones of broadly neutralizing antibodies (bNAbs) that targeted 3 non-overlapping epitopes on the HBV S antigen (HBsAg). Clones with the same immunoglobulin variable, diversity and joining heavy and light chain genes were shared among elite neutralizers. Single bNAbs protected humanized mice against infection, but selected for resistance mutations in mice with established infection. In contrast, infection was controlled in the absence of detectable escape mutations by a combination of bNAbs targeting non-overlapping epitopes with complementary sensitivity to mutations that commonly emerge during human infection. The co-crystal structure of one of the bNAbs with a peptide epitope containing residues frequently mutated in human immune escape variants revealed a loop anchored by oppositely charged residues. The structure provides a molecular explanation for why immunotherapy for chronic HBV infection may require combinations of complementary bNAbs, as described herein.
While the disclosure has been described through specific embodiments, routine modifications will be apparent to those skilled in the art and such modifications are intended to be within the scope of the present disclosure.
This application claims priority to U.S. provisional patent application No. 62/898,735, filed Sep. 11, 2019, and to U.S. provisional patent application no. 62/982,276, filed Feb. 27, 2020, the entire disclosures of each of which are incorporated herein by reference in their entireties.
This invention was made with government support under grant no. UL1TR001866 awarded by The National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/050509 | 9/11/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62982276 | Feb 2020 | US | |
| 62898735 | Sep 2019 | US |
